U.S. patent application number 16/130177 was filed with the patent office on 2019-03-28 for assays for detecting multiple tick-borne pathogens.
The applicant listed for this patent is The United States of America, as represented by the Secretary of Agriculture, The Texas A&M University System, The United States of America, as represented by the Secretary of Agriculture. Invention is credited to Maria D. Esteve-Gasent, Pamela J. Ferro, Joseph J. Modarelli, Adalberto A. Perez De Leon.
Application Number | 20190093149 16/130177 |
Document ID | / |
Family ID | 65806488 |
Filed Date | 2019-03-28 |
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United States Patent
Application |
20190093149 |
Kind Code |
A1 |
Perez De Leon; Adalberto A. ;
et al. |
March 28, 2019 |
ASSAYS FOR DETECTING MULTIPLE TICK-BORNE PATHOGENS
Abstract
Kits, methods, and material for use in detecting tick-borne
microorganism(s) are provided herein. One is able to detect DNA
from several different species of microorganisms from samples
(bodily fluid, tissue, etc.) from ticks and/or mammals to determine
if one or more of the microorganisms has infected the tick and/or
mammal. The assay uses PCR techniques with a multiple primers and
probes. The microorganisms can be in the Borrelial Layer, the
Rickettsiales Layer, and/or the Babesial Layer.
Inventors: |
Perez De Leon; Adalberto A.;
(Wake Forest, NC) ; Esteve-Gasent; Maria D.;
(College Station, TX) ; Ferro; Pamela J.; (Bryan,
TX) ; Modarelli; Joseph J.; (Bluffton, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The United States of America, as represented by the Secretary of
Agriculture
The Texas A&M University System |
Washington
College Station |
DC
TX |
US
US |
|
|
Family ID: |
65806488 |
Appl. No.: |
16/130177 |
Filed: |
September 13, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62563780 |
Sep 27, 2017 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6806 20130101;
C12Q 2600/112 20130101; C12Q 2600/166 20130101; C12Q 1/689
20130101; C12Q 1/686 20130101; C12Q 2600/16 20130101 |
International
Class: |
C12Q 1/689 20060101
C12Q001/689; C12Q 1/6806 20060101 C12Q001/6806; C12Q 1/686 20060101
C12Q001/686 |
Claims
1. A kit for detecting Borrelia spp., Rickettsia-like bacteria, or
Babesia spp. in a sample comprising at least one primer set for
detecting said Borrelia spp., Rickettsia-like bacteria, or Babesia
spp., an amplification control DNA, instructions for using said at
least one primer set and said amplification control plasmid,
optionally PCR reagents, and optionally reagents for isolating DNA
from a sample.
2. The kit of claim 1, wherein said at least one primer set for
said Borrelia spp. is selected from the primer sets of: (i) SEQ ID
NOs: 1 and 2, (ii) SEQ ID NOs: 5 and 7, (iii) SEQ ID NOs: 6 and 7,
(iv) SEQ ID NOs: 11 and 13, (v) SEQ ID NOs: 12 and 13, and (vi) SEQ
ID NOs: 17 and 18.
3. The kit of claim 2, further comprising at least one probe that
contains a DNA sequence that present within the amplicon generated
by each of said primer sets.
4. The kit of claim 3, wherein said at least one probe comprises
SEQ ID NOs: 3, 8, 14, or 19.
5. The kit of claim 1, wherein said at least one primer set for
said Rickettsia-like bacteria is selected from the primer sets of:
(vii) SEQ ID NOs: 21 and 22, (viii) SEQ ID NOs: 25 and 26, (ix) SEQ
ID NOs: 29 and 30, (x) SEQ ID NOs: 33 and 34, and (xi) SEQ ID NOs:
36 and 34.
6. The kit of claim 5, further comprising at least one probe that
contains a DNA sequence that present within the amplicon generated
by each of said primer sets.
7. The kit of claim 6, wherein said at least one probe comprises
SEQ ID NOs: 23, 27, or 31.
8. The kit of claim 1, wherein said at least one primer set for
said Babesia spp. is selected from the primer sets of: (xii) SEQ ID
NOs: 38 and 39, and (xiii) SEQ ID NOs: 77 and 39.
9. The kit of claim 8, further comprising at least one probe that
contains a DNA sequence that present within the amplicon generated
by each of said primer sets.
10. The kit of claim 9, wherein said at least one probe comprises
SEQ ID NO: 40.
11. The kit of claim 1; wherein said kit comprises said primer
sets: (i) SEQ ID NOs: 1 and 2, (ii) SEQ ID NOs: 5 and 7, (iii) SEQ
ID NOs: 6 and 7, (iv) SEQ ID NOs: 11 and 13, (v) SEQ ID NOs: 12 and
13, (vi) SEQ ID NOs: 17 and 18, (vii) SEQ ID NOs: 21 and 22, (viii)
SEQ ID NOs: 25 and 26, (ix) SEQ ID NOs: 29 and 30, (x) SEQ ID NOs:
33 and 34, (xi) SEQ ID NOs: 36 and 34, (xii) SEQ ID NOs: 38 and 39,
and (xiii) SEQ ID NOs: 77 and 39.
12. The kit of claim 11, wherein said Borrelia spp. is selected
from the group consisting of Borrelia burgdorferi, B. hermsii, B.
turicatae, and B. parkeri; wherein said Rickettsia-like bacteria is
selected from the group consisting of Ehrlichia canis, E.
chaffeensis, E. ewingii, Anaplasma phagocytophilum, and Rickettsia
rickettsii; and wherein said Babesia spp. is selected from the
group consisting of Babesia canis vogeli, B. gibsoni, B. canis
canis, B. canis rossi, B. caballi, B. odocoilei, B. divergens, and
B. bigemina.
13. The kit of claim 11, wherein said kit further comprises at
least one Borrelia spp. probe, at least one Rickettsia-like
bacteria probe, and at least one Babesia spp. probe; wherein said
at least one Borrelia spp. probe comprises at least one of SEQ ID
NOs: 3, 8, 14, and 19; wherein said at least one Rickettsia-like
bacteria probe comprises at least one of SEQ ID NOs: 23, 27, and
31; and wherein said at least Babesia spp. probe comprises SEQ ID
NO: 40.
14. The kit of claim 11, wherein said primer sets further comprise
(xiv) SEQ ID NOs: 46 and 47, (xv) SEQ ID NOs: 49 and 50, (xvi) SEQ
ID NOs: 52 and 53, (xvii) SEQ ID NOs: 56 and 57, (xviii) SEQ ID
NOs: 60 and 61, (xix) SEQ ID NOs: 64 and 65; and wherein said
Borrelia spp. is selected from the group consisting of Borrelia
burgdorferi, B. hermsii, B. turicatae, and B. parkeri; wherein said
Rickettsia-like bacteria is selected from the group consisting of
Ehrlichia canis, E. chaffeensis, E. ewingii, Anaplasma
phagocytophilum, and Rickettsia rickettsii; and wherein said
Babesia spp. is selected from the group consisting of Babesia canis
vogeli, B. gibsoni, B. canis canis, B. canis rossi, B. caballi, B.
odocoilei, B. divergens, B. bigemina, and B. conradae.
15. The kit of claim 14, wherein said kit further comprises at
least one Borrelia spp. probe, at least one Rickettsia-like
bacteria probe, and at least one Babesia spp. probe; wherein said
at least one Borrelia spp. probe comprises at least one of SEQ ID
NOs: 3, 8, 14, and 19; wherein said at least one Rickettsia-like
bacteria probe comprises at least one of SEQ ID NOs: 23, 27, and
31; and wherein said at least Babesia spp. probe comprises at least
one of SEQ ID NOs: 40, 54, 58, 62, and 66.
16. The kit of claim 1, wherein said amplification control plasmid
comprises SEQ ID NO: 68.
17. The kit of claim 1, wherein when said sample is obtained from a
canine, said amplification control plasmid comprises SEQ ID NO: 45,
and said kit further comprises canine specific primer set of SEQ ID
NOs: 42 and 43 and a canine specific probe comprising SEQ ID NO:
44.
18. The kit of claim 1, wherein said Borrelia spp. is B.
burgdorferi; wherein said Rickettsia-like bacteria is E. canis, E.
chaffeensis, and A. phagocytophilum; wherein said primer set for
detecting said B. burgdorferi comprises at least one of (a)(i) SEQ
ID NOs: 69 and 70 and (a)(ii) SEQ ID NOs: 1 and 2; wherein said
primer set for detecting E. canis comprises at least one of (b)(i)
SEQ ID NOs: 71 and 72 and (b)(ii) SEQ ID NOs: 29 and 30; wherein
said primer set for detecting E. chaffeensis comprises at least one
of (c)(i) SEQ ID NOs: 73 and 74 and (c)(ii) SEQ ID NOs: 33 and 34;
and wherein said primer set for detecting A. phagocytophilum
comprises at least one of (d)(i) SEQ ID NOs: 75 and 76 and (d)(ii)
SEQ ID NOs: 25 and 26.
19. The kit of claim 18, wherein when said sample is obtained from
a canine, said amplification control plasmid comprises SEQ ID NO:
45, and said kit further comprises canine specific primer set of
SEQ ID NOs: 42 and 43.
20. A method for detecting at least one tick-borne pathogen in a
sample from an animal comprising a) optionally, isolating DNA from
said sample to provided isolated DNA; b) contacting said isolated
DNA with a plurality of primer sets and DNA polymerase to provide a
first layer PCR mix, wherein said plurality of primer sets detect
at least one tick-borne pathogen; c) performing PCR on said first
layer PCR mix to generate at least one amplicon; and d) determining
the identify of said generated at least one amplicon, wherein said
at least one amplicon is specific for detecting said at least one
tick-borne pathogen in said sample.
21. The method of claim 20, wherein said animal from which said
sample is obtained is a tick or a mammal.
22. The method of claim 20, wherein said at least one tick-borne
pathogen is a Borrelia spp., Rickettsia-like bacteria, or a Babesia
spp.; wherein said primer sets for detecting said Borrelia spp. is
at least one of said primer sets of: (i) SEQ ID NOs: 1 and 2, (ii)
SEQ ID NOs: 5 and 7, (iii) SEQ ID NOs: 6 and 7, (iv) SEQ ID NOs: 11
and 13, (v) SEQ ID NOs: 12 and 13, and (vi) SEQ ID NOs: 17 and 18;
wherein said primer sets for detecting said Rickettsia-like
bacteria is at least one of said primer sets of: (vii) SEQ ID NOs:
21 and 22, (viii) SEQ ID NOs: 25 and 26, (ix) SEQ ID NOs: 29 and
30, (x) SEQ ID NOs: 33 and 34, and (xi) SEQ ID NOs: 36 and 34; and
wherein said primer set for detecting said Borrelia spp. is at
least one of the primer sets of: (xii) SEQ ID NOs: 38 and 39, and
(xiii) SEQ ID NOs: 77 and 39.
23. The method of claim 22, wherein said Borrelia spp. is selected
from the group consisting of Borrelia burgdorferi, B. hermsii, B.
turicatae, and B. parkeri; wherein said Rickettsia-like bacteria is
selected from the group consisting of Ehrlichia canis, E.
chaffeensis, E. ewingii, Anaplasma phagocytophilum, and Rickettsia
rickettsii; and wherein said Babesia spp. is selected from the
group consisting of Babesia canis vogeli, B. gibsoni, B. canis
canis, B. canis rossi, B. caballi, B. odocoilei, B. divergens, and
B. bigemina.
24. The method of claim 22, wherein said first layer PCR mix
further comprises at least one probe to detect Borrelia spp., at
least one probe to detect Rickettsia-like bacteria probe, and at
least one probe to detect Babesia spp.; wherein said at least one
Borrelia spp. probe comprises at least one of SEQ ID NOs: 3, 8, 14,
and 19; wherein said at least one Rickettsia-like bacteria probe
comprises at least one of SEQ ID NOs: 23, 27, and 31; and wherein
said at least Babesia spp. probe comprises SEQ ID NO: 40.
25. The method of claim 22, wherein said tick-borne pathogen is
Borrelia spp., said method further comprises e) contacting said
isolated DNA with at least one Borrelia spp. primer sets and DNA
polymerase to provide a second layer PCR mix; f) performing PCR on
said second layer PCR mix to generate at least one amplicon; and g)
determining the identify of said generated at least one amplicon,
wherein said at least one Borrelia spp. primer sets detect B.
burgdorferi, B. hermsii, B. turicatae, or B. parkeri wherein said
B. burgdorferi primer set is (i) SEQ ID NOs: 1 and 2; wherein said
B. hermsii set is at least one of (ii) SEQ ID NOs: 5 and 7, (iii)
SEQ ID NOs: 6 and 7, (xiv) SEQ ID NOs: 46 and 47, and (xv) SEQ ID
NOs: 49 and 50; wherein said B. turicatae is at least one of (iv)
SEQ ID NOs: 11 and 13 and (v) SEQ ID NOs: 12 and 13; and wherein
said B. parkeri primer set is (vi) SEQ ID NOs: 17 and 18.
26. The method of claim 25, wherein said second layer PCR mix
further comprises at least one probe to detect B. burgdorferi, B.
hermsii, B. turicatae, or B. parkeri; wherein said B. burgdorferi
probe comprises SEQ ID NO: 3; wherein said B. hermsii probe
comprises SEQ ID NO: 8; wherein said B. turicatae probe comprises
SEQ ID NO: 14; and wherein said B. parkeri probe comprises SEQ ID
NO: 19.
27. The method of claim 22, wherein said tick-borne pathogen is
Rickettsia-like bacteria, said method further comprises e)
contacting said isolated DNA with at least one Rickettsia-like
bacteria primer sets and DNA polymerase to provide a second layer
PCR mix; f) performing PCR on said second layer PCR mix to generate
at least one amplicon; and g) determining the identify of said
generated at least one amplicon; wherein said at least one
Rickettsia-like bacteria primer sets detect Rickettsia rickettsii,
Anaplasma phagocytophilum, Ehrlichia canis, E. chaffeensis, or E.
ewingii; wherein said R. rickettsii primer set is (vii) SEQ ID NOs:
21 and 22; wherein said A. phagocytophilum primer set is (viii) SEQ
ID NOs: 25 and 26; wherein said E. canis primer set is (ix) SEQ ID
NOs: 29 and 30; wherein said E. chaffeensis primer set is (x) SEQ
ID NOs: 33 and 34; and wherein said E. ewingii primer set is (xi)
SEQ ID NOs: 36 and 34.
28. The method of claim 27, wherein said second layer PCR mix
further comprises at least one probe to detect R. rickettsii, A.
phagocytophilum, E. canis, E. chaffeensis, or E. ewingii; wherein
said R. rickettsii probe comprises SEQ ID NO: 23; wherein said A.
phagocytophilum probe comprises SEQ ID NO: 27; and wherein said E.
canis probe, E. chaffeensis, and E. ewingii comprise SEQ ID NO:
31.
29. The method of claim 22, wherein said tick-borne pathogen is
Babesia spp., said method further comprises e) contacting said
isolated DNA with at least one Babesia primer sets and DNA
polymerase to provide a second layer PCR mix; f) performing PCR on
said second layer PCR mix to generate at least one amplicon; and g)
determining the identify of said generated at least one amplicon,
wherein said at least one Babesia primer sets detect Babesia canis
vogeli, B. gibsoni, or B. conradae; wherein said B. canis vogeli
primer set is at least one of (xvi) SEQ ID NOs: 52 and 53 and
(xvii) SEQ ID NOs: 56 and 57; wherein said B. gibsoni primer set is
(xviii) SEQ ID NOs: 60 and 61; and wherein said B. conradae primer
set is (xix) SEQ ID NOs: 64 and 65.
30. The method of claim 29, wherein said second layer PCR mix
further comprises at least one probe to detect B. canis vogeli, B.
gibsoni, or B. conradae; wherein said B. canis vogeli probe
comprises at least one of SEQ ID NO: 54 and SEQ ID NO: 58; wherein
said B. gibsoni probe comprises SEQ ID NO: 62; and wherein said B.
conradae probe comprises SEQ ID NO: 66.
31. The method of claim 20, wherein said at least one tick-borne
pathogen is selected from the group consisting of B. burgdorferi,
E. canis, E. chaffeensis, and A. phagocytophilum; wherein said
plurality of primer sets comprise a B. burgdorferi primer set, an
E. canis primer set, an E. chaffeensis primer set, and an A.
phagocytophilum primer set; wherein said B. burgdorferi primer set
comprises SEQ ID NOs: 69 and 70 or SEQ ID NOs: 1 and 2; wherein
said E. canis primer set comprises SEQ ID NOs: 71 and 72 or SEQ ID
NOs: 29 and 30; wherein E. chaffeensis primer set comprises SEQ ID
NOs: 73 and 74 or SEQ ID NOs: 33 and 34; and wherein said A.
phagocytophilum primer set comprises SEQ ID NOs: 75 and 76 or SEQ
ID NOs: 25 and 26.
32. The method of claim 31, wherein said step (d) determining the
identify of said generated at least one amplicon comprising running
said amplicon on a gel and determining the size of said amplicon;
wherein when said amplicon is 235 bp or 75 bp, B. burgdorferi is
present in said sample; wherein when said amplicon is 389 bp or 113
bp, E. canis is present in said sample; wherein when said amplicon
is 390 bp or 111 bp, E. chaffeensis is present in said sample; and
wherein when said amplicon is 644 bp or 95 bp, A. phagocytophilum
is present in said sample.
33. A plasmid comprising SEQ ID NO: 68.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This patent application claims priority to U.S. Patent
Application 62/563,780 filed on Sep. 27, 2017, contents of which
are expressly incorporated by reference herein.
SEQUENCE LISTING
[0002] The Sequence Listing submitted via EFS-Web as ASCII
compliant text file format (.txt) filed on Sep. 13, 2018, named
"SequenceListing_ST25", (created on Sep. 12, 2017, 19 KB), is
incorporated herein by reference. This Sequence Listing serves as
paper copy of the Sequence Listing required by 37 C.F.R. .sctn.
1.821(c) and the Sequence Listing in computer-readable form (CRF)
required by 37 C.F.R. .sctn. 1.821(e). A statement under 37 C.F.R.
.sctn. 1.821(f) is not necessary.
BACKGROUND OF THE INVENTION
Field of the Invention
[0003] This invention relates to kits for identifying tick-borne
pathogens in a sample, components for those kits, and methods of
identifying tick-borne pathogens.
Description of the Related Art
[0004] Ticks are obligate blood sucking ectoparasites of
vertebrates particularly mammals and birds. Tick bites cause
mechanical damage, irritation, inflammation and hypersensitivity.
Infestation with a large number of ticks may cause anemia and
reduced productivity. Salivary secretions of some ticks cause
toxicosis and paralysis. Hard ticks may affect human and animal
health by transmission of numerous tick-borne pathogens. The
tick-borne diseases can be caused by viruses, bacteria, or
parasites. Most people become infected through tick bites during
the spring and summer months. Tick-borne diseases are becoming a
serious problem. Lyme disease (LD) and human anaplasmosis have
emerged as two of the most common vector born bacterial illnesses
in the United States.
[0005] LD is the most prevalent arthropod borne disease in the US
with over 30,000 cases reported yearly to CDC. LD is transmitted by
Ixodes scapularis and I. pacificus. Mammalian reservoirs are small
rodents. LD is caused by the Borrelia burgdorferi (Bb), a
gram-negative bacterium, and is a multi-phase disorder in humans
Symptoms include fever, headache, fatigue and a characteristic skin
rash called erythema migrans. Early LD involves flu-like symptoms,
and chronic LD involves arthritis and carditis.
[0006] Ehrlichiosis is a zoonotic disease caused by a small,
gram-negative bacterium belonging to Ehrlichia spp., for example,
Ehrlichia canis (Ec) and Ehrlichia chaffeensis (Ech). Symptoms in
humans involve fever, headache, fatigue and muscle aches. These
symptoms occur within 1-2 weeks following infection. Anaplasmosis
is caused by Anaplasma phagocytophilium (Ap), and A. platys (which
mostly affects canids), a gram-negative, intracellular bacterium.
This bacterium targets neutrophils, alters their function in the
host and forms morulae within vacuoles. Symptoms in humans include
fever, headache, muscle pain, malaise, chills, nausea/abdominal
pain, cough and confusion. Severe clinical presentations may
include difficulty breathing, hemorrhage, renal failure or
neurological problems.
[0007] Rocky Mountain Spotted Fever (RMSF) is caused by a
gram-negative, intracellular, coccobacillus bacterium, called
Rickettsia rickettsii (Rr). Typical symptoms include fever,
lethargy, abdominal pain, vomiting and muscle pain. A rash is found
on 90% of patients with classic RMSF rash developing 2 to 5 days
post fever. In addition, other important tick-borne pathogens that
infect mammals include Borrelia hermsii, B. turicatae, and B.
parkeri which cause tick-borne relapsing fever (TBRF); and Babesia
canis vogeli, B. gibsoni, and B. conradae.
[0008] No diagnostic assay currently exists which allows for quick
and inexpensive detection and identification of TBRF pathogens.
Prior art qPCR assays are unable to distinguish between E. canis
and E. chaffeensis in a single qPCR assay; two distinct qPCR assays
are used to distinguish between these two bacteria. Further, prior
art qPCR assays are unable to detect and identify Borrelia
turnicatae. Most diagnostic laboratories run a "panel of tick-borne
pathogens" upon physician or veterinarian request. In most cases,
those panels are serological tests that will indirectly determine
whether a patient (human or animal) is or was exposed to a
particular pathogen.
[0009] Current assays to detect these pathogens are inadequate
either because the assays are not sensitive enough, not specific
enough, not fast enough, and/or are unable to determine if an
infection is currently occurring or if the infection occurred days,
weeks, or years ago. Jaulhac, et al., J. Clin. MicrobioL
38(5):1895-1900 (2000) describes an assay for identifying B.
burgdorferi sensu lato in synovial fluid from arthritic patients.
Anderson, et al., J. Clin. Microbio. 30(4):775-780 (1992) describes
detection of the etiologic agent of human ehrlichiosis using PCR.
Nikolausz, et al., Biochem. Soc. Trans. 37(2):454-459 (2009)
describes using single-nucleotide primer extension (SNuPE) in a
multiplex PCR assay to identify point mutations and microbial
organisms. Prior art qPCR assays conduct multiple qPCR panels in
multiple wells, and use multiple DNA samples in order to screen for
all of the pathogens--a process that is costly and extremely time
consuming.
[0010] An assay that overcomes the limitations of currently known
diagnostic assays is needed. A low-cost, quick, and sensitive assay
that allows one to differentially diagnose the disease and which
detects and allows identification of the causative pathogenic
microorganisms is needed.
BRIEF SUMMARY OF THE INVENTION
[0011] It is an object of this invention to have a kit for
detecting a tick-borne pathogen in a sample obtained from a tick or
a mammal. It is a further object of this invention that the kit can
detect Borrelia species (such as, B. burgdorferi, B. hermsii, B.
turicatae, and/or B. parkeri), Rickettsia-like bacteria (such as,
Ehrlichia canis, E. chaffeensis, E. ewingii, Anaplasma
phagocytophilum, and Rickettsia rickettsii), and/or Babesia species
(such as, Babesia canis vogeli, B. gibsoni, B. canis canis, B.
canis rossi, B. caballi, B. odocoilei, B. divergens, B. bigemina,
and B. conradae). The kit contains at least one primer set for only
one group of pathogens or, alternatively, at least one primer set
for each group (or layer) of pathogens, amplification control DNA,
optionally PCR reagents, optionally reagents for isolating DNA from
a sample, and instructions on using the kit. The amplification
control can be a plasmid containing SEQ ID NO: 68 and, if the
sample is obtained from a dog, a plasmid containing SEQ ID NO: 45
for which one uses primer set of SEQ ID NOs: 42 and 43 and,
optionally, a probe containing SEQ ID NO: 44.
[0012] The Borrelia group (layer) primer sets can be one or more of
the following: (i) SEQ ID NOs: 1 and 2, (ii) SEQ ID NOs: 5 and 7,
(iii) SEQ ID NOs: 6 and 7, (iv) SEQ ID NOs: 11 and 13, (v) SEQ ID
NOs: 12 and 13, (vi) SEQ ID NOs: 17 and 18, (xiv) SEQ ID NOs: 46
and 47, and (xv) SEQ ID NOs: 49 and 50.
[0013] The Rickettsia-like bacteria group (layer) primer sets can
be one or more of the following: (vii) SEQ ID NOs: 21 and 22,
(viii) SEQ ID NOs: 25 and 26, (ix) SEQ ID NOs: 29 and 30, (x) SEQ
ID NOs: 33 and 34, and (xi) SEQ ID NOs: 36 and 34.
[0014] The Babesia species group (layer) primer sets can be one or
more of the following: (xii) SEQ ID NOs: 38 and 39, (xiii) SEQ ID
NOs: 77 and 39, (xvi) SEQ ID NOs: 52 and 53, (xvii) SEQ ID NOs: 56
and 57, (xviii) SEQ ID NOs: 60 and 61, and (xix) SEQ ID NOs: 64 and
65.
[0015] Optionally, a kit contains probes for each layer; and
optionally, each probe in each layer contains the same fluorescent
label and quencher. For the Borrelia layer, the probe(s) contain(s)
one of SEQ ID NOs: 3, 8, 14, and 19. For the Rickettsia-like
bacteria layer, the probe(s) contain(s) one of SEQ ID NOs: 23, 27,
and 31. For the Babesia layer, the probe(s) contain(s) one of SEQ
ID NOs: 40, 54, 58, 62, and 66.
[0016] It is another object of this invention to have a kit for
detecting a tick-borne pathogen in a sample obtained from a tick or
a mammal. It is a further object of this invention that the kit can
detect B. burgdorferi, E. canis, E. chaffeensis, and/or A.
phagocytophilum in the sample. The kit contains at least one primer
set each pathogen, optionally PCR reagents, optionally reagents for
isolating DNA from a sample, and instructions on using the kit. The
primer sets for B. burgdorferi can be SEQ ID NOs: 69 and 70, or SEQ
ID NOs: 1 and 2. The primer set for E. canis can be SEQ ID NOs: 71
and 72 or SEQ ID NOs: 29 and 30. The primer set for E. chaffeensis
can be SEQ ID NOs: 73 and 74 or SEQ ID NOs: 33 and 34. The primer
set for A. phagocytophilum can be SEQ ID NOs: 75 and 76 or SEQ ID
NOs: 25 and 26. It is another object of this invention that this
kit can contain a label for detecting any amplicon generated by
PCR.
[0017] It is a further object of this invention to have methods for
detecting tick-borne pathogens in a sample by using some or all of
the primers, and optionally some or all of the probes described
above in PCR assays. The method can use an amplification control to
confirm the accuracy of the PCR assays. One amplification control
can be a plasmid containing SEQ ID NO: 68. If the sample is
obtained from a dog, another amplification control can be a plasmid
containing SEQ ID NO: 45 for which one uses primer set of SEQ ID
NOs: 42 and 43 and, optionally, a probe containing SEQ ID NO:
44.
[0018] It is another object of this invention to have a plasmid
containing SEQ ID NO: 68.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIGS. 1A-1D provides the names and sequences of forward and
reverse primers and probes for the indicated microorganisms and the
amplicon that is generated using the indicated forward and reverse
primers. The forward and reverse primers, probe, and amplicon for a
canine amplification control is also provided. SEQ ID NOs are
provided in FIGS. 1A-1D. The forward primers contain an "F" at the
end of their name; reverse primers contain an "R" at the end of
their name, and probes contain a "P" at the end of their name
"sense" means oligonucleotide sequences are on the "sense" strand
of DNA. "anti" means the oligonucleotide sequences are on the
"anti-sense" strand of DNA.
[0020] FIGS. 2A-2C provide the sequence of Tick10.PAC
polynucleotide (SEQ ID NO: 68) which contains the amplicons for the
indicated microorganisms, separated by linkers for ease of
cloning.
[0021] FIG. 3 is a comparison analysis of singleplex real-time PCR
(qPCR) assays quantification cycle (Cq) values against combined
Layerplex qPCR assay Cq values. Pathogens assayed are as follows:
Borrelia burgdorferi (Bb), B. hermsii (Bh), B. turicatae (Bt), B.
parkeri (Bp), Anaplasma phagocytophilum (Ap), Ehrlichia canis (Ec),
E. chaffeensis (Ech), E. ewingii (Ee), Rickettsia rickettsii (Rr),
Babesia species (Bab), and endogenous internal positive control
(NIPcK9). The primers and probes for these pathogens and NIPcK9 are
provided in FIGS. 1A-1C but do not include the primers and probes
listed "Additional Species Specific Assays" for Borrelia hermsii,
Babesia canis vogeli, B. gibsoni, and B. conradae.
DETAILED DESCRIPTION OF THE INVENTION
[0022] This invention involves two related PCR (polymerase chain
reaction) assays to detect and identify certain tick-borne
microorganisms in a sample. One PCR assay is single multiplex PCR
for which one separates any amplified DNA on a gel to detect and
identify if one, two, three or four different microorganisms are
present (referred to as "gel-based PCR" or "gel-based multiplex
PCR" assay). The other PCR assay is a single, multiplex, real-time,
quantitative PCR (qPCR) assay (uses a single well on a plate) which
detects if any of 17 tick-borne microorganisms are present in a
sample and identifies the group(s) to which the microorganism(s)
belong(s) (referred to as "LayerPlex" assay and as "LayerPlex qPCR"
assay). A single LayerPlex qPCR assay detects the presence of a
broad spectrum of tick-borne pathogens (17 pathogens) using a
single well on a multi-well plate which contrasts with prior art
qPCR assays that can screen for only 4-5 targets at one time in one
well. A LayerPlex assay utilizes multiple primer sets and one probe
(with a fluorescent label and quencher) to label more than one
microorganism in a given reaction (generally microorganisms of the
same family or genus are grouped together). In particular, in one
well of a multi-well plate, one places the primers and probes for
three layers (Borrelial, Rickettsiales, and Babesial) as well as
the primers and probes for the endogenous internal positive control
(NIPcK9, if the sample is obtained from a dog) thereby having a 4
layer assay in each well. LayerPlex permit large number of unique
target assays to be grouped under each probe label, up to the qPCR
machine's fluorogenic limit (e.g., 11 targets under 4 probe
labels). The LayerPlex qPCR assay can be performed in 60 minutes or
less, is highly sensitive and specific. Further, it costs much less
to perform than prior art assays, including prior art PCR
assays.
[0023] Based on the results of a LayerPlex assay, a veterinarian or
physician can initiate treatment of the patient (human or
non-human). However, one may want to further detect and identify
the species of the microorganism(s) by conducting additional qPCR
assays using the same or similar reaction conditions as the
LayerPlex assay and using many of the same primer sets but also a
few additional primer sets.
[0024] This LayerPlex assay determine if DNA from 17 different
tick-borne pathogens (in particular, bacteria and/or protozoa) are
present in a sample from an animal. The bacteria which can be
detected are Borrelia burgdorferi, B. hermsii, B. turicatae, B.
parkeri; Ehrlichia canis, E. chaffeensis, E. ewingii, Anaplasma
phagocytophilum, and Rickettsia rickettsii. The protozoa that can
be detected are Babesia canis vogeli, B. gibsoni, B. canis canis,
B. canis rossi, B. caballi, B. odocoilei, B. divergens, and B.
bigemina. This LayerPlex assay allows for differential diagnosis of
the animal's infection so that the person treating the animal can
use the appropriate antimicrobial compound(s) to kill the
microorganism present. The sample can be from one or more ticks or
a mammal (e.g., human, canine, feline, bovine, equine, ovine,
caprine, cervid, etc.). The sample can be blood or tissue. In one
embodiment, the pathogen is normally present in the particular
tissue or blood being tested when the pathogen infects the animal
(tick or mammal).
[0025] Until now, it has been extremely difficult and expensive to
differentiate between E. canis, E. ewingii, and E. chaffeensis in a
sample because of their highly similar genome. However, the methods
and the kits described herein permit one to distinguish between E.
canis, E. ewingii, and E. chaffeensis using a single qPCR assay. No
prior art single kit exists which can distinguish between these
three Ehrlichia species. Using the methods and kits described
herein, the medical/scientific community can now easily track the
spread of these three Ehrlichia species amongst various animals and
determine changes in bacteria's ability to infect different
animals.
[0026] Furthermore, the methods and kits described herein use and
contain a primer set that is highly specific and sensitive for
detecting Rickettsia rickettsii, enabling one to differentiate
between the bacteria that causes RMSF and non-pathogenic Rickettsia
species. This primer set (SEQ ID NOs: 21 and 22) are used to
amplify of a portion of Rr genomic DNA that is believed to encode a
protein with undetermined activity. This primer set has not
previously been used to detect and identify Rr in a sample. Also,
this LayerPlex assay uses primer sets that enable detection and
identification of B. hermsii, B. turicatae, and B. parkeri, the
causative agents of TBRF in North America; another unique aspect of
this LayerPlex assay. The combination of the primers used in the
methods and in the kits described herein are unique.
[0027] One embodiment of this invention involves methods to detect
and identify which of any of 17 microorganisms are present in a
sample by using the LayerPlex qPCR assay. In this embodiment, the
microorganisms are divided into the following three groups or
layers: Borrelial Layer (for detecting B. burgdorferi, B. hermsii,
B. turicatae, and/or B. parkeri), Rickettsiales Layer (for
detecting E. canis, E. chaffeensis, E. ewingii, A. phagocytophilum,
and/or R. rickettsia--also referred to as "Rickettsia-like"
bacteria), and Babesial Layer (for detecting many Babesia species,
including B. canis vogeli, B. gibsoni, B. canis canis, B. canis
rossi, B. caballi, B. odocoilei, B. divergens, and B. bigemina).
DNA is purified from a sample from an animal that is being tested
for the presence of one or more of these microorganism. The
purified DNA is combined with DNA polymerase, a plurality of
appropriate primer sets and probes, and other reagents for
performing the LayerPlex qPCR assay. The LayerPlex qPCR assay is
conducted, and the Cq values for each sample is measured. One can
detect the presence of one or more of these microorganisms' DNA
when the Cq value is within specific ranges (described below). In
this embodiment of the invention, the plurality of primer sets for
detecting Borrelia spp. can be one or more of the following: [0028]
(i) SEQ ID NOs: 1 and 2 which generate a 75 bp amplicon (SEQ ID NO:
4), [0029] (ii) SEQ ID NOs: 5 and 7 which generate a 117 bp
amplicon (SEQ ID NO: 9), [0030] (iii) SEQ ID NOs: 6 and 7 which
generate a 119 bp amplicon (SEQ ID NO: 10), [0031] (iv) SEQ ID NOs:
11 and 13 which generate a 142 bp amplicon (SEQ ID NO: 15), [0032]
(v) SEQ ID NOs: 12 and 13 which generate a 139 bp amplicon (SEQ ID
NO: 16), and [0033] (vi) SEQ ID NOs: 17 and 18 which generate a 117
bp amplicon (SEQ ID NO: 20). The plurality of primer sets for
detecting Rickettsia-like bacteria can be one or more of the
following: [0034] (vii) SEQ ID NOs: 21 and 22 which generate a 159
bp amplicon (SEQ ID NO: 24), [0035] (viii) SEQ ID NOs: 25 and 26
which generate a 95 bp amplicon (SEQ ID NO: 28), [0036] (ix) SEQ ID
NOs: 29 and 30 which generate a 113 bp amplicon (SEQ ID NO: 32),
[0037] (x) SEQ ID NOs: 33 and 34 which generate a 111 bp amplicon
(SEQ ID NO: 35), and [0038] (xi) SEQ ID NOs: 36 and 34 which
generate a 114 bp amplicon (SEQ ID NO: 37). The plurality of primer
set for detecting Babesia spp. can be one or more of the following:
[0039] (xii) SEQ ID NOs: 38 and 39 which generate a 94 bp amplicon
(SEQ ID NO: 41), and [0040] (xiii) SEQ ID NOs: 77 and 39 which
generate a 93 bp amplicon (SEQ ID NO: 78).
[0041] In this embodiment of this invention, the probes are
oligonucleotides labeled with a fluorescent label and a quencher.
In one embodiment, each oligonucleotide has a sequence of between
approximately eight nucleotides and approximately thirty
nucleotides of an amplicon defined by each of the plurality of the
primer sets (see above). In another embodiment, the probes have a
DNA sequence of any of SEQ ID NOs: 3, 8, 14, and 19 for the
Borrelial layer; SEQ ID NOs: 23, 27, and 31 for the Rickettsiales
layer; and SEQ ID NO: 40 for the Babesial Layer. Each probe within
each layer contains the same fluorescent label and quencher; thus
all generated amplicons for the microorganisms within each layer
will appear as one "signal". The LayerPlex qPCR assay allows one to
detect if one or more microorganisms of each layer (Borrelial,
Rickettsiales, or Babesial) are present in the sample, but does not
permit identification of the specific microorganism. One also
includes in the sample the primers and probe for the endogenous
internal positive control (NIPcK9, if sample is obtained from a
dog). Thus, four "layers" are being assayed in each well of a
multi-welled plate.
[0042] In an alternative embodiment of the LayerPlex qPCR methods,
one can additionally include the following primer sets to identify
Borrelia spp., [0043] (xiv) SEQ ID NOs: 46 and 47 to generate a 135
bp amplicon (SEQ ID NO: 48) and [0044] (xv) SEQ ID NOs: 49 and 50
to generate a 136 bp amplicon (SEQ ID NO: 51).
[0045] Alternatively, one can use primer sets (xiv) SEQ ID NOs: 46
and 47 and (xv) SEQ ID NOs: 49 and 50 instead of primer sets (ii)
SEQ ID NOs: 5 and 7 and (iii) SEQ ID NOs: 6 and 7. Alternatively,
one can use only primer set (xiv) SEQ ID NOs: 46 and 47 instead of
primer set (ii) SEQ ID NOs: 5 and 7; or only primer set (xv) SEQ ID
NOs: 49 and 50 instead of primer set (iii) SEQ ID NOs: 6 and 7.
When using primer set (xiv) SEQ ID NOs: 46 and 47 or primer set
(xv) SEQ ID NOs: 49 and 50, the probe can have oligonucleotide
sequence SEQ ID NO: 8.
[0046] Another embodiment of this invention enables one to detect
and identify the specific microorganisms for each layer by
optionally performing additional qPCR assays. As designed, each of
the individual, additional qPCR assays can utilize the same PCR
reaction conditions, DNA concentrations, and other parameters as
the LayerPlex qPCR assay, thereby lowering the cost, complexity,
time to perform these additional qPCR assays while having high
specificity and sensitivity. In an alternative embodiment, one can
use fluorescent dyes, such as SYBR.RTM. Green, instead of
probes.
[0047] If the Borrelial Layer is positive for the presence of
Borrelia spp., then one can perform multiple, distinct qPCR assays
with the DNA isolated from the sample. For each distinct qPCR
assay, one uses only one of the primer sets listed below. One can
use the probes having the sequences listed below, or use probes
with a different sequence that is present within the indicated
amplicon. The probes optionally contain 5'-FAM/BHQ1-3' as the
fluorescent label and quencher or other fluorescent labels and
quenchers. Because each qPCR reaction contains only one set of
primers and the accompanying probe, one can detect and identify the
particular species using the same fluorescent label. [0048] (i) SEQ
ID NOs: 1 and 2 to generate a 75 bp B. burgdorferi amplicon (SEQ ID
NO: 4) and, optionally, SEQ ID NO: 3 for the probe's
oligonucleotide, [0049] (ii) SEQ ID NOs: 5 and 7 to generate a 117
bp B. hermsii amplicon (SEQ ID NO: 9) and, optionally SEQ ID NO: 8
for the probe's oligonucleotide, or (iii) SEQ ID NOs: 6 and 7 to
generate a 119 bp B. hermsii amplicon (SEQ ID NO: 10) and,
optionally SEQ ID NO: 8 for the probe's oligonucleotide, or (xiv)
SEQ ID NOs: 46 and 47 to generate a 135 bp B. hermsii gene group I
(GG I) amplicon (SEQ ID NO: 48) and, optionally, SEQ ID NO: 8 for
the probe's oligonucleotide, or (xv) SEQ ID NOs: 49 and 50 to
generate a 136 bp B. hermsii gene group II (GG II) amplicon (SEQ ID
NO: 51) and, optionally, SEQ ID NO: 8 for the probe's
oligonucleotide, or two, three, or four of the primer sets and
probes in distinct qPCR assays, [0050] (iv) SEQ ID NOs: 11 and 13
to generate a 142 bp B. turicatae amplicon (SEQ ID NO: 15) and,
optionally, SEQ ID NO: 14 for the probe's oligonucleotide or (v)
SEQ ID NOs: 12 and 13 to generate a 139 bp B. turicatae amplicon
(SEQ ID NO: 16) and, optionally, SEQ ID NO: 14 for the probe's
oligonucleotide, or both sets of primers in distinct qPCR assays,
and [0051] (vi) SEQ ID NOs: 17 and 18 to generate a 117 bp B.
parkeri amplicon (SEQ ID NO: 20) and, optionally, SEQ ID NO: 19 for
the probe's oligonucleotide.
[0052] If the Rickettsiales Layer is positive for one or more
Rickettsia-like bacteria, one may conduct multiple, distinct qPCR
assays with the DNA isolated from the sample. For each distinct
qPCR assay, one uses only one of the primer sets listed below. One
can use the probes having the sequences provided below, or use
probes with a different sequence that is present within the
indicated amplicon. The probes optionally contain 5'-CF0560/BHQ1-3'
as the fluorescent label and quencher or other fluorescent labels
and quenchers. Because each qPCR reaction contains only one set of
primers and the accompanying probe, one can detect and identify the
particular species using the same fluorescent label. [0053] (vii)
SEQ ID NOs: 21 and 22 to generate a 159 bp R. rickettsii amplicon
(SEQ ID NO: 24) and, optionally, SEQ ID NO: 23 for the probe's
oligonucleotide; [0054] (viii) SEQ ID NOs: 25 and 26 to generate a
95 bp A. phagocytophilum amplicon (SEQ ID NO: 28) and, optionally,
SEQ ID NO: 27 for the probe's oligonucleotide; [0055] (ix) SEQ ID
NOs: 29 and 30 to generate a 113 bp E. canis amplicon (SEQ ID NO:
32) and, optionally, SEQ ID NO: 31 for the probe's oligonucleotide;
[0056] (x) SEQ ID NOs: 33 and 34 to generate a 111 bp E.
chaffeensis amplicon (SEQ ID NO: 35) and, optionally, SEQ ID NO: 31
for the probe's oligonucleotide; and [0057] (xi) SEQ ID NOs: 36 and
34 to generate a 114 bp E. ewingii amplicon (SEQ ID NO: 37) and,
optionally, SEQ ID NO: 31 for the probe's oligonucleotide.
[0058] If the Babesial Layer is positive for one or more
microorganism, one may conduct multiple, distinct qPCR assays with
the DNA isolated from the sample. For each distinct qPCR assay, one
uses only one of the primer sets listed below. One can use the
probes having the sequences provided below, or use probes with a
different sequence that is present within the indicated amplicon.
The probe optionally contains 5'-Q670/BHQ2-3' as the fluorescent
label and quencher or other fluorescent labels and quenchers.
Because each qPCR reaction contains only one set of primers and the
accompanying probe, one can detect and identify the particular
species using the same fluorescent label. It is noted that this
qPCR assay can detect and identify B. conradae in a sample by using
the primer set of SEQ ID NOs: 64 and 65; but that the LayerPlex
qPCR assay does not detect and identify B. conradae within the
Babesial Layer. [0059] (xvi) SEQ ID NOs: 52 and 53 to generate a 81
bp B. canis vogeli amplicon (SEQ ID NO: 55), and, optionally, SEQ
ID NO: 54 for the probe's oligonucleotide or (xvii) SEQ ID NOs: 56
and 57 to generate a 73 bp B. canis vogeli amplicon (SEQ ID NO:
59), and, optionally, SEQ ID NO: 58 for the probe's
oligonucleotide, or both sets of primers and probes in distinct
qPCR assays, [0060] (xviii) SEQ ID NOs: 60 and 61 to generate a 73
bp B. gibsoni amplicon (SEQ ID NO: 63), and, optionally, SEQ ID NO:
62 for the probe's oligonucleotide, and [0061] (xix) SEQ ID NOs: 64
and 65 to generate a 76 bp B. conradae amplicon (SEQ ID NO: 67),
and, optionally, SEQ ID NO: 66 for the probe's oligonucleotide.
[0062] In another embodiment of this LayerPlex qPCR assay and the
additional, follow-up single qPCR assays, one uses one or more
positive and/or negative controls (also referred to as
"amplification controls") to verify that the qPCR assay results are
accurate. One amplification control is specific for the animal from
which the sample was obtained. For example, if the sample is
obtained from a dog, cat, bovine, equine, cervid, etc., an
amplification control contains DNA specific for that animal and
confirms that the sample contains DNA from that animal. In one
embodiment, the amplification control for a canine sample utilizes
a NIPcK9 amplicon (SEQ ID NO: 45), primer set (SEQ ID NOs: 42 and
43), and probe (SEQ ID NO: 44). The other amplification control is
specific for the primer sets used and thus could be Tick10.PAC (SEQ
ID NO: 68) shown in FIGS. 2A-2C which contains some or all of the
amplicons of the tick-borne pathogens listed in FIGS. 1A-1D. For
samples from an animal that is not a canine (domestic dog, wolf,
coyote, etc.) one can use primers and a probe that are derived from
that animal's genome (nucleic or mitochondrial) and are specific
for that animal.
[0063] Another embodiment of this invention involves a kit for
detecting one or more tick-borne microorganisms (Borrelia spp.,
Rickettsia-like bacteria, and/or Babesia spp.) in a sample
containing a plurality of primers sets. This kit contains a
plurality of primer sets, instructions for use, and optionally,
probes. In this kit, the primer sets for detecting Borrelia spp.
can be one or more of the following: [0064] (i) SEQ ID NOs: 1 and
2, [0065] (ii) SEQ ID NOs: 5 and 7, [0066] (iii) SEQ ID NOs: 6 and
7, [0067] (iv) SEQ ID NOs: 11 and 13, [0068] (v) SEQ ID NOs: 12 and
13, and [0069] (vi) SEQ ID NOs: 17 and 18. The primer sets for
detecting Rickettsia-like bacteria can be one or more of the
following: [0070] (vii) SEQ ID NOs: 21 and 22, [0071] (viii) SEQ ID
NOs: 25 and 26, [0072] (ix) SEQ ID NOs: 29 and 30, [0073] (x) SEQ
ID NOs: 33 and 34, and [0074] (xi) SEQ ID NOs: 36 and 34. The
primer set for detecting Babesia spp. can be one or more of the
following: [0075] (xii) SEQ ID NOs: 38 and 39, and [0076] (xiii)
SEQ ID NOs: 77 and 39. The probes in this kit are oligonucleotides
labeled with a fluorescent label and a quencher. In one embodiment,
each oligonucleotide has a sequence of between approximately eight
nucleotides and approximately thirty nucleotides of an amplicon
defined by each of the plurality of the primer sets. The amplicons
are provided in FIGS. 1A-1D and discussed above. In another
embodiment, the probes have a DNA sequence of any of SEQ ID NOs: 3,
8, 14, 19, 23, 27, 31, and 40.
[0077] In another embodiment, the kit can contain additional primer
sets for detecting Borrelia spp. of at least one of the following:
[0078] (xiv) SEQ ID NOs: 46 and 47, and [0079] (xv) SEQ ID NOs: 49
and 50. The probes in this kit are oligonucleotides labeled with a
fluorescent label and a quencher. In one embodiment, each
oligonucleotide has a sequence of between approximately eight
nucleotides and approximately thirty nucleotides of an amplicon
defined by each of the plurality of the primer sets. The amplicons
are provided in FIGS. 1A-1D and discussed above. In another
embodiment, the probes have a DNA sequence of any of SEQ ID NOs: 48
and 51.
[0080] In another embodiment, the kit can contain additional primer
sets for detecting Babesia spp. of at least one of the following:
[0081] (xvi) SEQ ID NOs: 52 and 53, [0082] (xvii) SEQ ID NOs: 56
and 57, [0083] (xviii) SEQ ID NOs: 60 and 61, and [0084] (xix) SEQ
ID NOs: 64 and 65. The probes in this kit are oligonucleotides
labeled with a fluorescent label and a quencher. In one embodiment,
each oligonucleotide has a sequence of between approximately eight
nucleotides and approximately thirty nucleotides of an amplicon
defined by each of the plurality of the primer sets. The amplicons
are provided in FIGS. 1A-1D and discussed above. In yet another
embodiment, the probes have a DNA sequence of any of SEQ ID NOs:
54, 58, 62, and 66.
[0085] In another embodiment, the kit can contain a recombinant
plasmid that contains SEQ ID NO: 68 (Tick10.PAC plasmid).
Alternatively, the kit can contain Tick10.PAC (SEQ ID NO: 68) as an
amplicon. This kit can optionally contain a recombinant plasmid
containing the NIPcK9 amplicon (SEQ ID NO: 45) or the NIPcK9
amplicon itself, a primer set of SEQ ID NOs: 42 and 43, and
optionally a probe which contains an oligonucleotide containing
between approximately eight nucleotides and approximately thirty
nucleotides of SEQ ID NO: 45. Alternatively, the probe can have the
sequence of SEQ ID NO: 44. In another embodiment, the kit can
contain one or more amplification control plasmids for validating
the proper functioning of the qPCR assay. The amplification control
plasmids contain at least one sequence being tested and a sequence
that is specific from the animal from which a sample was obtained
(tick, dog, cat, equine, bovine, ovine, deer elk, moose, buffalo,
etc.). In another embodiment, a kit contains any of the above
described primers and probes sets so that one can be used to test
for specifically desired sets of microorganisms.
[0086] The primer sets, the amplicons generated, and the probes
useful for these LayerPlex PCR and follow-up multiplex qPCR assay
embodiments are contained in FIGS. 1A-1D and in FIGS. 2A-2C. In
this embodiment, one can use the fluorescent labels and quenchers
for the probes listed in FIGS. 1A-1D. In an alternative embodiment,
one can use known in the art fluorescent labels and quenchers for
the probes listed in FIGS. 1A-1D or other types of labels for
detecting the amplicons.
[0087] In another embodiment, a kit can also contain equipment and
instructions for collecting a sample (bodily fluids or tissue) from
a subject, reagents and instructions for isolating DNA from the
sample, and/or reagents and instructions for performing PCR. In
another embodiment, the generated amplicons can be isolated and
sequenced.
[0088] As discussed above, another embodiment of this invention
involves a multiplex PCR assay that detects and identifies B.
burgdorferi, E. canis, E. chaffeensis, and/or A. phagocytophilum in
a sample (bodily fluid, tissue, or combination thereof from a
mammal or tick). In one embodiment, DNA isolated from a sample is
mixed with primer sets that bind to genomic DNA from B.
burgdorferi, E. canis, E. chaffeensis, and/or A. phagocytophilum.
After amplification of DNA via PCR, the DNA is run on a gel. One
can stain the gel or use other types of molecules to identify the
size of any amplicons that are generated by the PCR assay, and thus
detect the presence of DNA from any one of these four
microorganisms, indicating that the sample contains those detected
microorganisms. In one embodiment of this gel-based multiplex PCR
assay method, one can use any combination of the primer sets as
follows: [0089] (a)(i) SEQ ID NOs: 69 and 70 to generate a 235 bp
B. burgdorferi amplicon (SEQ ID NO: 79) and/or (a)(ii) SEQ ID NOs:
1 and 2 to generate a 75 bp B. burgdorferi amplicon (SEQ ID NO: 4);
[0090] (b)(i) SEQ ID NOs: 71 and 72 to generate a 389 bp E. canis
amplicon (SEQ ID NO: 80) and/or (b)(ii) SEQ ID NOs: 29 and 30 to
generate a 113 bp E. canis amplicon (SEQ ID NO: 32); [0091] (c)(i)
SEQ ID NOs: 73 and 74 to generate a 390 bp E. chaffeensis amplicon
(SEQ ID NO: 81) and/or (c)(ii) SEQ ID NOs: 33 and 34 to generate a
111 bp E. chaffeensis amplicon (SEQ ID NO: 35); and [0092] (d)(i)
SEQ ID NOs: 75 and 76 to generate a 644 bp A. phagocytophilum
amplicon (SEQ ID NO: 82) and/or (d)(ii) SEQ ID NOs: 25 and 26 to
generate a 95 bp A. phagocytophilum amplicon (SEQ ID NO: 28).
[0093] In another embodiment of this invention, one can use
real-time qPCR to detect one or more of B. burgdorferi, E. canis,
E. chaffeensis, and/or A. phagocytophilum using the above sets of
primers and appropriate probes that contain fluorescent labels that
are distinct for each organism. Each probe would contain an
oligonucleotide having a sequence of between approximately eight to
approximately 30 nt in length and that is contained within the
above indicated amplicon. In one embodiment, the probes can contain
SEQ ID NOs: 3 (for B. burgdorferi), 31 (for E. canis and/or E.
chaffeensis), and 27 (for A. phagocytophilum) when one uses the
primer sets SEQ ID NOs: 1 and 2, SEQ ID NOs: 29 and 30, SEQ ID NOs:
33 and 34, and SEQ ID NOs: 25 and 26, respectively.
[0094] Another embodiment of this invention involves a kit
containing at least one of each primers set described below,
instructions for use of the primers to detect one or more of these
tick-borne bacteria (as well as for using any other optional
portions of this kit), optionally equipment for obtaining a sample
(bodily fluids or tissue) from an animal, optionally reagents for
purifying DNA from the sample obtained from an animal, optionally
reagents for performing PCR, optionally labels (dyes,
radioisotopes, probes, etc.) for detecting the amplicons generated,
and optionally the amplicons described below, which are all useful
for detecting B. burgdorferi, E. canis, E. chaffeensis, and A.
phagocytophilum; the primer sets and amplicons being as follows:
[0095] (a)(i) SEQ ID NOs: 69 and 70 to generate a 235 bp B.
burgdorferi amplicon (SEQ ID NO: 79) and/or (a)(ii) SEQ ID NOs: 1
and 2 to generate a 75 bp B. burgdorferi amplicon (SEQ ID NO: 4);
[0096] (b)(i) SEQ ID NOs: 71 and 72 to generate a 389 bp E. canis
amplicon (SEQ ID NO: 80) and/or (b)(ii) SEQ ID NOs: 29 and 30 to
generate a 113 bp E. canis amplicon (SEQ ID NO: 32); [0097] (c)(i)
SEQ ID NOs: 73 and 74 to generate a 390 bp E. chaffeensis amplicon
(SEQ ID NO: 81) and/or (c)(ii) SEQ ID NOs: 33 and 34 to generate a
111 bp E. chaffeensis amplicon (SEQ ID NO: 35); and [0098] (d)(i)
SEQ ID NOs: 75 and 76 to generate a 644 bp A. phagocytophilum
amplicon (SEQ ID NO: 82) and/or (d)(ii) SEQ ID NOs: 25 and 26 to
generate a 95 bp A. phagocytophilum amplicon (SEQ ID NO: 28).
[0099] In one embodiment, the probes can contain SEQ ID NOs: 3 (for
B. burgdorferi), 31 (for E. canis and/or E. chaffeensis), and 27
(for A. phagocytophilum) when one uses the primer sets SEQ ID NOs:
1 and 2, SEQ ID NOs: 29 and 30, SEQ ID NOs: 33 and 34, and SEQ ID
NOs: 25 and 26, respectively.
[0100] An embodiment of the invention involves assays to detect, in
a single reaction qPCR assay, multiple pathogens that cause a
plurality of diseases and provide fast and specific diagnostic
tools that allow the detection of co-infections. The assays
described herein can be performed on different samples, for
example, various tissues or bodily fluids obtained from an animal
(tick or mammal) The assays of the invention permit the detection
of the pathogens in less than one hour after the extraction of DNA
from the samples and initiating the qPCR assay. This period of time
is less than the period of time required to perform currently for
known in the art qPCR assays. As such, the invention advances
diagnostic medicine.
[0101] The sample being tested in this invention can be a tick
(fluid(s) and/or tissue(s)). In one embodiment, the tick fed on a
mammal, for example, a dog. In another embodiment, the tick is
obtained in areas where mammals live. The sample can also be a
tissue sample or bodily fluid sample from a human or a non-human
mammal, such as but not limited to, dog, cat, bovine, equine,
ovine, cervid, goat, primate, llama, buffalo, etc.
[0102] Non-limiting examples of tissue are rumen, reticulum, brain,
eye(s), pineal gland, pituitary gland, thyroid gland, parathyroid
glands, thorax, heart, lung, esophagus, thymus gland, pleura,
adrenal gland(s), appendix, gall bladder, urinary bladder, large
intestine, small intestine, kidney(s), liver, pancreas, spleen,
stoma, ovary/ovaries, uterus, testis, skin, and buffy coat sample
of blood. Additional examples of organs and tissue are well-known
to a person of ordinary skill in the art, and such embodiments are
within the purview of the invention. Similarly, non-limiting
examples of the bodily fluid samples include amniotic fluid,
aqueous humor, vitreous humor, bile, blood, cerebrospinal fluid,
chyle, endolymph, perilymph, lymph, mucus (including nasal drainage
and phlegm), pericardial fluid, peritoneal fluid, pleural fluid,
pus, rheum, saliva, sputum, synovial fluid, vaginal secretion,
semen, blood, serum or plasma. Additional examples of bodily fluids
are well known to a person of ordinary skill in the art, and such
embodiments are within the purview of the invention.
[0103] As used herein, the singular forms "a", "an" and "the" are
intended to include the plural forms as well, unless the context
clearly indicates otherwise. Further, to the extent that the terms
"including", "includes", "having", "has", "with", or variants
thereof are used in either the detailed description and/or the
claims, such terms are intended to be inclusive in a manner similar
to the term "comprising".
The term "about" or "approximately" means within an acceptable
error range for the particular value as determined by one of
ordinary skill in the art, which will depend in part on how the
value is measured or determined, i.e., the limitations of the
measurement system. For example, "about" can mean within 1 or more
than 1 standard deviation, per the practice in the art.
Alternatively, "about" can mean a range of up to 0 to 10%, 0 to 5%,
or up to 1% of a given value. Alternatively, particularly with
respect to biological systems or processes, the term can mean
within an order of magnitude, preferably within 5-fold, and more
preferably within 2-fold, of a value. Where particular values are
described in the application and claims, unless otherwise stated
the term "about" meaning within an acceptable error range for the
particular value should be assumed. In the context of compositions
containing amounts of ingredients where the terms "about" or
"approximately" are used, these compositions contain the stated
amount of the ingredient with a variation (error range) of 0-10%
around the value (X.+-.10%). Ranges are stated in shorthand, so as
to avoid having to set out at length and describe each and every
value within the range. Any appropriate value within the range can
be selected, where appropriate, as the upper value, lower value, or
the terminus of the range. For example, a range of 0.1-1.0
represents the terminal values of 0.1 and 1.0, as well as the
intermediate values of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and
all intermediate ranges encompassed within 0.1-1.0, such as
0.2-0.5, 0.2-0.8, 0.7-1.0, etc. When ranges are used herein, such
as for dose ranges, combinations and sub-combinations of ranges
(e.g., sub-ranges within the disclosed range), specific embodiments
therein are intended to be explicitly included.
[0104] One embodiment of PCR is performed with a template DNA or
RNA and one or more primers that are designed to amplify a target
amplicon, which is a portion of the template DNA or RNA, with a
polymerase. PCR involves temperature cycles that are designed to
facilitate different steps in a polymerase reaction leading to the
amplification of the target amplicon. The reagent mixture for PCR
typically contains deoxyribonucleotides (also called "nucleotides")
(dNTPs), metal ions (e.g., Mg.sup.2+, Mn.sup.2+), and a buffer.
Additional details, for example, additional reagents which may be
used in a PCR or temperature cycles that can be used in a
particular PCR can be known by a person of ordinary skill in the
art.
[0105] qPCR typically quantifies the amount of amplicon(s) present
in the sample. The amplicon can be detected by a number of
different methods, and such detection is typically carried out in
real-time ("RT"). As such, qPCR is also referred to "RT-qPCR".
Certain aspects of qPCR are described in "Real-Time PCR Application
Guide", Biorad 2006, the contents of which are herein incorporated
by reference in its entirety. The multiplex PCR assays of this
invention can also be multiplex qPCR assays. When performing qPCR,
one is monitoring DNA amplification (or amplicon production) during
each cycle of PCR using a label. When the DNA in the sample is in
the log linear phase of amplification, the amount of fluorescence
increases above the background. The point at which the fluorescence
becomes measurable is called the quantification cycle ("Cq") or
crossing point. By using multiple dilutions of a known amount of
standard DNA or amplification control DNA, a standard curve can be
generated of log concentration against Cq. The amount of nucleic
acids in an unknown sample can then be calculated from its Cq
value. For this invention, the higher the Cq value, the smaller the
quantity of a particular microorganism's DNA or DNA from a group of
microorganisms is present in the assayed liquid. Conversely, the
lower the Cq value, the higher the quantity of a microorganism's
DNA or DNA from a group of microorganisms is present in the assayed
liquid.
[0106] A multiplex PCR assay refers to a PCR assay designed to
amplify multiple amplicons from one or more template DNAs or RNAs
in one reaction. Typically, multiple pairs of primers are added to
a reaction that amplifies different target regions in the template
DNA or RNA with each primer pairs being used to generate a distinct
amplicon; that is, each amplicon generated by a primer pair has a
different sequence from the other amplicons generated by the other
primer pairs. One can determine if one or more of the distinct
amplicons are present in the PCR assay using different types of
identification modes (detection methods), such as gels, fluorescent
dyes, isotopes, etc. In one embodiment, the invention involves a
multiplex PCR assay for which the resultant amplicons are run on a
gel and then identified using an intercalating dye or sequenced or
by some other known in the art method.
[0107] As mentioned above, a LayerPlex qPCR assay is a type of
multiplex PCR assay that uses primer pairs to generate an amplicon
which determines if polynucleotides from any member of a particular
layer are present. For this invention, the LayerPlex identifies if
DNA from specific microorganisms within a group/layer of pathogens
are present in the sample. More particularly, the layers are (1)
Borrelial Layer which can identify if DNA from B. burgdorferi, B.
hermsii, B. turicatae, and/or B. parkeri are present in the sample,
(2) Rickettsiales Layer which can identify if DNA from E. canis, E.
chaffeensis, E. ewingii, A. phagocytophilum, and/or R. rickettsii
are present in the sample, and (3) Babesial Layer which can
identify if DNA from B. canis vogeli, B. gibsoni, B. canis canis,
B. canis rossi, B. caballi, B. odocoilei, B. divergens, and B.
bigemina are present in the sample.
[0108] In general, two types of labels can be used with qPCR. One
label is an intercalating dye that incorporates into
double-stranded DNA, such as SYBR.RTM. Green. The second type of
label is a "probe" that binds specifically to the target DNA, such
as TaqMan.RTM. probes, Molecular Beacons.TM., or Scorpion primers.
The probe is an oligonucleotide with a fluorescent dye (such as
Texas Red.RTM., FAM, TET, HEX, TAMRA, JOE, and ROX) and a quencher
(such as Black hole quencher (BHQ), Dabcyl, Dabsyl, and the minor
groove binding nonfluorescent quencher (MGBNFQ)) chemically
attached to the oligonucleotide. The oligonucleotide itself has no
significant fluorescence, but fluoresces either when annealed to
the template (as in Molecular Beacons.TM.) or when the dye is
clipped from the oligonucleotide during extension (as in
TaqMan.RTM. probes). The fluorescent compositions described herein
are simply examples of compositions for imaging, identifying,
and/or quantifying DNA. Instead of the fluorescent compositions
described herein, one can label DNA with compositions that are
known in the art (some of which are described infra) or that are
developed in the future. These labels can be used to image,
identify, and/or quantify DNA using similar methods as described
herein. The fluorescent compositions are simply one well-known and
well-accepted compositions for imaging, identifying, and/or
quantifying DNA for the methods described herein. The
oligonucleotide for a probe is at least approximately 8 nucleotides
in length. In some embodiments, the oligonucleotide for a probe is
between approximately 8 nucleotides and approximately 30
nucleotides long. In other embodiments, the oligonucleotide for a
probe is between approximately 15 nucleotides and approximately 30
nucleotides long. The sequence of the oligonucleotide is contained
within an amplicon and are contiguous nucleotides within the
amplicon. When performing multiplex or LayerPlex qPCR assays, one
tends to not use intercalating dyes because the intercalating dyes
typically do not distinguish between different amplicons. Instead
one typically uses probes containing different fluorescent
molecules so that one can distinguish which organism's DNA is being
amplified because of the distinct fluorescent emissions for each
probe. FIGS. 1A-1D provides the DNA sequence of various probes and
the fluorescent molecule and quencher molecule used with each
probes. One of ordinary skill in the art can easily use other
fluorescent molecules and quencher molecules with the probes and
can identify of sequences for the probes so that probe can still be
incorporated into an amplicon.
[0109] The LayerPlex qPCR assay described herein is a robust assay
because it is highly sensitive, highly specific, highly efficient,
and lacks inhibition. With a robust qPCR assay, the slope of the
standard curve, its linearity (represented by the correlation
coefficient, R.sup.2) and Y-intercept contain valuable quality
control information. High-quality qPCR assays are linear across the
entire dilution series (e.g., R.sup.2 close to 1), and the genomic
DNA concentrations of the samples to be tested should fall within
this linear range. The Y-intercept should be similar for all copy
number variant assays (e.g., typically within a two cycle
interval). See, D'haene, et al., Methods 50:262-270 (2010). The
data presented below demonstrate that the LayerPlex qPCR assay has
efficiencies between 90%-110%, R.sup.2 values >0.9, high
specificity, and high sensitivity. See, also, FIG. 3.
[0110] The gel-based PCR assay described herein is also highly
sensitive and highly specific, however, it takes more time to
perform than the Layerplex qPCR assay and does not detect as many
microorganisms as the Layerplex qPCR assay.
[0111] The term "nucleic acid" as used herein, refers to a polymer
of ribonucleotides or deoxyribonucleotides. Typically, "nucleic
acid" polymers occur in either single- or double-stranded form, but
are also known to form structures comprising three or more strands.
The term "nucleic acid" includes naturally occurring nucleic acid
polymers as well as nucleic acids comprising known nucleotide
analogs or modified backbone residues or linkages, which are
synthetic, naturally occurring, and non-naturally occurring, which
have similar binding properties as the reference nucleic acid, and
which are metabolized in a manner similar to the reference
nucleotides. Exemplary analogs include, without limitation,
phosphorothioates, phosphoramidates, methyl phosphonates,
chiral-methyl phosphonates, 2-O-methyl ribonucleotides,
peptide-nucleic acids (PNAs). A "nucleotide" encompasses a single
nucleotide or a base pair of nucleotides. "DNA", "RNA",
"polynucleotides", "polynucleotide sequence", "oligonucleotide",
"nucleotide", "nucleic acid", "nucleic acid molecule", "nucleic
acid sequence", "nucleic acid fragment", and "isolated nucleic acid
fragment" can be used interchangeably herein. For nucleic acids,
sizes are given in either kilobases (kb) or base pairs (bp), or
nucleotides (nt). Estimates are typically derived from agarose or
acrylamide gel electrophoresis, from sequenced nucleic acids, or
from published DNA sequences.
[0112] A "primer" is a single stranded polynucleotide, which can be
DNA or RNA, and which serves as a starting point for polynucleotide
synthesis, for example, in a PCR. A "primer set" is at least one
forward primer and at least one reverse primer capable of
generating at least one amplicon in a PCR assay. In some
embodiments of this invention, a primer set includes two forward
primers and one reverse primer, or, alternatively, two reverse
primers and one forward primer, which are capable of generating two
amplicons that have overlapping sequences in common.
[0113] The polynucleotides of the invention, for example, primers
and probes described herein, can be conjugated to a detectable
label. A label is a composition detectable by spectroscopic,
photochemical, biochemical, immunochemical, chemical, or other
means. Non-limiting examples of detectable labels include
chemiluminescent labels, fluorescent labels, radiolabels (.sup.32P
or other isotopes), electron-dense reagents, magnetic labels,
enzymes (e.g., as those commonly used in an ELISA), haptens, other
unique oligonucleotide sequences, and proteins for which antisera
or monoclonal antibodies are available. Additional examples of
labels suitable for the use in the claimed invention are well known
to a person of ordinary skill in the art and such embodiments are
within the purview of the invention.
[0114] A "target sequence" as used herein refers to a specific
sequence within the genome (nucleic or cellular) of an organism
being detected. In some embodiments, a target sequence is unique to
the organism and therefore, a target sequence can be used to
distinguish the organism being detected from the other organisms.
Therefore, the method of analyzing a DNA sample to detect two or
more organisms depends on identifying two or more specific
sequences that are unique to the organisms being detected. In some
embodiments, a target sequence is shared within a group of
organisms but different from organisms outside of that group and
can be used to distinguish the group of organisms from other groups
of organisms. Therefore, the method of analyzing a DNA sample to
the presence of organisms from two or more different groups depends
on identifying two or more specific sequences that are unique to
the organisms within each group being detected.
[0115] Prior to conducting the LayerPlex qPCR assay or the
gel-based PCR assay, one needs to isolate DNA from the sample. Any
known method of isolating and purifying DNA from a tissue and/or
bodily fluid sample obtained from an animal (mammal or tick) can be
used. Methods of isolating and purifying DNA from a tick sample and
a mammalian sample are provided in the examples below. In addition,
Schroeder, et al., J. Vet. Diag. Invest. 25:709-719 (2013)
describes an automated DNA extraction protocol for isolating and
purifying DNA from mammalian samples.
[0116] In a particular embodiment, the step of analyzing the DNA in
the methods of the invention involve conducting the LayerPlex qPCR
or a multiplex PCR assay (for use with a gel) designed to amplify
the target sequences from the organisms being detected. A multiplex
PCR assay or LayerPlex qPCR assay involves amplifying of one, two
or more target sequences using PCR primer pairs designed to amplify
the one, two or more target sequences from the genomes of the
organisms being detected. In one embodiment, the portion of the
target sequences amplified by the primer pairs specific for the
target sequences are different sizes. The amplified sequences are
referred to as "amplicons". As such, the amplicons can be
distinguished from each other, for example, based on their sizes as
determined by different techniques, for example, agarose gel
electrophoresis. The presence of an amplified DNA of a specific
size indicates the presence of the organism containing the
corresponding target sequence in its genome. In another embodiment,
the two or more amplicons can be the size. In this embodiment, one
may need to use a different mechanism for distinguishing the
amplicons of the same size from each other. But one of ordinary
skill in the art can use the methods described herein to help
distinguish the amplicons of the same size, if that is required to
differentially diagnosis the microorganism present in the sample.
Many different types of equipment for performing the PCR assays are
known to one of ordinary skill in the art.
[0117] In another embodiment, the amplicons produced by PCR can be
sequenced to confirm the presence of the target sequences from the
microorganism(s) being detected. In some embodiments, the amplicons
can range in size from approximately 15 bp to several hundred base
pairs. As seen in FIGS. 1A-1D, the amplicons for the LayerPlex qPCR
assay can range from 73 bp to 159 bp. Further, one uses the primers
described in FIGS. 1A-1D to generate the indicated amplicons and
the probes also described in FIGS. 1A-1D to identify the
presence/absence of the amplicons in the LayerPlex qPCR assay. As
discussed below, the amplicons for the gel-based PCR assay can
range from approximately 230 bp to approximately 650 bp using the
primers described below in Table 1. In another embodiment, one can
use the appropriate primer sets to generate the indicated amplicons
in FIGS. 1A-1C (which are smaller than the amplicons in Table 1
below). One can use any dye or other type of marker that is
effective for identifying DNA on an agarose gel, polyacrylamide
gel, or other types of gels.
[0118] In another embodiment, the step of analyzing the DNA using
the methods of the invention include conducting DNA hybridization
with at least one probe that hybridizes with at least one amplicon
from the organisms being detected. Therefore, a probe specific for
an organism hybridizes to the amplicon (and are complementary to a
portion of the amplicon's sequence), if the specific organism's DNA
is present in the sample, and an amplicon is produced. If the
organism's DNA is not present in the sample, then no amplicon is
produced, and the probe does not bind to the amplicon(s) that are
produced by the PCR assay for the other microorganisms that are
being detected.
[0119] A person of ordinary skill in the art can design various
probes for specific binding to the target sequences in each of the
organisms being detected and such embodiments are within the
purview of the invention. For example, in one embodiment, FIGS.
1A-1D lists probes and their sequences for the indicated
microorganisms that are useful in this invention.
[0120] The methods for detecting the binding between a probe and a
target sequence are well known in the art and such methods are
within the purview of the invention. For example, the probes that
hybridize to the sequences in the genomes of the organisms being
detected can be conjugated to a detectable label. The detectable
label can be a fluorescent label, radioactive label, enzyme label,
chemiluminescent label or magnetic label. Additional examples of
detectable labels are known to a person of ordinary skill in the
art and such embodiments are within the purview of the
invention.
[0121] In one embodiment, different labels conjugated to different
probes designed to bind to target sequences in different organisms
to be detected are distinguishable from each other. For example,
different labels can be of different types, i.e., a first label can
be a radioactive label, a second label can be an enzyme label, and
a third label can be a fluorescent label. Alternately, different
probes can be of the same type; however, distinguishable from each
other. For example, three labels can be fluorescent labels emitting
fluorescence of different frequencies. A skilled artisan can design
different probes that enable distinguishing the different probes
from each other and such embodiments are within the purview of the
invention.
[0122] In a further embodiment, the step of analyzing the DNA in
the methods of the invention can include conducting a molecular
beacon assay using two or more molecular beacons that each
hybridizes to target sequences from the organisms being detected. A
molecular beacon is a polynucleotide that hybridizes to a target
sequence. Typically, a molecular beacon is a hairpin shaped
polynucleotide with an internally quenched fluorophore whose
fluorescence is restored when the beacon hybridizes to a target
sequence. Certain aspects of molecular beacon assay are described
in Marras, et al., Clinica Chimica Acta 363:48-60 (2006), which is
incorporated herein by reference in its entirety. The beacons
specific for the organisms being detected are designed to hybridize
to a target sequences within the genomes of the organisms.
Therefore, a beacon specific for an organism hybridizes to the
genomic DNA isolated from the sample only when the specific
organism is present in the sample, and the beacon does not bind to
the genomic DNA isolated from the sample when a different or
related organism is present in the sample. When a beacon hybridizes
to a target sequence, the fluorophore and the quencher are
separated from each other resulting in fluorescence. As such,
fluorescence corresponding to a fluorophore attached to a beacon
indicates the presence of the corresponding target sequence and the
presence of the corresponding organism in the sample. The methods
of detecting the fluorescence from a beacon are well known in the
art and such methods are within the purview of the invention. In
one embodiment, different fluorophores are conjugated to different
beacons designed to bind to sequences from different organisms
being detected. For example, three fluorophores can emit
fluorescence of different frequencies. A skilled artisan can design
different beacons that enable distinguishing different beacons from
each other and such embodiments are within the purview of the
invention.
[0123] In a particular embodiment, the step of analyzing the DNA in
the methods of the invention involve conducting a multiplex PCR
assay designed to identify target sequences from the organisms
suspected of being present in a sample. A multiplex PCR assay
involves amplifying one, two, or more target sequences using PCR
primer pairs designed to bind to specific sequences within the DNA
that could be present in the sample to generate one, two, or more
amplicons, and detecting one, two, or more labels bound to the one,
two or more amplicons generated which allows one to determine which
microorganism's DNA is present in a sample. As such, designing a
multiplex qPCR involves designing primers specific for
amplification of target sequences from the organisms being detected
as well as designing molecular beacons, probes or primers that
detect the amplified amplicons.
[0124] In a further embodiment, the step of analyzing the DNA in
the methods of the invention include conducting a multiplex or
single nucleotide primer extension assay based on different
nucleotides added to the primers designed to hybridize to target
sequences from the organisms being detected.
[0125] For both the gel-based multiplex PCR assay and the LayerPlex
qPCR assay, the concentrations of the primers and probes, the
temperatures of the assay and the amount of time spent at the
various temperatures, the concentration of the various components
of the PCR master mix, and the method of isolating DNA from a
sample, can be important to successful detection of microorganisms'
DNA in the sample. The concentration of each primer for the
gel-based multiplex PCR can range from approximately 0.04 .mu.M to
approximately 4 .mu.M, or from approximately 0.1 .mu.M to
approximately 1 .mu.M, approximately 0.4 .mu.M, approximately 0.3
.mu.M, or a combination thereof. The concentration of DNA isolated
from a sample for the gel-based multiplex PCR can range from
approximately 0.00012 pg/.mu.l to approximately 1.2 pg/.mu.l, from
approximately 0.0012 pg/.mu.l to approximately 0.12 pg/.mu.l, or
approximately 0.012 pg/.mu.l.
[0126] For the LayerPlex qPCR assay and the gel-based multiplex PCR
assay, in one embodiment, the temperatures and times for
denaturation, annealing, and extension of the PCR assay can range
as follows: for denaturation, approximately 85.degree. C. to
approximately 98.degree. C., for approximately 10 seconds to
approximately 10 minutes; for annealing, approximately 50.degree.
C. to approximately 80.degree. C. for approximately 10 seconds to
approximately 10 minutes; for extensions, from approximately
60.degree. C. to approximately 80.degree. C. for approximately 10
seconds to approximately 10 minutes. The number of PCR cycles can
range from approximately 10 to approximately 50. In another
embodiment, the temperatures and times for denaturation, annealing,
and extension cycles of the PCR assay are as follows: approximately
85.0.degree. C. to approximately 98.0.degree. C. for approximately
10 seconds to approximately 30 minutes) (Stage 1); approximately
85.0.degree. C. to approximately 98.0.degree. C. for approximately
1 second to approximately 10 minutes followed by approximately
40.0.degree. C. to approximately 80.0.degree. C. for approximately
1 second to approximately 10 minutes with approximately 30 to
approximately 50 repetitions (Stage 2). Data are collected at the
last step of Stage 2. In another embodiment, the temperatures and
times for denaturation, annealing, and extension cycles of the PCR
assay are as follows: approximately 90.0.degree. C. to
approximately 96.0.degree. C. for approximately 1 minute to
approximately 20 minutes (Stage 1); approximately 90.0.degree. C.
to approximately 96.0.degree. C. for approximately 1 second to
approximately 5 minutes followed by approximately 50.0.degree. C.
to approximately 70.0.degree. C. for approximately 1 second to
approximately 1 minute with approximately 35 to approximately 45
repetitions (Stage 2).
[0127] The concentration of each microorganism's primers for the
LayerPlex qPCR assay can range from approximately 0.05 .mu.M to
approximately 500 .mu.M, or from approximately 0.5 .mu.M to
approximately 50 .mu.M, approximately 5 .mu.M or approximately 5.6
.mu.M. The concentration of each microorganism's probe for the
LayerPlex qPCR assay can range from approximately 0.01 .mu.M to
approximately 100 .mu.M, or from approximately 0.1 .mu.M to
approximately 10 .mu.M, approximately 1 .mu.M or approximately 1.6
.mu.M. The concentration of DNA isolated from a sample for the
LayerPlex qPCR can range from approximately 9.49E-10 ng/.mu.l to
approximately 7.59E+5 ng/.mu.l. The temperatures and times
denaturation, annealing, and extension parts of the PCR assay for
the LayerPlex qPCR assay can range as follows: for denaturation,
approximately 85.degree. C. to approximately 98.degree. C., for
approximately 10 seconds to approximately 30 minutes; for
annealing, approximately 85.degree. C. to approximately 98.degree.
C. for approximately 0.1 seconds to approximately 10 minutes; for
extensions, from approximately 50.degree. C. to approximately
80.degree. C. for approximately 0.1 seconds to approximately 10
minutes. The number of PCR cycles can range from approximately 20
to approximately 60.
[0128] For both the gel-based multiplex PCR assay and the LayerPlex
qPCR assay, in an optional embodiment, one could have at least one
portion of the PCR assay to include primers (and a probe for the
LayerPlex qPCR assay) that is specific for the animal from which
the sample was obtained. Further, if the sample is a tick (or its
bodily fluids and/or tissue), one may want another portion of the
PCR assay to include primers (and a probe for the LayerPlex qPCR
assay) that is specific for the mammal from which the tick was
obtained (if the tick was obtained while feeding on an animal) The
goal would be to confirm that the sample was obtained from that
type of animal. In the examples below and in FIG. 1C, a primer set
and a probe specific for a dog is described (see NIPcK9 primers and
probe). One of ordinary skill in the art can generate a primer set
and a probe (if applicable) specific for a feline, bovine, ovine,
equine, cervid, etc., using mitochondrial or nucleic DNA from that
animal.
[0129] Following are examples which illustrate procedures for
practicing the invention. These examples should not be construed as
limiting. All percentages are by weight and all solvent mixture
proportions are by volume unless otherwise noted.
Example 1--Assaying Specimens for Presence of Ehrlichia canis (Ec),
Ehrlichia chaffeensis (Ech), Anaplasma phagocytophilum (Ap), and
Borrelia burgdorferi (Bb)
[0130] A total of 1,182 specimens (namely, 650 ticks, 525 wildlife
tissues, and 7 dog blood samples) were analyzed in this study. All
sample submissions were voluntary. Upon arrival, ticks were
identified using dichotomous key (e.g., Keirans and Litwak, J.
Medical Entomology 26(5):435-448 (1989); and Strickland and
Diamant, Ticks of Veterinary Importance, Washington: Animal and
Plant Health Inspection Service, U.S. Dept. of Agriculture (1976)).
The identified ticks were placed in a 70% ethanol solution and
submitted for DNA extraction. When tick morphology was not enough
to determine the species, 12S rRNA PCR and sequencing of the PCR
product was used to determine the species (Williamson, et al.,
Emerg. Infect. Dis. 16(3):441-446 (2010)).
[0131] DNA was extracted from whole ticks using the High Pure PCR
Template Preparation Kit (Roche, Indianapolis, Ind.) following
manufacturers recommendations with modifications to adapt the
protocol to ticks. Ticks were placed in screw cap microfuge tubes
containing 1.4 mm ceramic beads for flat ticks, or 2.8 mm ceramic
beads for engorged ticks. 200 .mu.l phosphate-buffered saline
(PBS), 200 .mu.l of tissue lysis buffer, and 40 .mu.l of Proteinase
K were added to the tube; the tubes were then placed in a bead mill
BeadRuptor 24 (Omni International, Inc., Kennesaw Ga.) and
homogenized for 5 minutes at a 5.65 m/s intensity (equivalent to
210 g). The supernatant was separated into a clean microfuge tube,
and 200 .mu.l of binding buffer was added to each sample. This
mixture was incubated for 10 minutes at 70.degree. C. After
incubation, 100 .mu.l of isopropanol was mixed into the solution.
This solution was then transferred to a high pure filter tube and
collection tube assembly. This assembly was then centrifuged for 1
minute at 8,000 g. After centrifugation the collection tube and
flow-through were discarded, and a new collection tube was
assembled with the same pure filter tube. 500 .mu.l of wash buffer
was added to the reservoir of the pure filter tube, and the
assembly was centrifuged for 1 minute at 8,000 g. The washing
procedure was then repeated. After the second washing, the assembly
was centrifuged at full speed for 10 seconds to remove any residual
ethanol, and the collection tube was then discarded. The high pure
filter tube was then assembled with a clean microfuge tube, and 100
.mu.l of pre-warmed elution buffer (70.degree. C.) was added to the
high pure filter tube. This assembly was centrifuged for 1 minute
at 8,000 g. Following elution, the extracted DNA was analyzed using
a Nanodrop (ThermoFisher Scientific, Waltham, Mass.) for quantity
and quality. The extracted DNA was then stored at -20.degree. C.
until further analysis.
[0132] DNA was extracted from blood samples using the High Pure PCR
Template Preparation Kit (Roche, Indianapolis, Ind.) following
manufacturers recommendations. As described above, the extracted
DNA was analyzed using a Nanodrop (ThermoFisher Scientific,
Waltham, Mass.) for quantity and quality. The extracted DNA was
then stored at -20.degree. C. until ready for further analysis.
[0133] Multiplex PCR for Pathogen Detection:
[0134] The amplification controls for Ec, Ech, Ap, and Bb utilized
in this study were synthetically generated. The genomes for these
organisms are available at GenBank.RTM. (NCBI). The primers used in
the multiplex reaction are provided in Table 1 below and generated
an amplicon having the indicated SEQ ID NOs. and size.
TABLE-US-00001 TABLE 1 Bacteria Amplicon (gene) Forward primer (SEQ
ID NO) Reverse primer (SEQ ID NO) (size) Bb AACACACCAGCATCACTTTC
GAGAATTAACTCCGCCTTG SEQ ID NO: (fla B) AGG (SEQ ID NO: 69) AGAAGG
(SEQ ID NO: 70) 79 (235 bp) Ec (16S CAATTATTTATAGCCTCTGG
TATAGGTACCGTCATTATCT SEQ ID NO: rRNA) CTATAGGAA (SEQ ID NO: 71)
TCCCTAT (SEQ ID NO: 72) 80 (389 bp) Ech 16S CAATTGCTTATAACCTTTTG
TATAGGTACCGTCATTATCT SEQ ID NO: rRNA) GTTATAAAT (SEQ ID NO: 73)
TCCCTAT (SEQ ID NO: 74) 81 (390 bp) Ap CCGCTTTAGTGGCACATTAT
CCGAAATTCTAAAATCTGG SEQ ID NO: (msp2) AG (SEQ ID NO: 75) GT (SEQ ID
NO: 76) 82 (644 bp)
[0135] These amplicons were synthesized (Integrated DNA
Technologies, Inc.) and cloned in a vector that was later
transformed into E. coli TOP10 cells. After transformation, plasmid
DNA was extracted from this culture and used as the amplification
control for the PCR tests. Plasmid DNA was extracted by using the
PureYield.TM. Plasmid Midiprep System (Promega Corp., Madison Wis.)
following manufacturer's recommendations. Briefly, 250 ml overnight
cultures of E. coli TOP10 cells containing the amplification
control vectors were harvested by centrifugation at 5,000 g for 10
minutes. Cells were lysed and placed in a column assembly to
extract and purify DNA. DNA was eluted in 600 .mu.l of nuclease
free water.
[0136] All samples were tested for Bb, Ec, Ech and Ap in a
multiplex PCR platform. The PCR reaction composition and the
amplification conditions are provided in Tables 2 and 3 below.
TABLE-US-00002 TABLE 2 PCR composition 25 .mu.l Final reaction
Concentration 10X Standard Taq Reaction Buffer 2.5 .mu.l 1 unit Taq
DNA Polymerase (AccuStart) 0.1 .mu.l 200 .mu.M Forward Primer fla B
(SEQ ID NO: 69) 0.5 .mu.l 0.4 .mu.M (Bb) Reverse Primer fla B (SEQ
ID NO: 70) 0.5 .mu.l 0.4 .mu.M (Bb) Forward Primer msp2 (SEQ ID NO:
75) 0.5 .mu.l 0.4 .mu.M (Ap) Reverse Primer msp2 (SEQ ID NO: 76)
0.5 .mu.l 0.4 .mu.M (Ap) Forward Primer 16S rRNA (SEQ ID NOs: 0.5
.mu.l 0.3 .mu.M 71 & 73) (Ec & Ech) Reverse Primer 16S rRNA
(SEQ ID NOs: 0.5 .mu.l 0.3 .mu.M 72 & 74) (Ec & Ech) DNA 2
.mu.l 0.012 pg/.mu.l Nuclease-free water To 25 .mu.l
TABLE-US-00003 TABLE 3 Thermocycler conditions for multiplex PCR
STEP TEMP TIME Initial Denaturation 94.degree. C. 3 min
Denaturation 30 cycles 94.degree. C. 1 min Annealing 63.degree. C.
1 min Extension 72.degree. C. 1 min Final extension 72.degree. C. 1
min Hold 4.degree. C.
[0137] After the PCR reaction, 5 .mu.L of the product was mixed
with Orange G loading buffer and placed into wells of a 1% agarose
gel containing 0.4 .mu.g/ml of ethidium bromide (Bio-Rad
Laboratories Inc., Hercules, Calif.). The gel was run at 90 volts
for 40-60 minutes. After electrophoresis the gels were imaged using
ChemiDoc.TM. Touch Imaging System (Bio-Rad Laboratories, Inc.,
Hercules, Calif.). Any positive bands were then cut from the gel
and purified using the Wizard.RTM. SV Gel and PCR Clean-up System
(Promega Corp., Madison Wis.) per the manufacturer's
recommendations. Clean PCR amplicons were then sent to Eton
Biosciences Ltd. (San Diego, Calif.) for sequencing to confirm the
PCR result.
[0138] Sequences were cleaned individually before assembly using
MacVector Version 13.0.7 (MacVector Inc., North Carolina) as
follows. First, the 5'- and 3'-ends were removed from each sequence
to avoid utilization of unclean and noisy sections obtained during
sequencing. After cleaning the ends, each peak in the chromatograms
was checked for accuracy of the corresponding nucleotide to make
sure listed nucleotides were correct. Once all sequencing results
were cleaned, the forward and reverse sequences were assembled
using MacVector Assembler 13.0.7 (MacVector Inc., North Carolina).
The consensus sequence produced was then used for further alignment
analyses and Neighbor-Joint phylogenetic tree assembly.
Example 2--Construction of Amplification Control Plasmids
[0139] The amplification control plasmid for multiplex PCR assays
were cloned individually utilizing the cloning vector pCR2.1 TOPO
system (ThermoFischerScientific, Waltham, Mass.) using the primers
disclosed in Table 1. PCR amplicons corresponding to each
amplification control were ligated into pCR2.1 TOPO plasmid vector
following manufacturer's recommendations using conditions provided
in Table 4.
TABLE-US-00004 TABLE 4 Ligation reaction used for TOPO-TA cloning
Reagent Vol Fresh PCR product 4 .mu.l Salt Solution 1 .mu.l TOPO
.TM. Vector 1 .mu.l Final Volume 6 .mu.l
[0140] For Ap, a synthetic fragment of the gene msp2 (locus
APH_1017, gene location: 1076720-1075449 A. phagocytophilum HZ)
cloned into the plasmid pIDTSMARTkan (Integrated DNA Technologies,
Inc.) was ordered. msp2 was subcloned with EcoRV flanking cut sites
into the cloning vector pCR2.1, TOPO system
(ThermoFischerScientific, Waltham, Mass.).
[0141] Vectors containing the fragments of interest were chemically
transformed into E. coli Top10 cells following manufacturer's
recommendations. Transformed cells were plated on LB plates
containing 100 .mu.g/ml ampicillin (Amp100) and 50 .mu.g/ml
kanamycin (Kan50) at 37.degree. C. overnight. Individual colonies
were selected and grown overnight in 7 ml LB media supplemented
with Amp100 and Kan50 at 37.degree. C. and 200 rpm. Plasmid
constructs were isolated using the Promega PureYield.TM. Midi
System kit following manufacturer's recommendations with little
modification. 600 .mu.l of overnight bacterial cultures were
transferred to 1.5 ml microfuge tube. Cells were lysed by adding
100 .mu.l of cell lysis buffer, and mixed by inverting the tube 6
times. Then 350 .mu.l of cold (4-8.degree. C.) neutralization
solution was added and mixed thoroughly by inverting the tube 9
times to ensure complete neutralization. The tubes were centrifuged
at 20,000 g for 3 minutes. Supernatants were transferred to a
PureYield.TM. minicolumn after placing into a PureYield.TM.
collection tube, and centrifuged at 20,000 g for 30 seconds. The
flow through was discarded, and 200 .mu.l of endotoxin removal wash
buffer was added to the column followed by centrifugation at 20,000
g for 30 seconds. Finally, 400 .mu.l of column wash solution was
added to the column and centrifuged at maximum speed for 30
seconds; the columns were transferred to clean 1.5 ml microfuge
tubes, and 25 .mu.l of elution buffer was added to the column
matrix and incubated for 3 minute at room temperature before
eluting the clean plasmid DNA by centrifugation at 20,000 g for 1
minute.
[0142] Constructs were confirmed by PCR using primers specific for
the fragments cloned as well as by restriction enzyme (RE)
digestion using specific RE depending on the construct. EcoRI was
used to check for the flaB gene fragment, NotI was used to check
for either Ec or Ech 16S rRNA gene fragments, and EcoRV was used to
check the insertion of the Ap msp2 gene fragment.
[0143] Constructs were also submitted for sequencing (Eton
Biosciences, San Diego, Calif.), and sequences were analyzed
through BLAST using MacVector 12.6 software (MacVector Inc., Cary,
N.C.). Once initial constructs were confirmed, individual clones
for each construct were grown in 250 ml of LB complemented with
Amp100 and Kan50 overnight at 37.degree. C. under shaking
conditions (200 rpm).
[0144] Plasmid DNA was purified using Promega PureYield.TM. Plasmid
Midiprep kit following manufacturer's recommendations with
modification. Overnight cultures were pelleted by centrifugation at
5,000 g for 10 minutes, and supernatants were discarded. The cell
pellets were resuspended in 3 ml cell resuspension solution
followed by the addition of 3 ml of cell lysis solution and mixed
by gently inverting the tube 3-5 times. Tubes were incubated for 3
minutes at room temperature followed by the addition of 5 ml
neutralization solution, and the lysates were centrifuged at room
temperature and 5,000 g for 15 minutes to eliminate cellular
debris. Remaining debris was removed using the PureYield.TM.
clearing; followed by the DNA elution step. Both the steps were
performed following manufacturer's recommendations with additional
incubation of the elution mix for 5 minutes at room temperature
before eluting by centrifugation. Plasmid DNA concentration and
quality were measured using a NanoDrop (ThermoFisher Scientific,
Waltham, Mass.) prior to the following cloning steps.
[0145] To construct the different multiplex amplification controls
containing three genetic markers, 20 .mu.g of plasmid DNA from each
of the clones (those containing Bb, Ec, Ech and Ap markers) were
digested with the corresponding restriction enzyme and subcloned
into the final pCR2.1 plasmid to generate two combinations of
amplification controls (Bc-Ap or Bch-Ap). To sub-clone all
amplification controls in one plasmid, one sub-cloning at a time
was performed. Ec marker was cloned into pCR2.1 containing Bb flaB
amplicon. The same approach was taken for Ech amplification
control.
[0146] Ap marker was then cloned. Briefly, the vector containing
the Bb flaB fragment was digested with NotI to clone the 16S rRNA
from Ec or Ech. EcoRV was used to clone the Ap msp2 fragment in
between the Bb flaB and the Ec or Ech 16S rRNA genes. All digestion
fragments were confirmed by running DNA electrophoresis using 1.5%
agarose gels at 80 volts for 45 min.
[0147] DNA purification for agarose gels was done using PCR and Gel
Clean Up.TM. kit (Promega Inc.). DNA concentration and purity was
measured using NanoDrop (ThermoFisher Scientific, Waltham, Mass.).
In this process digested plasmids were dephosphorylated following
manufacturer's recommendations (Table 5, Antarctic Phosphatase,
NEB) at 37.degree. C. for 30 minutes. This dephosphorylation was
done to prevent sticky ends from re-ligating during the following
steps, and to optimize the cloning process. Following
dephosphorylation of the digested plasmids, the corresponding
inserts were added, and the ligation reaction using T4 DNA ligase
(NEB Inc.) was performed as indicated in Table 6.
TABLE-US-00005 TABLE 5 Plasmid Dephosphorylation Protocol (NEB
Inc.) Reagent Volume Plasmid 50 .mu.l 10x Antarctic Phosphatase
buffer 1 .mu.l Dd water 6 .mu.l Antarctic Phosphatase 3 .mu.l
TABLE-US-00006 TABLE 6 Ligation Protocol with T4 DNA Ligase (NEB
Inc.) Reagent Volume Insert 10 .mu.l Vector 3 .mu.l T4 DNA ligase 1
.mu.l 10x buffer 2 .mu.l Dd water 4 .mu.l Incubated at
14-16.degree. C. overnight
[0148] Ligation reactions were precipitated prior to their
transformation into E. coli Top10 electro-competent cells. To this
end, each ligation reaction was incubated at -20.degree. C. for a
minimum of 2 hours with 0.5 volume of 7.5M ammonium hydroxide and
2.5 volume 95% ethanol. After incubation, constructs were
centrifuged at 20,000 g for 15 minutes and pellets were cleaned
with 75% ethanol. All constructs were re-suspended in water and
electroporated into E. coli Top10 cells utilizing the following
conditions: 2 .mu.l of ligation reaction was added to the volume of
cells recommended by the manufacturer. For each ligation reaction,
the following protocol was used.
[0149] The ligation and the cells were mixed gently with the
pipette tip, and the mix was transferred to an ice-cold
electroporation cuvette. For electroporation, Eppendorf
electroporator was set at 25 .mu.F capacitator, 2.5 kV and
200.OMEGA.. To recuperate the cells after the pulse, 250 .mu.l room
temperature S.O.C. medium was added to the cells and incubated at
37.degree. C. for 1 hour under shaking conditions (200 rpm). After
the preliminary incubation without antibiotics, the transformation
was spread on a pre-warmed LB plate containing Amp100 and Kan50 and
incubated overnight at 37.degree. C. for colony confirmation.
[0150] After overnight growth, single colonies were selected and
grown overnight in LB broth complemented with Amp100 and Kan50 and
incubated at 37.degree. C. overnight under shaking conditions (200
rpm) to identify amplification clones from each cloning step. Cells
from the cultures were pelleted at described above, and plasmid DNA
purified using PureYield.TM. Plasmid Miniprep kit (Promega, Inc.)
following the manufacturer's recommendations and modifications
described above. Plasmid DNA samples were evaluated for DNA
concentration and quality using a NanoDrop (ThermoFisher
Scientific, Waltham, Mass.). Constructs were confirmed by PCR using
specific primers for each marker. Amplification products were
separated in 1.5% agarose electrophoresis and sequenced for
confirmation (Eton Biosciences, San Diego, Calif.). Sequences were
analyzed through BLAST using MacVector 12.6 software (MacVector
Inc., Cary, N.C.).
Example 3--DNA Extraction
[0151] DNA was extracted from the tick samples using Wizard.TM. SV
Genomic DNA Purification kit (Promega Corp., Madison, Wis.)
following manufacturer's recommendations with modifications. For
optimal DNA extraction, adult individual ticks were homogenized
utilizing the bead mill Bead Ruptor 24 (Omni International, Inc.,
Kennesaw, Ga.) in 200 .mu.l of Nuclei Lysis Solution, plus 50 .mu.l
of 0.5M EDTA, 40 .mu.l of a 20 mg/ml Proteinase K solution, and 5
.mu.l RNase A solution. The mixture was incubated at 70.degree. C.
for 10 minutes. After the initial incubation, un-engorged ticks
were homogenized with 1.4 mm ceramic beads while 2.8 mm ceramic
beads were used with engorged ticks. After homogenization, tubes
were centrifuged at 10,000 g to eliminate tick debris. Supernatants
were collected, and 250 .mu.l of Wizard.TM. SV Lysis Buffer was
added to each sample and the mixture. The mixture was run through
filter columns at 13,000 g for 3 minutes. DNA bound to the filter
was washed and eluted following manufacturer's recommendations. To
extract DNA from the tick immature stages (nymphs and larvae),
pools of a maximum of 15 nymphs or 50 larvae were made. Specimens
received a code indicating the type of pool generated.
[0152] All immature specimens were stored at -80.degree. C. with
100 .mu.l TE buffer for at least one hour. Specimens were
homogenized utilizing pestles while the samples were frozen,
followed by DNA extraction procedures using the prepGEM.TM. DNA
Extraction Insect following manufacturer's recommendations by
adding to the tick sample 35 .mu.l of PCR-grade water, 4 .mu.l of
10.times. buffer BLACK and 1 .mu.l prepGEM.TM.. The mixture was
incubated at 75.degree. C. for 15 minutes, and then at 95.degree.
C. for 15 minutes. The extracted DNA concentration and purity were
measured using a NanoDrop (ThermoFisher Scientific, Waltham,
Mass.). The extracted DNA was stored at -20.degree. C. until use.
Tissue and blood samples were processed following manufacturer's
recommendations with no modifications.
Example 4--Multiplex PCR
[0153] The PCR was done in 25 .mu.l volume, using AccuStart.TM.
SuperMix (Quanta Bioscience Inc., Gaithersburg, Md.). Table 2,
supra, shows the composition and concentration of the reagents
utilized in such reaction. A master mix was prepared in a PCR clean
bench to avoid contamination. After the reaction tubes were
prepared, PCR was performed in an Eppendorf AG Master Cycler
following the cycling condition described in Table 3, supra. The
amplification products were separated on a 1.5% agarose gel
(Bio-Rad Laboratories Inc., Hercules, Calif.) at 90 volts for 40-60
minutes and imaged using the ChemiDoc touch Imaging System (Bio-Rad
Laboratories Inc., Hercules, Calif.). Positive bands were excised
from the gel and purified using the Wizard.TM. SV Gel and PCR clean
up system (Promega Corp., Madison, Wis.) following manufacturer's
recommendations. The purified products were sent for sequencing
(Eton Biosciences, San Diego, Calif.). Sequences were analyzed
through BLAST using MacVector 12.6 software (MacVector Inc., Cary,
N.C.).
Example 5--Gel-Based Multiplex & qPCR Assays to Identify
Tick-Borne Pathogens
[0154] 211 Rhipicephalus sanguineus (brown dog ticks) gathered from
160 domestic dogs and environmental locations from La Paz, Baja
California, Mexico were utilized for this study. Ticks from each
animal and location were grouped individually into tubes ranging
from 1-20 ticks per tube, depending on the number found on/in the
respective collection locations. Each tube was assessed for tick
engorgement level, where at least one engorged tick was sampled
from each tube if applicable. Ticks sampled from each tube depended
on amount of ticks residing in respective tube (i.e. in the case of
a tube containing at least five ticks, one engorged and one flat
tick was sampled). In most cases only one engorged tick was sampled
from each eligible tube under the assumption of pool feeding, where
all surrounding ticks on the same animal are expected to maintain
equal pathogen distribution through engorgement. Individual ticks
selected from each tube were assessed for species identification
(R. sanguineus, n=211), developmental stage (noted as adult/nymph),
and sex.
[0155] Whole ticks utilized for this study were submitted for DNA
extraction. Standardized procedures were followed for tick
handling, and subsequent DNA extraction with the MagMAX.TM.-96
Nucleic Acid (NA) Isolation Kit (ThermoFisher Scientific, Waltham,
Mass.) following manufacturer's recommendations adopted from a
previous publication (Schroeder, et al., J. Vet. Diag. Invest.
25:709-719 (2013)), with modifications to adapt the protocol to
ticks. Select ticks were placed in screw cap microfuge tubes
containing 1.4 mm ceramic beads (Omni International, Inc.,
Kennesaw, Ga.). 400 .mu.l of Phosphate-Buffered Saline (PBS) was
added to each tube; the tubes were then placed in a bead mill
BeadRuptor 24 (Omni International, Inc., Kennesaw, Ga.) and
homogenized for 5 minutes at a 5.65 m/s intensity. 200 .mu.l of
homogenate suspension was then transferred to a 96-well deep-well
plate containing 20 .mu.l of magnetic bead mix (10 .mu.l of
lysis/binding enhancer and 10 .mu.l of NA binding beads), 300 .mu.l
of lysis solution, 1 .mu.l of carrier NA (1 .mu.g/.mu.l), and 300
.mu.l of isopropanol). This plate represents the sample plate. The
following 96-well plates were loaded into an automated
KingFisher.TM. Flex Magnetic Particle Processor (ThermoFisher
Scientific, Waltham, Mass.) for nucleic acid purification: sample
plate, wash solution 1 plate (300 .mu.l/well), wash solution 2
plate (300 .mu.l/well), and elution buffer plate (90 .mu.l/well),
reagents supplied with kit. The nucleic acid purification procedure
consisted of the following steps: lysis/binding for 5 min, one
60-sec wash 1, one 15-sec wash 2, 1-min dry step, and a 3-min
heated elution step at 95.degree. C. All samples were immediately
placed on ice following denaturation and prior to qPCR
amplification. After amplification, the extracted DNA was then
stored at -20.degree. C. To minimize the potential risks for
contamination, DNA extraction, (q)PCR amplification, and agarose
gel electrophoresis were performed in physically separate rooms.
Positive controls and negative controls (water) were included in
qPCR multiplex assays.
[0156] Bacterial cell substrate DNA extraction provided genomic DNA
controls. IFA bacterial substrate slides (VMRD, Pullman, Wash.)
were utilized to represent pure genomic DNA of Bb, Rr and Ec. These
inactivated bacterial slides were prepared through rehydration with
10 .mu.l PBS onto a single slide well and then suspended in 90
.mu.l PBS, followed by DNA extraction through automated KingFisher
DNA extraction.
[0157] Molecular Analysis PCR:
[0158] The TaqMan qPCR (25 .mu.l total volume) used for this study
utilized the components of a commercial qPCR kit (following
manufacturer's recommendations) and primers and probes designed for
Bb, Ec, Rr and an endogenous canine amplification control. The qPCR
was performed in an Applied Biosystems.RTM. 7500 Fast Real-Time PCR
System (ThermoFisher Scientific, Waltham, Mass.). Amplification
continued for 40 cycles, where samples with a quantification cycle
(Cq).ltoreq.36.0 cycles were considered positive.
[0159] For suspect verification, positive results from the qPCR
multiplex assays were compared with those obtained by conventional
PCR. A conventional PCR protocol for the detection of the 16S RNA
gene of Ehrlichia spp. was utilized for the confirmation of Ec as
previously described (Wen, et al., J. Clin. Microbiol. 35:1852-1855
(1997)). The respective PCR was standardized as a nested PCR
reaction, utilizing primers common to Ehrlichia spp., followed by a
nested PCR to detect the specific species of Ehrlichia. Another
conventional PCR protocol for the detection of the ompA gene of
Rickettsia spp. was utilized for the confirmation of R. rickettsii
as previously described (Ishikura, et al., Microbiol. Immunol.
47:823-832 (2003)). The respective PCR was standardized as a nested
PCR reaction, utilizing primers common to Rickettsia spp., followed
by a nested PCR to detect the specific species of Rickettsia.
Positive (genomic) controls and negative controls (water) were
included in all PCR assays.
[0160] Sequencing:
[0161] After completion of nested PCR reactions on the positive Ec
suspects, 10 .mu.l of the product was mixed with OrangeG loading
buffer (NEB Inc., Ipswich, Mass.) and placed into wells of a 1.5%
agarose gel containing 0.4 .mu.g/ml of GelRed.TM. (Biotium,
Hayward, Calif.). Gel electrophoresis was performed at 90 volts for
60-90 minutes. After electrophoresis the gels were imaged using
GelDoc.TM. Imaging System (Bio-Rad Laboratories, Inc., Hercules,
Calif.). Positive bands were then cut directly from the gel and
purified using the Wizard.RTM. SV Gel and PCR Clean-up System
(Promega Corp., Madison Wis.) per the manufacturer's
recommendations. Purified products were then sent to Eton
Biosciences Ltd. (San Diego, Calif.) for sequencing to confirm the
PCR results. Sequences were determined on both forward and reverse
strands to obtain maximal data accuracy.
[0162] Contig sequences from each Ec suspects were assembled
individually using CLC MainWorkBench 7.7 (CLCbio, Aarhus, Denmark)
by utilizing the associated sequences assembly tool. Consensus
sequences produced were then aligned to an in-house curated
Ehrlichia spp. 16S rRNA gene sequence reference database containing
Ehrlichia spp. type strains Oklahoma (E. canis), Welgevonden (E.
ruminantium), Arkansas (E. chaffeensis), Stillwater (E. ewingii),
and AS145 (E. muris), through the alignment creation tool based on
standard parameters set by CLC MainWorkbench 7.7. Each end of the
raw alignment was trimmed to remove positions containing gaps.
Finally the reformatted sequences were re-sorted by similarity to
achieve a completed data set. Trees were generated for the suspect
sequence set against Ehrlichia spp. based on the Neighbor Joining
tree construction method and the nucleotide distance measure
Jukes-Cantor. In addition, bootstrap analysis was performed at 1000
replicates. Trees were then re-rooted against an outgroup species
type strain (Ap strain Webster).
Example 6--Specificity of Gel-Based Multiplex PCR Assay
[0163] The specificity of a gel-based multiplex PCR assay was
tested with 1,182 specimens, particularly, 650 ticks, 525 wildlife
tissues, and 7 dog blood samples. Target sequences were identified
using between 25 ng and 100 ng total DNA from the sample. The test
was 95.7% specific for Bb using ticks, and 83.3% when using tissues
(Tables 7 and 8). Blood samples that tested positive (1/7) were
confirmed positive by sequencing.
TABLE-US-00007 TABLE 7 PCR specificity by sample type Bacterial
species Ticks Small mammals tissue Blood samples Bb 95.7% 83.3%
100% Ec 85.18% 87.09% 100% Ech 47.82% 100% 0
TABLE-US-00008 TABLE 8 PCR specificity by sample type Bacterial
species No. positive sequencing/No. positive PCR Percent Bb 54/51
94.4 Ec 51/59 86.4 Ech 24/36 66.6
[0164] Ap infection was not detected in any of the samples tested.
Nevertheless, the invention was tested with samples containing
other Anaplasma spp. and was shown not to cross react. The
detection of Ec was 85.18% specific when analyzing tick samples,
and 87.09% specific when testing tissues. All positive blood
samples were confirmed by sequencing (1/7). Ech showed 100%
specificity when testing tissues and only 50% specificity when
testing ticks.
[0165] The apparent low specificity of the test in tissues for Ec
and Ech is mostly because these bacteria are intracellular
pathogens that infect white blood cells, in particular monocytes.
Ap infects granulocytes. These tests will allow physicians and
veterinarians to differentiate whether Ec, Ech and/or Ap caused the
infection. The current technologies do not permit such
differentiation because conducting serological diagnosis involves
significant cross-reaction between all Rickettsiales microorganisms
(Anaplasma, Ehrlichia and Rickettsia species). Tick samples
infected with both pathogens were also detected (Table 9).
TABLE-US-00009 TABLE 9 Ehrlichia and Borrelia co-infection Positive
Samples (n) Ec and Bb Ech and Bb Ticks Samples (45) 18 9 Small
mammals tissues samples (5) 0 0 Blood samples (1) 0 0
Example 7--LayerPlex Assay to Identify Tick-Borne Pathogens
[0166] This LayerPlex assay using qPCR to identify one or more
tick-borne pathogens in a sample. The LayerPlex assay has the
following three layers; (1) Borrelial Layer which identifies if any
of the following are present: B. burgdorferi, B. hermsii, B.
turicatae, and B. parkeri; (2) Rickettsiales Layer which identifies
if any of the following are present: R. rickettsii, E. canis, E.
chaffeensis, E. ewingii, and A. phagocytophilum; and (3) Babesial
Layer which identifies if any of the following are present: B.
canis vogeli, B. gibsoni, B. canis canis, B. canis rossi, B.
caballi, B. odocoilei, B. divergens, and B. bigemina. Each layer in
this assay utilize its own distinct fluorescent labels for
determining the presence of one or more of the microbial organisms
in that layer. Then to identify the specific pathogen present, one
must conduct a second qPCR assay with different fluorescent labels
to identify the particular microorganism.
[0167] As mentioned above, one performs the LayerPlex qPCR assay on
a sample to determine if any pathogens are identified by the three
layers (Borrelial, Rickettsiales, and Babesial) and thus are
present in the sample. In one well of a multi-well plate, one
places the primers and probes for the three layers (Borrelial,
Rickettsiales, and Babesial) as well as the primers and probes for
the endogenous internal positive control (NIPcK9 if the sample is
obtain from a dog) (thus, each well contains 4 layers). FIG. 1A
shows the names and sequences for the forward and reverse primers,
the probes, and the amplicons for each pathogen in the Borrelial
Layer. In this example, the Borrelial Layer probes use FAM/BHQ1
(fluorescein (5' reporter) and Black Hole Quencher 1.TM. (3'
acceptor)) fluorescent label. FIG. 1B shows the names and sequences
for the forward and reverse primers, the probes, and the amplicons
for each pathogen in the Rickettsiales Layer. In this example, the
Rickettsiales Layer probes use CF0560/BHQ1 (Cal-Fluor Orange (5'
reporter) and Black Hole Quencher 1.TM. (3' acceptor)) fluorescent
label. FIG. 1C shows the names and sequences for the two forward
primers, one reverse primer, one probe, and amplicon that are using
to determine if any of the three Babesia species identified by the
Babesial Layer are present in the sample. In this example, the
Babesial Layer probe uses Q670/BHQ2 (Quasar.RTM. 670 (5' reporter)
and Black Hole Quencher 2.TM. (3' acceptor)) fluorescent label. If
one is testing samples from dogs, one can use NIPcK9, a canine
mitochondrial DNA sequence, as a positive internal amplification
control for this LayerPlex qPCR assay and to confirm that the
sample was obtained from a dog because the sample should be
positive for the NIPcK9 amplicon. For NIPcK9, the probe and the
forward and reverse primers which generate an amplicon are shown in
FIG. 1C. In this example, one can use TAMRA/BHQ2
(tetramethylrhodamine (5' reporter) and Black Hole Quencher 2.TM.
(3' acceptor)) fluorescent label for NIPcK9. One also uses a
positive internal amplification control plasmid, referred to as
Tick10.PAC (see FIGS. 2A-2C), which contains each of the amplicons
generated by the above discussed primers. The sequences of
Tick10.PAC (SEQ ID NO: 68) and NIPcK9 amplicon (SEQ ID NO: 45) can
be cloned into any appropriate plasmid such as pUC57. One can use
bodily fluids and/or tissue samples from a mammal or a tick with
this LayerPlex qPCR diagnostic assay. To isolate the DNA from the
sample, one uses the methods described above.
[0168] For the qPCR assays, Applied Biosystems.TM. 7500 Fast
Real-Time PCR System (ThermoFisher Scientific, Waltham, Mass.) and
MicroAmp.TM. Fast Optical 96-Well Reaction Plate and optical plate
seals (ThermoFisher Scientific, Waltham, Mass.) are used. Stock
solutions of the primers/probes and the plasmids are made using
nuclease free water and IDTE pH 8.0. The Tick10.PAC and NIPcK9
plasmids are diluted to 1,000 copies and 10,000 copies which
generate a Cq value of 23-32 and 20-29, respectively. Path-ID qPCR
Master Mix (ThermoFisher Scientific, Waltham, Mass.) is used. A
12.5.times. LayerPlex and NIPcK9 primers and probes mix is
generated that contains all of the above described primers and
probes for the Borrelial, Rickettsiales, and Babesial layers and
the positive control (NIPcK9). qPCR master mix (17 .mu.l) is
generated by combining 2.5 .mu.l nuclease-free water, 12.5 .mu.l
2.times. Path-ID qPCR Master Mix, and 2.0 .mu.l 12.5.times.
LayerPlex and NIPcK9 primers and probes mix. The concentrations of
NIPcK9 primers and probe are 0.4 .mu.M. The concentrations of all
other primers are 5.6 .mu.M, and the concentrations of all other
probes are 1.6 .mu.M. 17 .mu.l qPCR master mix is distributed into
qPCR plate wells. 8 .mu.l of sample DNA in water (at concentration
of between 2 ng DNA/.mu.l and 100 ng DNA/.mu.l water) are added to
the wells, mixed, and plates sealed. qPCR cycles are 95.0.degree.
C. for 10 minutes (Stage 1, denaturation); 95.0.degree. C. for 1
second and 60.0.degree. C. for 30 seconds with 40 repetitions
(Stage 2, annealing & extension). Fluorescence data is acquired
at the conclusion of each annealing step to measure amplification
quantity which is used to generate the Cq value at the conclusion
of the final extension cycle.
[0169] For the Borrelial Layer (i.e., to detect Borrelia spp. in
the sample), one uses the following primer sets (forward and
reverse) to generate the indicated amplicon and probes containing
the indicated oligonucleotide sequence: [0170] (i) SEQ ID NOs: 1
and 2 to generate a 75 bp B. burgdorferi amplicon (SEQ ID NO: 4)
and use SEQ ID NO: 3 for the probe, [0171] (ii) SEQ ID NOs: 5 and 7
to generate a 117 bp B. hermsii amplicon (SEQ ID NO: 9) and use SEQ
ID NO: 8 for the probe, [0172] (iii) SEQ ID NOs: 6 and 7 to
generate a 119 bp B. hermsii amplicon (SEQ ID NO: 10) and use SEQ
ID NO: 8 for the probe, [0173] (iv) SEQ ID NOs: 11 and 13 to
generate a 142 bp B. turicatae amplicon (SEQ ID NO: 15) and use SEQ
ID NO: 14 for the probe, [0174] (v) SEQ ID NOs: 12 and 13 to
generate a 139 bp B. turicatae amplicon (SEQ ID NO: 16) and use SEQ
ID NO: 14 for the probe, and [0175] (vi) SEQ ID NOs: 17 and 18 to
generate a 117 bp B. parkeri amplicon (SEQ ID NO: 20) and use SEQ
ID NO: 19 for the probe. These Borrelial Layer probes contain
5'-FAM/BHQ1-3' as the fluorescent label and quencher.
[0176] For the Rickettsiales Layer (i.e., to detect Rickettsia spp.
or Anaplasma spp. or Ehrlichia spp. in the sample), one uses the
following primer sets (forward and reverse) to generate the
indicated amplicon and probes containing the indicated
oligonucleotide sequence: [0177] (vii) SEQ ID NOs: 21 and 22 to
generate a 159 bp R. rickettsii amplicon (SEQ ID NO: 24) and use
SEQ ID NO: 23 for the probe, [0178] (viii) SEQ ID NOs: 25 and 26 to
generate a 95 bp A. phagocytophilum amplicon (SEQ ID NO: 28) and
use SEQ ID NO: 27 for the probe, [0179] (ix) SEQ ID NOs: 29 and 30
to generate a 113 bp E. canis amplicon (SEQ ID NO: 32) and use SEQ
ID NO: 31 for the probe, [0180] (x) SEQ ID NOs: 33 and 34 to
generate a 111 bp E. chaffeensis amplicon (SEQ ID NO: 35) and use
SEQ ID NO: 31 for the probe, and [0181] (xi) SEQ ID NOs: 36 and 34
to generate a 114 bp E. ewingii amplicon (SEQ ID NO: 37) and use
SEQ ID NO: 31 for the probe. These Rickettsiales Layer probes
contain 5'-CF0560/BHQ1-3' as the fluorescent label and
quencher.
[0182] For the Babesial Layer (i.e., to detect Babesia spp. in the
sample), one uses the following primer sets (forward and reverse)
to generate the indicated amplicon and probes containing the
indicated oligonucleotide sequence: [0183] (xii) SEQ ID NOs: 38 and
39 to generate a 94 bp Babesia spp. amplicon (SEQ ID NO: 41) and
use SEQ ID NO: 40 for the probe, and [0184] (xiii) SEQ ID NOs: 77
and 39 to generate a 93 bp Babesia spp. amplicon (SEQ ID NO: 78)
and use SEQ ID NO: 40 for the probe. These Babesial Layer probes
contains 5'-Q670/BHQ2-3' as the fluorescent label and quencher.
[0185] For this LayerPlex qPCR assay, for an amplification control
one uses (1) a plasmid that contains canine specific NIPcK9
amplicon (SEQ ID NO: 45) which is generated using primer set SEQ ID
NOs: 42 and 43 and using probe (SEQ ID NO: 44) containing
5'-TAMRA/BHQ2-3'; and (2) Tick10.PAC (SEQ ID NO: 68) which contains
the sequence for each amplicon in the Borrelial Layer,
Rickettsiales Layer, and Babesial Layer, as well as additional
nucleotides for ease of cloning.
[0186] A particular layer from this LayerPlex qPCR is positive for
a microorganism within that layer if the Cq value for that layer is
36.0 or less for all types of samples, then the sample is positive
for at least one microorganism in that layer; when the Cq value for
a layer is between 36.1 and 38.0, then the sample is suspect
positive for at least one microorganism within that layer (and one
can repeat the qPCR assay using 1/4 or 1/2 of the sample size); and
when the Cq value for a layer is 38.1 or greater, then the sample
is negative for any of the possible microorganism within that
layer; so long as the amplification controls' Cq values are as
follows: (1) If one used NIPcK9 for amplification control and to
conclusively identify the sample as contained dog mDNA, then
NIPcK9's Cq value is 25.0 or less for a dog blood sample or 36.0 or
less for any other type of sample from a dog, the LayerPlex assay
is accurate. (2) If one is using Tick10.PAC plasmid as an
amplification control (instead of NIPcK9), then Tick10.PAC's Cq
value is 36.0 or less, regardless of the type of sample being
tested, the LayerPlex assay is accurate. But, if the Cq value for
NIPcK9 and/or Tick10.PAC indicates an invalid result, then one
should repeat the LayerPlex qPCR assay by using 1/4 or 1/2 sample
volume.
[0187] When one wants to know the specific microorganism present in
a positive layer, one can then conduct individual, distinct qPCR
assays for that positive layer. The probes for these individual and
distinct qPCR assays use the same fluorescent labels and quenchers
that they used for the LayerPlex PCR test because each qPCR will be
in different wells of a plate and one can distinguish which wells
are positive and which are negative.
[0188] To identify the species within the Borrelial Layer, one uses
the following primer sets to generate the indicated amplicon and
probes having the indicated oligonucleotide sequence: [0189] (i)
SEQ ID NOs: 1 and 2 to generate a 75 bp B. burgdorferi amplicon
(SEQ ID NO: 4) and use SEQ ID NO: 3 for the probe's
oligonucleotide, [0190] (xiv) SEQ ID NOs: 46 and 47 to generate a
135 bp B. hermsii gene group I (GG I) amplicon (SEQ ID NO: 48) and
use SEQ ID NO: 8 for the probe's oligonucleotide, [0191] (xv) SEQ
ID NOs: 49 and 50 to generate a 136 bp B. hermsii gene group II (GG
II) amplicon (SEQ ID NO: 51) and use SEQ ID NO: 8 for the probe's
oligonucleotide, [0192] (iv) SEQ ID NOs: 11 and 13 to generate a
142 bp B. turicatae amplicon (SEQ ID NO: 15) and use SEQ ID NO: 14
for the probe's oligonucleotide, [0193] (v) SEQ ID NOs: 12 and 13
to generate a 139 bp B. turicatae amplicon (SEQ ID NO: 16) and use
SEQ ID NO: 14 for the probe's oligonucleotide, and [0194] (vi) SEQ
ID NOs: 17 and 18 to generate a 117 bp B. parkeri amplicon (SEQ ID
NO: 20) and use SEQ ID NO: 19 for the probe's oligonucleotide.
[0195] To identify the species within the Rickettsiales Layer, one
uses the following primer sets to generate the indicated amplicon
and probes having the indicated oligonucleotide sequence: [0196]
(vii) SEQ ID NOs: 21 and 22 to generate a 159 bp R. rickettsii
amplicon (SEQ ID NO: 24) and use SEQ ID NO: 23 for the probe's
oligonucleotide, [0197] (viii) SEQ ID NOs: 25 and 26 to generate a
95 bp A. phagocytophilum amplicon (SEQ ID NO: 28) and use SEQ ID
NO: 27 for the probe's oligonucleotide, [0198] (ix) SEQ ID NOs: 29
and 30 to generate a 113 bp E. canis amplicon (SEQ ID NO: 32) and
use SEQ ID NO: 31 for the probe's oligonucleotide, [0199] (x) SEQ
ID NOs: 33 and 34 to generate a 111 bp E. chaffeensis amplicon (SEQ
ID NO: 35) and use SEQ ID NO: 31 for the probe's oligonucleotide;
and [0200] (xi) SEQ ID NOs: 36 and 34 to generate a 114 bp E.
ewingii amplicon (SEQ ID NO: 37) and use SEQ ID NO: 31 for the
probe's oligonucleotide.
[0201] To identify the species within the Babesial Layer, one uses
the following primer sets to generate the indicated amplicon and
probes having the indicated oligonucleotide sequence: [0202] (xvi)
SEQ ID NOs: 52 and 53 to generate a 81 bp B. canis vogeli amplicon
(SEQ ID NO: 55), and use SEQ ID NO: 54 for the probe's
oligonucleotide or (xvii) SEQ ID NOs: 56 and 57 to generate a 73 bp
B. canis vogeli amplicon (SEQ ID NO: 59), and use SEQ ID NO: 58 for
the probe's oligonucleotide, [0203] (xviii) SEQ ID NOs: 60 and 61
to generate a 73 bp B. gibsoni amplicon (SEQ ID NO: 63), and use
SEQ ID NO: 62 for the probe's oligonucleotide, and [0204] (xix) SEQ
ID NOs: 64 and 65 to generate a 76 bp B. conradae amplicon (SEQ ID
NO: 67), and use SEQ ID NO: 66 for the probe's oligonucleotide.
[0205] One also conducts qPCR assays to confirm that the sample is
obtained from the animal one believes from which the sample was
obtain and an amplification control for the tick-borne pathogen
sequences. If the sample was believed obtain from a canine, one
uses a plasmid that contains canine specific NIPcK9 amplicon (SEQ
ID NO: 45) which is generated using SEQ ID NO: 42 and 43 (forward
and reverse primers) and using probe (SEQ ID NO: 44) containing
5'-TAMRA/BHQ2-3'. For the microorganism amplification control, one
uses a plasmid containing Tick10.PAC (SEQ ID NO: 68) which contains
the sequence for each amplicon in the Borrelial Layer,
Rickettsiales Layer, and Babesial Layer as well as additional
nucleotides for ease of cloning.
[0206] For all of these individual qPCR assays to detect and
identify the particular species of microorganism(s) present in a
sample, a sample is positive for the microorganism when the Cq
value for a qPCR assay is 36.0 or less. When the Cq value of a qPCR
assay is between 36.1 and 38.0, then the sample is suspect
positive, and one can repeat the qPCR assay using 1/4 or 1/2 of the
sample size. When the Cq value of a qPCR assay is 38.1 or greater,
then the sample is negative; so long as the amplification controls'
Cq values are as follows: (1) If one uses NIPcK9, then NIPcK9's Cq
value is 25.0 or less for a dog blood sample or 36.0 or less for
any other type of sample from a dog, the qPCR assay is accurate.
(2) If one uses Tick10.PAC plasmid, then Tick10.PAC's Cq value is
36.0 or less, regardless of the type of sample being tested, the
qPCR assay is accurate. But, if the Cq value for NIPcK9 and/or
Tick10.PAC indicates an invalid result, then one should repeat the
qPCR assay by using 1/4 or 1/2 sample volume.
[0207] Tables 10, 11, 12, and 13, below, details the specificity of
each primer set in the LayerPlex PCR assay for the Rickettsial
Layer (Table 10), Borrelial Layer (Table 11), and Babesial Layer
(Table 12), and the primer set for the NIPcK9 PCR assay (Table 13).
Each specific species targeted by each primer set for each layer of
the LayerPlex PCR is detailed along the x-axis, and all closely
related species that were tested for potential cross amplification
are detailed along the y-axis. The tables demonstrate that only the
intended species were detected by each primer set in each
respective LayerPlex PCR, and no closely related species were
subsequently detected. See FIGS. 1A-1D for the primer sets used for
the species listed in the top row in the tables below.
TABLE-US-00010 TABLE 10 Rickettsial Assays Specificity E. canis E.
chaffeensis E. ewingii A. phagocytophilum R. rickettsii E. canis
Positive Negative Negative Negative Negative E. chaffeensis
Negative Positive Negative Negative Negative E. ewingii Negative
Negative Positive Negative Negative E. ruminantium Negative
Negative Negative Negative Negative E. muris Negative Negative
Negative Negative Negative A. phagocytophilum Negative Negative
Negative Positive Negative A. marginale Negative Negative Negative
Negative Negative A. ovis Negative Negative Negative Negative
Negative A. centrale Negative Negative Negative Negative Negative
R. rickettsii Negative Negative Negative Negative Positive
TABLE-US-00011 TABLE 11 Borrelial Assays Specificity B. hermsii GGI
B. hermsii GGII B. parkeri B. turicatae B. burgdorferi B. hermsii
GGI Positive Negative Negative Negative Negative B. hermsii GGII
Negative Positive Negative Negative Negative B. parkeri Negative
Negative Positive Negative Negative B. turicatae Negative Negative
Negative Positive Negative B. miyamotoi Negative Negative Negative
Negative Negative B. coriaceae Negative Negative Negative Negative
Negative B. anserina Negative Negative Negative Negative Negative
B. crocidurae Negative Negative Negative Negative Negative B.
recurrentis Negative Negative Negative Negative Negative B.
burgdorferi Negative Negative Negative Negative Positive B. garinii
Negative Negative Negative Negative Negative B. afzelii Negative
Negative Negative Negative Negative
TABLE-US-00012 TABLE 12 Babesial Assay Specificity Babesia Assay B.
canis vogeli Positive B. canis canis Positive B. canis rossi
Positive B. gibsoni Positive B. caballi Positive B. odocoilei
Positive B. divergens Positive B. bigemina Positive B. microti
Negative B. bovis Negative B. duncani Negative B. conradae Negative
T. equi Negative
TABLE-US-00013 TABLE 13 NIPcK9 Assay Specificity NIPcK9 Canis lupus
familiaris dog) Positive Canis lupus gray-wolf) Positive Canis
latrans coyote) Positive Equus caballus horse) Negative Felis catus
cat) Negative Capra aegagrus hircus goat) Negative Ovis aries
sheep) Negative Odocoileus virginianus Negative white-tailed deer)
Mephitis mephitis skunk) Negative Bos taurus cow) Negative Sus
scrofa pig) Negative Gallus gallus chicken) Negative Procyon lotor
raccoon) Negative Vulpes vulpes fox) Negative
[0208] Tables 14, 15, 16, and 17 below, demonstrate the sensitivity
analysis for the LayerPlex PCR assay, and the LayerPlex PCR assay's
robustness for the target microorganisms using the primers
discussed above each layer. The efficiency, R.sup.2, and limit of
detection data for each assay are provided.
TABLE-US-00014 TABLE 14 qPCR Assays Efficiency R.sup.2 Limit of
detection (copy #) B. hermsii 99% 1.000 ~16 B. turicatae 101% 0.988
~16 B. parkeri 99% 0.998 ~16 B. burgdorferi 97% 0.996 ~16
TABLE-US-00015 TABLE 15 qPCR Assays Efficiency R.sup.2 Limit of
detection (copy #) E. canis 97% 0.996 ~16 E. ewingii 91% 1.000 ~16
E. chaffeensis 94% 0.997 ~16 A. phagocytophilum 95% 0.996 ~16 R.
rickettsii 97% 0.995 ~16
TABLE-US-00016 TABLE 16 qPCR Assays Efficiency R.sup.2 Limit of
detection (copy #) Babesia spp. 91% 0.995 ~16
TABLE-US-00017 TABLE 17 qPCR Assays Efficiency R.sup.2 Limit of
detection (copy #) Borrelial Layer 105% 0.998 ~16 Rickettsial Layer
104% 0.999 ~16 Babesial Layer 105% 0.999 ~16
[0209] Table 18, below, shows the results of testing 654 samples
with the Borrelial and Rickettsial layers of the LayerPlex qPCR
assay to screen for the following pathogens: B. burgdorferi, B.
hermsii, B. turicatae, B. parkeri, R. ricketsii, E. canis, E.
chaffeensis, E. ewingii, and A. phagocytophilum.
TABLE-US-00018 TABLE 18 Rickettsial layer Borrelial layer Sample +
- % positive + - % positive Human blood 2 165 1.2 9 115 7.5
Domestic dog blood 47 118 28.4 3 164 2.67 Ticks from domestic 11
154 6.6 15 138 11.19 dogs Ticks from humans 0 0 0 1 16 6.2 White
tailed deer 14 33 29.7 0 0 0 (liver/spleen) Temazate deer 2 2 50 0
0 0 (liver/spleen) Feral hog (liver/spleen) 1 1 50 0 0 0 Mexican
badger 0 0 0 1 1 50 (liver/spleen) Ticks from deer 3 63 5.4 1 65
1.5 Ticks from weasel 1 0 100 0 0 0 Total 81 573 12.38% 30 624
4.59%
[0210] A random subset of 94 samples were selected for species
level identification. Of the 94 samples, 7 (7.4%) and 5 (5.3%) were
detected as positive for Rickettsial and Borrelial Layers
respectively. Further species level analysis revealed that of the
Rickettsial Layer, 5 positives samples were determined to be
positive for E. canis, 1 suspect positive for R. rickettsii, and 1
suspect positive for A. phagocytophilum. No samples were determined
to be positive for E. ewingii or E. chaffeensis. The Borrelial
Layer positive samples are pending species level identification.
Out of the 654 samples tested by LayerPlex qPCR, 5 samples were
positive for both Borrelial and Rickettsial layers and were
subsequently suspected as co-infections of the respective
microorganisms.
[0211] A random set of 22 blood samples deriving from dogs
suspected of tick-borne infection were screened by the LayerPlex
qPCR assay using the Borrelial, Rickettsial, and Babesial layers
using the primers and probes in FIGS. 1A-1D. Then, the samples that
tested positive for at least one layer was subjected to qPCR for
the microorganisms within that layer using the primers and probes
of FIGS. 1A-1D (but the probes contain different fluorescent
molecules so that the microbial species could be distinguished from
each other as discussed previously). The results revealed that 3
samples (13.6%) were positive for B. turicatae, 3 samples (13.6%)
were positive for E. canis, 1 sample (4.5%) was positive for B.
gibsoni, and zero samples were positive for B. hermsii, B. parkeri,
B. burgdorferi, A. phagocytophilum, E. chaffeensis, E. ewingii, or
R. rickettsii.
[0212] All patents, patent applications, provisional applications,
and publications referred to or cited herein are incorporated by
reference in their entirety, including all figures and tables, to
the extent they are not inconsistent with the explicit teachings of
this specification. Also, all non-patent publications referred to
or cited herein are incorporated by reference in their
entirety.
[0213] It should be understood that the examples and embodiments
described herein are for illustrative purposes only and that
various modifications or changes in light thereof will be suggested
to persons skilled in the art and are to be included within the
spirit and purview of this application and the scope of the
appended claims. In addition, any elements or limitations of any
invention or embodiment thereof disclosed herein can be combined
with any and/or all other elements or limitations (individually or
in any combination) or any other invention or embodiment thereof
disclosed herein, and all such combinations are contemplated within
the scope of the invention without limitation thereto.
Sequence CWU 1
1
82122DNABorrelia burgdorferi 1aagagggtgt tcaacaggaa gg
22223DNABorrelia burgdorferi 2gagaattaac tccgccttga gaa
23325DNABorrelia burgdorferi 3tcaacagcca gcacctgcta cagca
25475DNABorrelia burgdorferi 4aagagggtgt tcaacaggaa ggagctcaac
agccagcacc tgctacagca ccttctcaag 60gcggagttaa ttctc
75519DNABorrelia hermsii 5gggcgcaaat caggatgag 19621DNABorrelia
hermsii 6gtgggagcaa atcaggatga g 21724DNABorrelia hermsii
7tcctcttgct gtcctatctc ttgc 24828DNAArtificial Sequencechemically
synthesized 8agcctgagcr ccttcacctg caaaaaga 289117DNABorrelia
hermsii 9gggcgcaaat caggatgagg caattgctgt taatatttat gcatctaatg
ttgcaaatct 60ttttgcaggt gaaggcgctc aggctgctcc agtgcaagag ataggacagc
aagagga 11710119DNABorrelia hermsii 10gtgggagcaa atcaggatga
ggcaattgct gttaatattt atgcatctaa tgttgcaaat 60ctttttgcag gtgaaggtgc
tcaggctgct ccagagcaag agataggaca gcaagagga 1191125DNABorrelia
turicatae 11agaccggtac acaggattct aaagc 251222DNABorrelia turicatae
12ccggcacaca ggattctaaa ac 221326DNABorrelia turicatae 13gttcctgctc
cctgaataac attatc 261427DNABorrelia turicatae 14agttttggga
agtgttgttg gtggcgt 2715142DNABorrelia turicatae 15agaccggtac
acaggattct aaagcacaat attctgattt ttctgatcaa gatataagag 60ataaagtttt
gggaagtgtt gttggtggcg ttgtagacaa tgttatgagt ggaattgata
120atgttattca gggagcagga ac 14216139DNABorrelia turicatae
16ccggcacaca ggattctaaa acacaatatt ctgatttttc tgatcaagat ataagagata
60aagttttggg aagtgttgtt ggtggcgttg tagacaatgt tatgagtgga attgataatg
120ttattcaggg agcaggaac 1391724DNABorrelia parkeri 17ttgtccaata
agtcagctgc tcag 241824DNABorrelia parkeri 18tcttaatgtc catgaagctt
gtgc 241926DNABorrelia parkeri 19ctgaagagct tggaatgcaa cctgca
2620117DNABorrelia parkeri 20ttgtccaata agtcagctgc tcagaatata
aaaacagctg aagagcttgg aatgcaacct 60gcaaaaatta acacaccagc atcattagct
ggagcacaag cttcatggac attaaga 1172126DNARickettsia rickettsii
21agagtaaatc aacggaagag caaaac 262221DNARickettsia rickettsii
22cccctccact acctgcatca t 212325DNARickettsia rickettsii
23tcctctccaa tcagcgattc aggca 2524159DNARickettsia rickettsii
24agagtaaatc aacggaagag caaaactttc aatcagaagg agtggtaagt tcagtaagta
60agatgccaac cataaattct agtgagtcct ctccaatcag cgattcaggc agtaagtcag
120actctgactc aaaagatgat gatgcaggta gtggagggg 1592524DNAAnaplasma
phagocytophilum 25gactttccta gcatggagtt ggtt 242625DNAAnaplasma
phagocytophilum 26gcgtgccctt ttgtaatacc tataa 252725DNAAnaplasma
phagocytophilum 27catttcacct tacacatgcg ccgga 252895DNAAnaplasma
phagocytophilum 28gactttccta gcatggagtt ggttaaactc aacatttcac
cttacacatg cgccggaata 60ggtggaagcg ttataggtat tacaaaaggg cacgc
952926DNAEhrlichia canis 29gcctctggct ataggaaatt gttagt
263026DNAEhrlichia canis 30ctcggggatt atacagtatt acccac
263129DNAEhrlichia canis 31agattcctac gcattactca cccgtctgc
2932113DNAEhrlichia canis 32gcctctggct ataggaaatt gttagtggca
gacgggtgag taatgcgtag gaatctacct 60agtagtacgg aatagccatt agaaatggtg
ggtaatactg tataatcccc gag 1133326DNAEhrlichia chaffeensis
33gaacggacaa ttgcttataa cctttt 263428DNAEhrlichia chaffeensis
34ccatcatttc taatggctat tccatact 2835111DNAEhrlichia chaffeensis
35gaacggacaa ttgcttataa ccttttggtt ataaataatt gttagtggca gacgggtgag
60taatgcgtag gaatctacct agtagtatgg aatagccatt agaaatgatg g
1113627DNAEhrlichia ewingii 36cgaacgaaca attcctaaat agtctct
2737114DNAEhrlichia ewingii 37cgaacgaaca attcctaaat agtctctgac
tatttagata gttgttagtg gcagacgggt 60gagtaatgcg taggaatcta cctagtagta
tggaatagcc attagaaatg atgg 1143819DNABabesia spp. 38cgcatttagc
gatggacca 193921DNABabesia spp. 39cctaattccc cgttacccgt t
214027DNABabesia spp. 40catcagcttg acggtagggt attggcc
274194DNABabesia spp. 41cgcatttagc gatggaccat tcaagtttct gacccatcag
cttgacggta gggtattggc 60ctaccgaggc agcaacgggt aacggggaat tagg
944225DNACanis lupus 42ggattctact ccaaagacct gatca 254320DNACanis
lupus 43ggttagggat gtggcaacga 204425DNACanis lupus 44cacgtcgaat
accaacgcct gagcc 254596DNACanis lupus 45ggattctact ccaaagacct
gatcatcgag tccgctaaca cgtcgaatac caacgcctga 60gccctcttaa ttacactcgt
tgccacatcc ctaacc 964623DNABorrelia hermsii 46ggacattgag agtacatgtg
ggc 234724DNABorrelia hermsii 47cctcttgctg tcctatctct tgca
2448135DNABorrelia hermsii 48ggacattgag agtacatgtg ggcgcaaatc
aggatgaggc aattgctgtt aatatttatg 60catctaatgt tgcaaatctt tttgcaggtg
aaggcgctca ggctgctcca gtgcaagaga 120taggacagca agagg
1354924DNABorrelia hermsii 49tggacattga gagtacatgt ggga
245024DNABorrelia hermsii 50cctcttgctg tcctatctct tgct
2451136DNABorrelia hermsii 51tggacattga gagtacatgt gggagcaaat
caggatgagg caattgctgt taatatttat 60gcatctaatg ttgcaaatct ttttgcaggt
gaaggtgctc aggctgctcc agagcaagag 120ataggacagc aagagg
1365220DNABabesia canis vogeli 52ggaaatcgcc gagctctacc
205321DNABabesia canis vogeli 53cgctgggagt cgttgaagta c
215423DNABabesia canis vogeli 54tcggcaagac cgtcaaggat gcc
235581DNABabesia canis vogeli 55ggaaatcgcc gagctctacc tcggcaagac
cgtcaaggat gccgtcatca ctgtgcctgc 60gtacttcaac gactcccagc g
815622DNABabesia canis vogeli 56catcaagatg aaggaaatcg cc
225719DNABabesia canis vogeli 57cgcaggcaca gtgatgacg
195827DNABabesia canis vogeli 58atccttgacg gtcttgccga ggtagag
275973DNABabesia canis vogeli 59catcaagatg aaggaaatcg ccgagctcta
cctcggcaag accgtcaagg atgccgtcat 60cactgtgcct gcg 736022DNABabesia
gibsoni 60caatgaccag ggaaacagga ct 226123DNABabesia gibsoni
61agcatcacca ataagacgct cag 236225DNABabesia gibsoni 62actccctcat
acgtcgcctt caccg 256373DNABabesia gibsoni 63caatgaccag ggaaacagga
ctactccctc atacgtcgcc ttcaccgaca ctgagcgtct 60tattggtgat gct
736416DNABabesia conradae 64tctgccgccg ggactt 166518DNABabesia
conradae 65ggccgaggcc caaattaa 186626DNABabesia conradae
66cttcgttttc tcggtggcat ccctct 266776DNABabesia conradae
67tctgccgccg ggacttcgtt cccttcgggg cttcgttttc tcggtggcat ccctctggtt
60aatttgggcc tcggcc 76681416DNAArtificial Sequencechemically
synthesized 68atcgcggatc cgcgggacat tgagagtaca tgtgggcgca
aatcaggatg aggcaattgc 60tgttaatatt tatgcatcta atgttgcaaa tctttttgca
ggtgaaggcg ctcaggctgc 120tccagtgcaa gagataggac agcaagagga
agcatggaca ttgagagtac atgtgggagc 180aaatcaggat gaggcaattg
ctgttaatat ttatgcatct aatgttgcaa atctttttgc 240aggtgaaggt
gctcaggctg ctccagagca agagatagga cagcaagagg aagtgttgtc
300caataagtca gctgctcaga atataaaaac agctgaagag cttggaatgc
aacctgcaaa 360aattaacaca ccagcatcat tagctggagc acaagcttca
tggacattaa gagtatgaga 420ccggtacaca ggattctaaa gcacaatatt
ctgatttttc tgatcaagat ataagagata 480aagttttggg aagtgttgtt
ggtggcgttg tagacaatgt tatgagtgga attgataatg 540ttattcaggg
agcaggaact gttcaagagg gtgttcaaca ggaaggagct caacagccag
600cacctgctac agcaccttct caaggcggag ttaattctcc gcggatccgc
gatccggaat 660tccgcgcctc tggctatagg aaattgttag tggcagacgg
gtgagtaatg cgtaggaatc 720tacctagtag tacggaatag ccattagaaa
tggtgggtaa tactgtataa tccccgagga 780gtcgaacgga caattgctta
taaccttttg gttataaata attgttagtg gcagacgggt 840gagtaatgcg
taggaatcta cctagtagta tggaatagcc attagaaatg atgggtagtc
900gaacgaacaa ttcctaaata gtctctgact atttagatag ttgttagtgg
cagacgggtg 960agtaatgcgt aggaatctac ctagtagtat ggaatagcca
ttagaaatga tgggttatga 1020ctttcctagc atggagttgg ttaaactcaa
catttcacct tacacatgcg ccggaatagg 1080tggaagcgtt ataggtatta
caaaagggca cgccaaagag agtaaatcaa cggaagagca 1140aaactttcaa
tcagaaggag tggtaagttc agtaagtaag atgccaacca taaattctag
1200tgagtcctct ccaatcagcg attcaggcag taagtcagac tctgactcaa
aagatgatga 1260tgcaggtagt ggaggggccg gaattccggc cggatatccg
gcgaatcgct tttagcgatg 1320gaccattcaa gtttctgacc catcagcttg
acggtagggt attggcctac cgaggcagca 1380acgggtaacg gggaattagg
ccggatatcc ggaatt 14166923DNABorrelia burgdorferi 69aacacaccag
catcactttc agg 237025DNABorrelia burgdorferi 70gagaattaac
tccgccttga gaagg 257129DNAEhrlichia canis 71caattattta tagcctctgg
ctataggaa 297227DNAEhrlichia canis 72tataggtacc gtcattatct tccctat
277329DNAEhrlichia chaffeensis 73caattgctta taaccttttg gttataaat
297427DNAEhrlichia chaffeensis 74tataggtacc gtcattatct tccctat
277522DNAAnaplasma phagocytophilum 75ccgctttagt ggcacattat ag
227621DNAAnaplasma phagocytophilum 76ccgaaattct aaaatctggg t
217722DNABabesia spp. 77gcttttagcg atggaccatt ca 227893DNABabesia
spp. 78gcttttagcg atggaccatt caagtttctg acccatcagc ttgacggtag
ggtattggcc 60taccgaggca gcaacgggta acggggaatt agg
9379235DNABorrelia burgdorferi 79aacacaccag catcactttc agggtctcaa
gcgtcttgga ctttaagagt tcatgttgga 60gcaacccaag atgaagctat tgctgtaaat
atttatgcag ctaatgttgc aaatcttttc 120tctggtgagg gagctcaaac
tgctcaggct gcaccggttc aagagggtgt tcaacaggaa 180ggagctcaac
agccagcacc tgctacagca ccttctcaag gcggagttaa ttctc
23580389DNAEhrlichia canis 80caattattta tagcctctgg ctataggaaa
ttgttagtgg cagacgggtg agtaatgcgt 60aggaatctac ctagtagtac ggaatagcca
ttagaaatgg tgggtaatac tgtataatcc 120ccgaggggga aagatttatc
gctattagat gagcctacgt tagattagct agttggtgag 180gtaatggctt
accaaggcta tgatctatag ctggtctgag aggacgatca gccacactgg
240aactgagata cggtccagac tcctacggga ggcagcagtg gggaatattg
gacaatgggc 300gaaagcctga tccagctatg ccgcgtgagt gaagaagscc
ttcgggttgt aaaactcttt 360caatagggaa gataatgacg gtacctata
38981390DNAEhrlichia chaffeensis 81caattgctta taaccttttg gttataaata
attgttagtg gcagacgggt gagtaatgcg 60taggaatcta cctagtagta tggaatagcc
attagaaatg atgggtaata ctgtataatc 120cctgcggggg aaagatttat
cgctattaga tgagcctacg ttagattagc tagttggtaa 180ggtaatggct
taccaaggct atgatctata gctggtctga gaggacgatc agccacactg
240gaactgagat acggtccaga ctcctacggg aggcagcagt ggggaatatt
ggacaatggg 300cgaaagcctg atccagctat gccgcgtgag tgaagaaggc
cttcgggttg taaaactctt 360tcaataggga agataatgac ggtacctata
39082644DNAAnaplasma phagocytophilum 82ccgctttagt ggcacattat
agtactccga ccctaaaact ttctgatatg aagtacctat 60atatgccaca gcatttgaga
ggaaacggta atttaaacca agtttgagct tgtatgaaag 120ttgtaaattg
gcgtgccctt ttgtaatacc tataacgctt ccacctattc cggcgcatgt
180gtaaggtgaa atgttgagtt taaccaactc catgctagga aagtcataac
acaaattcaa 240cgtagcagtg gtattcctta tggccataat ctctatcact
tcagcaccct caaccgtcaa 300agcaagagct tttgtgaaaa gaacgctttg
cacaatcatc agatccagta aattaaaaaa 360attatcgctc ctatttctgg
gaaaagcccc aacaactttc ttagttttat taactctctt 420gagttcttct
tcctgcatta ccgaaagtct tctcagttga tcatatccta gttctgccac
480catgtatttt tctatagcga taacattttc acgcgaaaga cttccgatta
aatcaatacg 540atcatagccc ctctgtaaag acaccatcgc tttgtgtttt
cctaatacaa agggcatacc 600accacctttg cggcccttat gtaacccaga
ttttagaatt tcgg 644
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