U.S. patent application number 10/138162 was filed with the patent office on 2005-08-04 for methods for detecting ehrlichia canis and ehrlichia chaffensis in vertebrate and invertebrate hosts.
Invention is credited to Rikihisa, Yasuko, Stich, Roger William.
Application Number | 20050170341 10/138162 |
Document ID | / |
Family ID | 24601110 |
Filed Date | 2005-08-04 |
United States Patent
Application |
20050170341 |
Kind Code |
A1 |
Stich, Roger William ; et
al. |
August 4, 2005 |
Methods for detecting Ehrlichia canis and Ehrlichia chaffensis in
vertebrate and invertebrate hosts
Abstract
Tools and methods for detecting the presence of E. canis and E.
chaffeensis in a sample obtained from an animal are provided. The
methods employ a polymerase chain reaction and primer sets that are
based on the p30 gene of E. canis and the p28 gene of E.
chaffeensis. The present invention also relates to the p30 and the
p28 primer sets. Each p30 primer set comprises a first primer and
the second primer, both of which are from 15 to 35 nucleotides in
length. The first p30 primer comprises a sequence which is
complementary to a consecutive sequence, within the following
sequence: CCA AGTGTCTCAC ATTTTGGTAG CTTCTCAGCT AAAGAAGAAA
GCAAATCAAC TGTTGGAGTTTTTGGATTAA AACATGATTG GGATGGAAGT CCAATACTTA
AGAATAAACA CGCTGACTTTACTGTTCCAA AC. SEQ ID NO.1. The second p30
primer comprises a sequence which is complementary to the inverse
complement of a consecutive sequence contained within the following
sequence: GTTACT CAATGGGTGG CCCAAGAATA GAATTCGAAA TATCTTATGA
AGCATTCGAC GTAAAAAGTC CTAATATCAA TTATCAAAAT GACGCGCACA GGTACTGCGC
TCTATCTCAT CACACATCGG CAGCCAT, SEQ ID NO.2. The first p28 comprises
a sequence which is complementary to a consecutive sequenc, within
the following sequence: A GTTTTCATAA CAAGTGCATT GATATCACTA
ATATCTTCTC TACCTGGAGT ATCATTTTCC GACCCAACAG GTAGTGGTAT TAACGG, SEQ
ID NO. 3. The second p28 primercomprises a sequence which is
complementary to the inverse complement of a consecutive
sequencewithin one of the following two sequences: CAT TTCTAGGTTT
TGCAGGAGCT ATTGGCTACT CAATGGATGG TCCAAGAATA GAGCTTGAAG TATCTTATGA,
SEQ ID NO. 4, or C AAGGAAAGTT AGGTTTAAGC TACTCTATAA GCCCAGA, SEQ ID
NO. 5.
Inventors: |
Stich, Roger William;
(Columbus, OH) ; Rikihisa, Yasuko; (Worthington,
OH) |
Correspondence
Address: |
CALFEE HALTER & GRISWOLD, LLP
800 SUPERIOR AVENUE
SUITE 1400
CLEVELAND
OH
44114
US
|
Family ID: |
24601110 |
Appl. No.: |
10/138162 |
Filed: |
May 2, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10138162 |
May 2, 2002 |
|
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09648520 |
Aug 25, 2000 |
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6432649 |
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Current U.S.
Class: |
435/6.12 ;
435/91.2 |
Current CPC
Class: |
C12Q 1/689 20130101 |
Class at
Publication: |
435/006 ;
435/091.2 |
International
Class: |
C12Q 001/68; C12P
019/34 |
Claims
What is claimed is:
1. A method for detecting E. canis in a sample obtained from an
animal, comprising (a) providing a primer set comprising: (i) a
forward primer of from 15 to 35 nucleotides in length, said forward
primer comprising a sequence which is the complement of a
consecutive sequence within the following sequence:
6 SEQ ID NO.1 CCA AGTGTCTCAC ATTTTGGTAG CTTCTCAGCT AAAGAAGAAA
GCAAATCAAC TGTTGGAGTTTTTGGATTAA AACATGATTG GGATGGAAGT CCAATACTTA
AGAATAAACA CGCTGACTTTACTGTTCCAA AC., and
(ii) a reverse primer of from 15 to 35 nucleotides in length, said
reverse primer comprising a sequence which is complementary to the
inverse complement of a consecutive sequence within the following
sequence:
7 SEQ ID NO.2 GTTACT CAATGGGTGG CCCAAGAATA GAATTCGAAA TATCTTATGA
AGCATTCGAC GTAAAAAGTC CTAATATCAA TTATCAAAAT GACGCGCACA GGTACTGCGC
TCTATCTCAT CACACATCGG CAGCCAT,.;
(b) amplifying DNA in the sample with the said primer set and a
polymerase chain reaction, and (c) determining the length or
sequence of the PCR products of step (b), wherein the presence of a
PCR product having a length or sequence which corresponds to the
length or sequence, respectively, of that region of the E. canis
p30 gene which is located between the regions to which the forward
primer and the reverse primer bind is indicative of the presence of
E. canis in the sample.
2. The method of claim 1 wherein the consecutive sequence is at
least 14 nucleotides in length.
3. The method of claim 1 wherein the forward primer and the reverse
primer, respectively, comprise one of the following pairs of
sequences:
8 PAIR 1: ATAAACACGCTGACTTTACTGTTCC, S, SEQ ID NO. 6
GTGATGAGATAGAGCGCAGTACC,.; SEQ ID NO. 7 PAIR 2
AACACGCTGACTTTACTGTTCC,, SEQ ID NO. 8 ATGGCTGCCGATGTGTGATG,, SEQ ID
NO. 9 PAIR 3: ACGCTGACTTTACTGTTCCAAAC,, SEQ ID NO. 10
ATGGCTGCCGATGTGTGATG,, SEQ ID NO. 11 PAIR 4:
AACATGATTGGGATGGAAGTC,, SEQ ID NO. 12 GCCGATGTGTGATGAGATAG,; SEQ ID
NO. 13 PAIR 5: AAACATGATTGGGATGGAAGTC,, SEQ. ID NO. 12
GCCGATGTGTGATGAGATAGAG,; SEQ ID NO. 14 PAIR 6:
GATTGGGATGGAAGTCCAATAC,, SEQ. ID. NO. 13 GCCGATGTGTGATGAGATAG,; SEQ
ID NO. 11 PAIR 7: GATTGGGATGGAAGTCCAATAC,, SEQ ID NO. 13
GCCGATGTGTGATGAGATAGAG,; SEQ ID NO. 14 PAIR 8:
ACACGCTGACTTTACTGTTCCAAAC,; SEQ ID NO. 15 ATGGCTGCCGATGTGTGATGAG,-
; SEQ ID NO. 16 PAIR 9: GTGTCTCACATTTTGGTAGCTTCT- C,, SEQ ID NO. 17
CTTGGGCCACCCATTGAGTAAC,; SEQ ID NO. 18 PAIR 10
GATTGGGATGGAAGTCCAATAC,, SEQ. ID. NO. 13 CGATGTGTGATGAGATAGAGC,;
SEQ ID NO. 19 PAIR 11: TGATTGGGATGGAAGTCCAATAC,, SEQ ID NO. 20
CGATGTGTGATGAGATAGAGCG,, SEQ ID NO. 21 PAIR 12:
CATGATTGGGATGGAAGTCCAATA- C,, SEQ ID NO. 21 ATGGCTGCCGATGTGTGATG,;
SEQ ID NO. 11 PAIR 13: CCAAGTGTCTCACATTTTGGTAGC,, SEQ ID NO. 22
TGGGCCACCCATTGAGTAAC,. SEQ ID NO. 23
4. The method of claim 3 wherein the primer set is pair 12.
5. The method of claim 3 wherein the primer set is pair 1.
6. A method for detecting E. canis in a sample obtained from an
animal, comprising (a) providing a first primer set comprising: (i)
a forward primer of from 15 to 35 nucleotides in length, said
forward primer comprising a sequence which is the complement of a
consecutive sequence within the following sequence:
9 SEQ ID NO.1 CCA AGTGTCTCAC ATTTTGGTAG CTTCTCAGCT AAAGAAGAAA
GCAAATCAAC TGTTGGAGTTTTTGGATTAA AACATGATTG GGATGGAAGT CCAATACTTA
AGAATAAACA CGCTGACTTTACTGTTCCAA AC., and
(ii) a reverse primer of from 15 to 35 nucleotides in length, said
reverse primer comprising a sequence which is complementary to the
inverse complement of a consecutive sequence within the following
sequence:
10 SEQ ID NO.2 GTTACT CAATGGGTGG CCCAAGAATA GAATTCGAAA TATCTTATGA
AGCATTCGAC GTAAAAAGTC CTAATATCAA TTATCAAAAT GACGCGCACA GGTACTGCGC
TCTATCTCAT CACACATCGG CAGCCAT,.;
(b) amplifying DNA in the sample with the said primer set and a
polymerase chain reaction to provide a pool of PCR products, (c)
amplifying the products of step (b) using a polymerase chain
reaction and a second primer set comprising: (i) a forward primer
of from 15 to 35 nucleotides in length, said forward primer
comprising a sequence which is the complement of a consecutive
sequence within the following sequence:
11 SEQ ID NO.1 CCA AGTGTCTCAC ATTTTGGTAG CTTCTCAGCT AAAGAAGAAA
GCAAATCAAC TGTTGGAGTTTTTGGATTAA AACATGATTG GGATGGAAGT CCAATACTTA
AGAATAAACA CGCTGACTTTACTGTTCCAA AC., and
(ii) a reverse primer of from 15 to 35 nucleotides in length, said
reverse primer comprising a sequence which is complementary to the
inverse complement of a consecutive sequence within the following
sequence:
12 SEQ ID NO.2 GTTACT CAATGGGTGG CCCAAGAATA GAATTCGAAA TATCTTATGA
AGCATTCGAC GTAAAAAGTC CTAATATCAA TTATCAAAAT GACGCGCACA GGTACTGCGC
TCTATCTCAT CACACATCGG CAGCCAT,.;
wherein one or both of the primers of the second primer set are
internal to the primers of the first primer set; and (d)
determining the length or sequence of the PCR products of step (c),
wherein the presence of a PCR product having a length or sequence
which corresponds to the length or sequence, respectively, of that
region of the E. canis p30 gene which is located between the
regions to which the forward primer of the second primer set and
the reverse primer of the second primer set bind is indicative of
the presence of E. canis in the sample.
7. A method for detecting E. chaffeensis in a sample obtained from
an animal, comprising (a) providing a primer set comprising: (i) a
forward primer of from 15 to 35 nucleotides in length, said forward
primer comprising a sequence which is the complement of a
consecutive sequence within the following sequence:
13 SEQ ID NO.3 A GTTTTCATAA CAAGTGCATT GATATCACTA ATATCTTCTC
TACCTGGAGT ATCATTTTCC GACCCAACAG GTAGTGGTAT TAACGG,; and
(ii) a reverse primer of from 15 to 35 nucleotides in length, said
reverse primer comprising a sequence which is complementary to the
inverse complement of a consecutive sequence within one of the
following two sequences:
14 SEQ ID NO.4 CAT TTCTAGGTTT TGCAGGAGCT ATTGGCTACT CAATGGATGG
TCCAAGAATA GAGCTTGAAG TATCTTATGA,, or SEQ ID NO. 5 C AAGGAAAGTT
AGGTTTAAGC TACTCTATAA GCCCAGA,
(b) amplifying DNA in the sample with the said primer set and a
polymerase chain reaction, and (c) determining the length or
sequence of the PCR products of step (b), wherein the presence of a
PCR product having a length or sequence which corresponds to the
length or sequence, respectively, of that region of the E.
chaffeensis p28 gene which is located between the regions to which
the forward primer and the reverse primer bind is indicative of the
presence of E. chaffeensis in the sample.
8. The method of claim 7 wherein the consecutive sequence is at
least 14 nucleotides in length.
9. The method of claim 7 wherein the forward primer and the reverse
primer, respectively, comprise one of the following pairs of
sequences:
10. The method of claim 9 wherein the primer set is pair 1.
11. The method of claim 9 wherein the primer set is pair 3.
12. A method for detecting E. chaffeensis in a sample obtained from
an animal, comprising (a) providing a first primer set comprising:
providing a primer set comprising: (i) a forward primer of from 15
to 35 nucleotides in length, said forward primer comprising a
sequence which is the complement of a consecutive sequence within
the following sequence:
15 SEQ ID NO. 3 A GTTTTCATAA CAAGTGCATT GATATCACTA ATATCTTCTC
TACCTGGAGT ATCATTTTCC GACCCAACAG GTAGTGGTAT TAACGG,; and
(ii) a reverse primer of from 15 to 35 nucleotides in length, said
reverse primer comprising a sequence which is complementary to the
inverse complement of a consecutive sequence within one of the
following two sequences:
16 SEQ ID NO.4 CAT TTCTAGGTTT TGCAGGAGCT ATTGGCTACT CAATGGATGG
TCCAAGAATA GAGCTTGAAG TATCTTATGA,, or SEQ ID NO. 5 C AAGGAAAGTT
AGGTTTAAGC TACTCTATAA GCCCAGA,
(b) amplifying DNA in the sample with the said primer set and a
polymerase chain reaction to provide a pool of PCR products, (c)
amplifying the products of step (b) using a polymerase chain
reaction and a second primer set comprising: (i) a forward primer
of from 15 to 35 nucleotides in length, said forward primer
comprising a sequence which is the complement of a consecutive
sequence within the following sequence:
17 SEQ ID NO. 3 A GTTTTCATAA CAAGTGCATT GATATCACTA ATATCTTCTC
TACCTGGAGT ATCATTTTCC GACCCAACAG GTAGTGGTAT TAACGG,; and
(ii) a reverse primer of from 15 to 35 nucleotides in length, said
reverse primer comprising a sequence which is complementary to the
inverse complement of a consecutive sequence within one of the
following two sequences:
18 SEQ ID NO.4 CAT TTCTAGGTTT TGCAGGAGCT ATTGGCTACT CAATGGATGG
TCCAAGAATA GAGCTTGAAG TATCTTATGA,, or SEQ ID NO. 5 C AAGGAAAGTT
AGGTTTAAGC TACTCTATAA GCCCAGA,
wherein one or both of the primers of the second primer set are
internal to the primers of the first primer set; and (d)
determining the length or sequence of the PCR products of step (c),
wherein the presence of a PCR product having a length or sequence
which corresponds to the length or sequence, respectively, of that
region of the E. chaffeensis p28gene which is located between the
regions to which the forward primer of the second primer set and
the reverse primer of the second primer set bind is indicative of
the presence of E. chaffeensis in the sample.
13. A primer set for detecting E. canis in a sample, said primer
set comprising: (a) a forward primer of from 15 to 35 nucleotides
in length, said forward primer comprising a sequence which is the
complement of a consecutive sequence within the following
sequence:
19 SEQ ID NO.1 CCA AGTGTCTCAC ATTTTGGTAG CTTCTCAGCT AAAGAAGAAA
GCAAATCAAC TGTTGGAGTTTTTGGATTAA AACATGATTG GGATGGAAGT CCAATACTTA
AGAATAAACA CGCTGACTTTACTGTTCCAA AC., and
(ii) a reverse primer of from 15 to 35 nucleotides in length, said
reverse primer comprising a sequence which is complementary to the
inverse complement of a consecutive sequence within the following
sequence:
20 SEQ ID NO.2 GTTACT CAATGGGTGG CCCAAGAATA GAATTCGAAA TATCTTATGA
AGCATTCGAC GTAAAAAGTC CTAATATCAA TTATCAAAAT GACGCGCACA GGTACTGCGC
TCTATCTCAT CACACATCGG CAGCCAT,.;
14. The primer set of claim 13 wherein forward primer and the
reverse primer, respectively, comprise one of the following pairs
of sequences:
21 PAIR 1: ATAAACACGCTGACTTTACTGTTCC, S, SEQ ID NO. 6
GTGATGAGATAGAGCGCAGTACC,.; SEQ ID NO. 7 PAIR 2
AACACGCTGACTTTACTGTTCC,, SEQ ID NO. 8 ATGGCTGCCGATGTGTGATG,, SEQ ID
NO. 9 PAIR 3: ACGCTGACTTTACTGTTCCAAAC,, SEQ ID NO. 10
ATGGCTGCCGATGTGTGATG,, SEQ ID NO. 11 PAIR 4:
AACATGATTGGGATGGAAGTC,, SEQ ID NO. 12 GCCGATGTGTGATGAGATAG,, SEQ ID
NO. 13 PAIR 5: AAACATGATTGGGATGGAAGTC,, SEQ. ID NO. 12
GCCGATGTGTGATGAGATAGAG,; SEQ ID NO. 14 PAIR 6:
GATTGGGATGGAAGTCCAATAC,, SEQ. ID. NO. 13 GCCGATGTGTGATGAGATAG,; SEQ
ID NO. 11 PAIR 7: GATTGGGATGGAAGTCCAATAC,, SEQ ID NO. 13
GCCGATGTGTGATGAGATAGAG,; SEQ ID NO. 14 PAIR 8:
ACACGCTGACTTTACTGTTCCAAAC,; SEQ ID NO. 15 ATGGCTGCCGATGTGTGATGAG,-
; SEQ ID NO. 16 PAIR 9: GTGTCTCACATTTTGGTAGCTTCT- C,, SEQ ID NO. 17
CTTGGGCCACCCATTGAGTAAC,; SEQ ID NO. 18 PAIR 10
GATTGGGATGGAAGTCCAATAC,, SEQ. ID. NO. 13 CGATGTGTGATGAGATAGAGC,;
SEQ ID NO. 1; PAIR 11: TGATTGGGATGGAAGTCCAATAC,, SEQ ID NO. 20
CGATGTGTGATGAGATAGAGCG,, SEQ ID NO. 21 PAIR 12:
CATGATTGGGATGGAAGTCCAATA- C,, SEQ ID NO. 21 ATGGCTGCCGATGTGTGATG,;
SEQ ID NO. 11 PAIR 13: CCAAGTGTCTCACATTTTGGTAGC,, SEQ ID NO. 22
TGGGCCACCCATTGAGTAAC,. SEQ ID NO. 23
15. The primer set of claim 14 wherein the primer set comprises
pair 1.
16. The primer set of claim 14 wherein the primer set comprises
pair 12.
17. A primer set for detecting E. chaffeensis in a sample, said
primer set comprising a forward primer of from 15 to 35 nucleotides
in length, said forward primer comprising a sequence which is the
complement of a consecutive sequence within the following
sequence:
22 SEQ ID NO. 3 (a) A GTTTTCATAA CAAGTGCATT GATATCACTA ATATCTTCTC
TACCTGGAGT ATCATTTTCC GACCCAACAG GTAGTGGTAT TAACGG,; and +P1 (b) a
reverse primer of from 15 to 35 nucleotides in length, said reverse
primer comprising a sequence which is complementary to the inverse
complement of a consecutive sequence within one of the following
two sequences: SEQ ID NO. 4 CAT TTCTAGGTTT TGCAGGAGCT ATTGGCTACT
CAATGGATGG TCCAAGAATA GAGCTTGAAG TATCTTATGA,, or SEQ ID NO. 5 C
AAGGAAAGTT AGGTTTAAGC TACTCTATAA GCCCAGA,.
18. The primer set of claim 17 wherein the forward primer and the
reverse primer, respectively, comprise one of the following pairs
of sequences:
23 PAIR 1 AGGTAGTGGTATTAACGG, SEQ ID NO 24 AGATACTTCAAGCTCTATTC,;
SEQ ID NO. 25 PAIR 2: AGGTAGTGGTATTAACGG,, SEQ ID NO
TCATAAGATACTTCAAGCTC,; SEQ ID NO PAIR 3 CTTCTCTACCTGGAGTATC,, SEQ
ID NO GCTTATAGAGTAGCTTAAACC,; SEQ ID NO PAIR 4:
CAGGTAGTGGTATTAACG,, SEQ ID NO CATAAGATACTTCAAGCTC,; SEQ ID NO PAIR
5 CAGGTAGTGGTATTAACG,, SEQ ID NO GATACTTCAAGCTCTATTC,; SEQ ID NO
PAIR 6 CTTCTCTACCTGGAGTATC,, SEQ ID NO GCTTATAGAGTAGCTTAAAC,; SEQ
ID NO PAIR 7 CTACCTGGAGTATCATTTTC,, SEQ ID NO
GGCTTATAGAGTAGCTTAAAC,; SEQ ID NO PAIR 8 AATATCTTCTCTACCTGG,, SEQ
ID NO GATACTTCAAGCTCTATTC,; SEQ ID NO PAIR 9 AGTTTTCATAACAAGTGC,,
SEQ ID NO. CATAAGATACTTCAAGCTC,; SEQ ID NO PAIR 10:
AGTTTTCATAACAAGTGC,, SEQ ID NO GATACTTCAAGCTCTATTC,; SEQ ID NO PAIR
11 CTTCTCTACCTGGAGTATCATTTTC, SEQ ID NO
GAGTAGCTTAAACCTAACTTTCCTTG,; SEQ ID NO PAIR 12:
CTTCTCTACCTGGAGTATC,, SEQ ID NO GAGTAGCTTAAACCTAACTTTC,; SEQ ID NO
PAIR 13 AATATCTTCTCTACCTGG, SEQ ID NO TCATAAGATACTTCAAGC,; SEQ ID
NO PAIR 14: AATATCTTCTCTACCTGG, SEQ ID NO 64 CATAAGATACTTCAAGCTC,;
SEQ ID NO PAIR 15 AATATCTTCTCTACCTGG, SEQ ID NO
CATAAGATACTTCAAGCTC,; SEQ ID NO PAIR 16 CTCTACCTGGAGTATCATTTTC, SEQ
ID NO GGCTTATAGAGTAGCTTAAACC; SEQ ID NO PAIR 17:
CTCTACCTGGAGTATCATTTTC, SEQ ID NO GAGTAGCTTAAACCTAACTTTC; SEQ ID NO
PAIR 18: ACCTGGAGTATCATTTTC, SEQ ID NO TCTGGGCTTATAGAGTAG,; SEQ ID
NO PAIR 19 CTTCTCTACCTGGAGTATC, SEQ ID NO CTGGGCTTATAGAGTAGC,; SEQ
ID NO PAIR 20 CTTCTCTACCTGGAGTATC, SEQ ID NO CTGGGCTTATAGAGTAGC,
SEQ ID NO
19. The primer set of claim 18 wherein the primer set is pair
1.
20. The primer set of claim 18 wherein the primer set is pair 3.
Description
BACKGROUND OF THE INVENTION
[0001] The Ehrlichiae are obligate intracellular bacteria found in
circulating leukocytes of infected animals. Ehrlichia canis (E.
canis) infects monocytes and causes ehrlichiosis in animals
belonging to the family Canidae. E. canis is transmitted by the
brown dog tick, Rhipicephalus sanguineus.
[0002] Canine monocytic ehrlichiosis (CME) consists of an acute and
a chronic phase. The acute phase is characterized by fever, serous
nasal and ocular discharges, anorexia, depression, and loss of
weight. The chronic phase is characterized by severe pancytopenia,
epistaxis, hematuria, blood in feces in addition to more severe
clinical signs of the acute disease. If treated early during the
course of the disease, dogs respond well to doxycycline. However,
chronically infected dogs do not respond well to the antibiotic.
Therefore, early diagnosis is very important for treating canine
ehrlichiosis.
[0003] Human monocytic ehrlichiosis (HME) is a tick-borne, emerging
infectious disease that is caused by the rickettsial pathogen,
Ehrlichia chaffeensis (E. chaffeensis). The course of HME begins
with an asymptomatic pre-patent period, followed by an acute phase
where the vertebrate host suffers pyrexia, anorexia, weight loss,
cytopenia and even death. Non-human hosts that survive the acute
phase typically undergo partial recovery and suffer mild chronic
infections, during which they could be persistent carriers that are
capable of infecting tick vectors. Both dogs and white tailed deer
are susceptible to infection with E. chaffeensis, and both of these
hosts are suspected reservoirs of this pathogen. E. chaffeensis
appears to be transmitted by Amblyomma. americanum, and appears to
be endemic to the southern U.S. where this tick is indigenous.
Symptoms of human monocytic ehrlichiosis (HME) are similar to those
of canine monocytic ehrlichiosis (CME) that is also known as
tropical canine pancytopenia. (Hildebrandt, P. K., D. L. Huxsoll,
et al. (1973 (1973). Pathology of canine ehrlichiosis (tropical
canine pancytopenia). Am J Vet Res 34(10): 1309-20.; Kuehn, N. F.
and S. D. Gaunt (1985). Clinical and hematologic findings in canine
ehrlichiosis. J Am Vet Med Assoc 186(4): 355-8.; Eng, T. R., J. R.
Harkess, et al. (1990). Epidemiologic, clinical, and laboratory
findings of human ehrlichiosis in the United States, 1988. Jama
264(17): 2251-8.; McDade, J. E. (1990). Ehrlichiosis--a disease of
animals and humans. J Infect Dis 161(4): 609-17.) The etiologic
agents of CME and HME, E. canis and E. chaffeensis, respectively,
have been placed in the same genogroup based on 16S rRNA sequences
and antigenic cross-reactivity Anderson, B. E., J. E. Dawson, et
al. (1991). Ehrlichia chaffeensis, a new species associated with
human ehrlichiosis. J Clin Microbiol 29(12): 2838-42.
[0004] The primary test for diagnosing CME or HME is the indirect
fluorescent antibody (IFA) test. This test uses the etiologic
agents Ehrlichia canis or E. chaffeensis, respectively, to diagnose
infection. The IFA test, however, has serious limitations. The IFA
test is subject to false positives because the antigens are whole
infected cells which comprise many nonspecific proteins that can
cross-react with sera from some patients. The IFA test is also
subject to false negatives because IFA antigens are unstable and
may become inactivated during storage. In addition the IFA test
requires a special equipment to perform the test. For example, the
IFA test requires a tissue culture system for growing the bacterium
that are used to prepare the antigen slides, a fluorescent
microscope, and trained persons to evaluate the serum reactivity to
the bacterial antigen on the slide.
[0005] Serodiagnosis is another method which has been developed to
diagnose canine or human ehrlichiosis. The method involves testing
the blood of the animal for antibodies immunoreactive with outer
membrane proteins of these pathogens. Serodiagnosis cannot be used
until the infected subject has produced such antibodies.
Accordingly, serodianosis cannot be used early during the course of
infection. Moreover, serodiagnosis cannot be used for detecting an
ongoing infection.
[0006] Accordingly, it is desirable to have additional methods and
tools which can be used for diagnosing canine and human
ehrlichiosis, particularly methods and tools which can be used to
detect an ongoing infection. Methods and tools which can be used to
detect E. canis and E. chaffeensis in the invertebrate vectors
which transmit these pathogens to their respective vertebrate hosts
are also desirable.
SUMMARY OF THE INVENTION
[0007] The present invention provides tools and methods for
detecting the presence of E. canis and E. chaffeensis in a sample
obtained from an animal, particularly from a member of the Canidae
family. The method for detecting E. canis comprises providing a p30
primer set comprising a first primer having a sequence which is
complementary to a sequence on the E. canis p30 gene sense strand
and a second primer which is complementary to a sequence which is
complementary to a sequence on the E. canis p30 gene of antisense
strand, amplifying DNA in the sample using a polymerase chain
reaction and the p30 primer set, and determining the length or
sequence of the PCR products, wherein the presence of a PCR product
having a sequence or length which corresponds to the sequence or
length of the region of the p30 gene which is located between the
nucleotide sequences to which the first p30 primer and the second
p30 primer bind is indicative of the presence of E. canis in the
sample.
[0008] The present invention also relates to the p30 primer sets.
Each p30 primer set comprises a first p30 primer and the second p30
primer, both of which are from 15 to 35 nucleotides in length. The
first p30 primer, i.e., the forward primer, comprises a sequence
which is complementary to a consecutive sequence, preferably of at
least 14 nucleotides in length, within the following sequence: CCA
AGTGTCTCAC ATTTTGGTAG CTTCTCAGCT AAAGAAGAAA GCAAATCAAC
TGTTGGAGTTTTTGGATTAA AACATGATTG GGATGGAAGT CCAATACTTA AGAATAAACA
CGCTGACTTTACTGTTCCAA AC. SEQ ID NO.1. The second p30 primer, i.e.
the reverse primer, comprises a sequence which is complementary to
the inverse complement of a consecutive sequence, preferably of at
least 14 nucleotides in length, contained within the following
sequence: GTTACT CAATGGGTGG CCCAAGAATA GAATTCGAAA TATCTTATGA
AGCATTCGAC GTAAAAAGTC CTAATATCAA TTATCAAAAT GACGCGCACA GGTACTGCGC
TCTATCTCAT CACACATCGG CAGCCAT , SEQ ID NO.2. Such primers are
useful for detecting the presence of E. canis in members of the
Canidae family. Such primers are also useful for detecting the
presence of E. canis DNA in samples obtained from ticks or other
invertebrate carriers which feed on the vertebrate hosts.
[0009] The method for detecting E. chaffeensis comprises providing
a p28 primer set comprising a first primer comprising a sequence
which is complementary to a sequence on the E. chaffeensis p28 gene
sense strand and a second primer which is complementary to a
sequence on the E. chaffeensis p28 gene antisense strand,
amplifying DNA in the sample using a polymerase chain reaction and
the p28 primer set, and determining the length or sequence of the
PCR products, wherein the presence of a PCR product having a
sequence or length which corresponds to the sequence or length of
that portion of the p28 gene which is located between the sequence
to which the first p28 primer and second p28 primer bind is
indicative of the presence of E. chaffeensis DNA in the sample.
[0010] The present invention also relates to the primers in the p28
primer set. The first p28 and second p28 primers are from 15 to 35
nucleotides in length. The first p28 primer, i.e. the forward
primer, comprises a sequence which is substantially identical to
the complement of a consecutive sequence, preferably of at least 14
nucleotides in length, within the following sequence: A GTTTTCATAA
CAAGTGCATT GATATCACTA ATATCTTCTC TACCTGGAGT ATCATTTTCC GACCCAACAG
GTAGTGGTAT TAACGG, SEQ ID NO. 3.
[0011] The second primer, i.e. the reverse primer, comprises a
sequence which is complementary to the inverse complement of a
consecutive sequence, preferably of at least 14 nucleotides in
length, within one of the following two sequences: CAT TTCTAGGTTT
TGCAGGAGCT ATTGGCTACT CAATGGATGG TCCAAGAATA GAGCTTGAAG TATCTTATGA,
SEQ ID NO.4, or C AAGGAAAGTT AGGTTTAAGC TACTCTATAA GCCCAGA, SEQ ID
NO.5. Such primers are useful for detecting the presence of E.
chaffeensis in samples obtained from vertebrate animals such as
humans or dogs, or from the invertebrate vectors which transmit
this pathogen from one vertebrate animal to another.
BRIEF DESCRIPTION OF THE FIGURES
[0012] FIG. 1 shows the nucleotide sequence of the p30 gene of the
Jake strain of E. canis. (Genbank Accession No. AF082744.)
[0013] FIG. 2 is a consensus open reading frame (ORF) sequence for
the E. chaffeensis p28 gene. The consensus sequence was derived
from the p28 genes of the Sapulpa, Jacksonville and Arkansas
isolates.
[0014] FIG. 3 shows the results of a PCR assay of peripheral blood
from dogs during acute and chronic experimental CME. Three dogs
were tested with the nested p30-based PCR described in the text.
Buffy coat DNA preparations were assayed from two dogs prior to
(lanes 1 and 3) and 21 d after (Lanes 3 and 4) inoculation with E.
canis Ebony isolate-infected blood from an experimentally infected
dog. The third dog (lane 5) was from the experimentally infected
donor blood. The molecular size standard (M) is a 100 bp ladder
(Life Technologies).
[0015] FIG. 4. Confirmation of the E. canis nested target amplicon
sequence. Multiple sequence alignment of the amplicon derived from
nested p30-based PCR of DNA from E. canis (Ebony isolate), the E.
canis p30 target DNA sequence (Jake Isolate) and primer set 1
(ECA30-384S and ECA30-583A). The DNA sequence for ECA30-583A is the
reverse complement of that oligonucleotide. The sequences were
aligned and compared with the Pileup (GCG, version 10) and Boxshade
programs. White letters surrounded by a black box represent bases
that are identical in each sequence of the alignment.
[0016] FIG. 5. Sensitivity of the p30-based nested PCR assay. The
16S rDNA (panel A) and p30 (panel B) PCR assays were compared to
determine relative sensitivities. Buffy coat DNA was isolated from
an E. canis carrier dog and subjected to a dilution series for
simultaneous amplification with the two assays. Lanes 1-5,
respectively, are assays of the template dilutions of
1.times.10.sup.0, 10.sup.-1, 10.sup.-2, 10.sup.-3 and 10.sup.-4
both panels.
[0017] FIG. 6. Specificity of the p30-based nested PCR assay. Lane
1 is the template-free control, lanes 2-3 represent amplification
of 50 ng of E. canis Ebony and Oklahoma isolates, respectively, and
lanes 4-6 represent assay of 50 ng of DNA isolated from E.
chaffeensis, HGE agent and E. muris, respectively. The 100 bp
ladder (M) serves as the molecular size standard.
[0018] FIG. 7. Preparation of blood samples for the p30-based
nested PCR assay. Blood was collected with heparin from an E.
canis-infected carrier dog, divided into 0.5 ml aliquots and
subjected to different preparation procedures performed in
duplicate as described in the text. Samples assayed with the nested
p30-based PCR included buffy coats treated with DNAzol (Lanes 1 and
2), buffy coats treated with DNAzol BD (Lanes 3 and 4), whole blood
treated with DNAzol BD (Lanes 5 and 6), buffy coats subjected to
proteinase K digestion followed by phenol extraction and ethanol
precipitation (Lanes 7 and 8) and DNA extracted from PBMC with
Qiagen spin columns (Lanes 9 and 10). A 100 bp DNA ladder (M) was
used as a molecular size standard.
[0019] FIG. 8. Analysis of oligonucleotide primer sets derived from
the E. canis p30 gene sequence. In the primer site plot (A), the
horizontal lines represent the portion of the p30 open reading
frame analyzed for optimal primer pairs. Each horizontal line
represents the p30 ORF; vertical bars above the line represent the
location of forward primers and vertical bars below each line
represent reverse primers. The truncated multiple sequence (B)
demonstrates similarity between the sequences of the forward
primers and the E. chaffeensis p28 sequence clusters I, II and III
to the E. canis p30 sequence. Letters surrounded by a black box
represent bases identical to the E. canis sequence. Gray letters
surrounded by a black box represent bases identical to E. canis and
all three E. chaffeensis gene clusters. The same analysis was
performed for reverse primers. The percent identical bases between
the different primers and the E. chaffeensis cluster sequences were
determined and are displayed in Table 1.
[0020] FIG. 9. Analysis of oligonucleotide primer sets derived from
the E. chaffeensis p28 consensus sequence. The primer site plot (A)
represents the location of forward and reverse primers within p28;
vertical bars above the line represent the location of forward
primers and vertical bars below each line represent reverse
primers. The truncated multiple sequence alignments demonstrate
identity between the sequences of the forward (B) and reverse (C)
primers. Gray letters surrounded by a black box represent bases
identical to E. canis and all three E. chaffeensis gene clusters.
The percent identical bases between the different primers and the
E. canis sequences were determined and are displayed in Table
5.
[0021] FIG. 10. Detection of E. chaffeensis (Arkansas isolate) with
the p28-based PCR assay. Panel A demonstrates amplicons from
duplicate non-optimized PCR in the presence of 50 ng of DNA from E.
chaffeensis-infected DH82 cells and various primer sets that are
specific to the Arkansas isolate (11.sub.A, 13.sub.A and 15.sub.A)
or universal to all three p28 clusters (3.sub.U and 1.sub.U), which
are described in Table X. Panel B consists of male ticks of four
species known to parasitize dogs that were intrastadially exposed
to E. chaffeensis by allowing them to feed on on a rickettsemic dog
followed by holding for 10 days in a humidity chamber. Both
1.degree. and 2.degree. optimized PCR assays are shown for A.
americanum. PCR-positive ticks are indicated by a white "+" at the
top of each lane. Note that E. chaffeensis infection was detected
in 20-40% of each tick species tested. A 100 bp ladder served as
the molecular size standard (M).
DETAILED DESCRIPTION OF THE INVENTION
[0022] In one aspect,the present invention provides methods and
tools for detecting the presence of Ehrlichia canis in samples
obtained from a vertebrate or invertebrate animal. The method
comprises amplifying the DNA contained within the sample using a
primer set comprising primers which comprise sequences that are
complementary to select regions of the p30 gene of E. canis and a
polymerase chain reaction (PCR) to provide a pool of PCR products,
and then assaying the pool for the presence or absence of a PCR
product whose length or sequence indicates that PCR product
corresponds to the region of the p30 gene that is flanked by the
nucleotide sequences which are complementary to the first and
second members of the p30 primer set. The tools are the members of
the p30 primer sets. The E. canis p 30 gene belongs to the omp-1
multiple gene family and has the sequence which is set forth in
Genbank as Accession No. AF082744.1 (Jake isolate), Accession No.
AF082750.1 (Florida isolate), Accession No. AF082749.1(Fuzzy
isolate), Accession No. AF082748.1m (DJ isolate), Accession No.
AF082747.1 (Arkansas isolate), Accession No. AF082746.1 (Oklahoma
isolate) and Accession No. AF082745.1 (Louisiana isolate of E.
canis.) The E. canis p30 gene encodes the immunodominants
ehrlichial outer membrane protein P30.
[0023] In another aspect, the present invention provides methods
and tools for detecting the presence of Ehrlichia chaffeensis in
samples obtained from vertebrate or invertebrate animals. The
method comprises amplifying the DNA contained within the sample
using a p28 primer set whose members comprise a sequence which is
complementary to select regions of the p28 gene of E. chaffeensis
and a polymerase chain reaction (PCR) to provide a pool of PCR
products, and then assaying the pool for the presence of a PCR
product whose length or sequence indicates that PCR product
corresponds to the region of the p28 gene that comprises and is
flanked by the nucleotide sequences to which the members of the p28
primer set bind. The tools are the members of the p28 primer sets.
The E. chaffeensis p28 gene belongs to the omp-1 multiple gene
family and has the sequence which is set forth in Genbank as
(Accession No. AF077735.1 (St. Vincent isolate), Accession No.
AF077734.1 (Sapulpa isolate), Accession No. AF077733.1 (Jax
isolate), Accession No. AF077732.1 (91HE17 isolate) and Accession
No. AF068234 (Arkansas isolate). The E. chaffeensis p28 gene
encodes the immunodominants ehrlichial outer membrane protein
P28.
[0024] Primers
[0025] The primers in the p30 primer set are based upon select
sequences in the p30 gene of E. canis. The p30 gene encodes the
major outer membrane protein of E. canis. The sequences of the
first and second primers in the p30 primer set are distinct from
sequences found in the closely related p2.sup.8 gene in E.
chaffeensis. The first primer in the p30 primer set is an
oligonucleotide of from 15 to 35 nucleotides in length, preferably
from 18 to 30 nucleotides in length. The second p30 primer in the
E. canis primer set is an oligonucleotide of from 15 to 35
nucleotides, preferably from 18 to 30 nucleotides in length. The
first p30 primer, i.e., the forward primer, comprises a sequence
which is substantially identical to the complement of consecutive
sequence located between nucleotides 278 and 412 of the sense
strand of the open reading frame sequence of the p30 gene of E.
canis. The sequence of the sense strand in this region is as
follows: CCA AGTGTCTCAC ATTTTGGTAG CTTCTCAGCT AAAGAAGAAA GCAAATCAAC
TGTTGGAGTT TTTGGATTAA AACATGATTG GGATGGAAGT CCAATACTTA AGAATAAACA
CGCTGACTTT ACTGTTCCAA AC. SEQ ID NO.1 As used herein the term
"substantially identical" means that the sequence is at least 90%
identical, preferably at least 95% identical, more preferably 100%
identical to a particular reference sequence, e.g., to the
complement of a consecutive sequence contained within SEQ ID NO. 1.
The second p30 primer, i.e., the reverse primer, comprises a
sequence which is substantially identical to and the inverse of a
consecutive sequence located between nucleotides 465 through 597 of
the sense strand of the p30 gene of E. canis. The sequence of the
second p30 primer is substantially identical to the complement of
the inverse complement of a consecutive sequence contained within
the following sequence: GTTACT CAATGGGTGG CCCAAGAATA GAATTCGAAA
TATCTTATGA AGCATTCGAC GTAAAAAGTC CTAATATCAA TTATCAAAAT GACGCGCACA
GGTACTGCGC TCTATCTCAT CACACATCGG CAGCCAT, SEQ ID NO.2.
[0026] In specific embodiments, the first and second primers in the
p30 primer set comprise the sequences shown in Table 1 below. The
first and second primers may also comprise sequences which are
shorter by one or two oligonucleotides than the sequences shown in
Table 1 below. The first and second primers of the E. canis primer
set may also comprise a sequence which is longer than the sequences
shown in Table 1 below. Such sequences have one or two additional
nucleotides attached to the 5' end of the above-listed sequences.
The additional nucleotides are selected from the group consisting
of adenylic acid, guanylic acid, and combinations thereof
[0027] The sequence of the thirteen E. canis p30 primer sets shown
in Table 1 below are based upon a comparison of the open-reading
frame sequences of the seven E. canis and five E. chaffeensis
isolates. It is expected that such primer sets will specifically
amplify the target sequence of multiple E. canis isolates, but not
the E. chaffeensis isolates. It is expected that the primers shown
in Table 1 below are both species-universal and species-specific
for E. canis. In accordance with the present invention, it has been
determined that amplification of DNA isolated from E. chaffeensis,
E. muris, and the HGE agent with these primer sets failed to
produce a PCR product that is diagnostic of E. canis.
1TABLE 1 Oligonucleotide sequences of E. canis p30 primer sets. Set
Forward Reverse 1 ECA30-384S ECA30-583A (ATAAACACGCTGACTTTACTGTTCC)
(GTGATGAGATAGAGCGCA GTACC) 2 ECA30-387S ECA30-578A
(AACACGCTGACTTTACTGTTCC) (ATGGCTGCCGATGTGTGA TG) 3 ECA30-390S
ECA30-597A (ACGCTGACTTTACTGTTCCAAAC) (ATGGCTGCCGATGTGTGA TG) 4
ECA30-351S ECA30-591A (AACATGATTGGGATGGAAGTC) (GCCGATGTGTGATGAGAT
AG) 5 ECA30-350S ECA30-591bA (AAACATGATTGGGATGGAAGTC)
(GCCGATGTGTGATGAGAT AGAG) 6 ECA30-356S ECA30-591A
(GATTGGGATGGAAGTCCAATAC) 7 ECA30-356S ECA30-591bA 8 ECA30-388S
ECA30-597bA (ACACGCTGACTTTACTGTTCCAAAC) (ATGGCTGCCGATGTGTGA TGAG) 9
ECA30-282S ECA30-486A (GTGTCTCACATTTTGGTAGCTTCTC)
(CTTGGGCCACCCATTGAG TAAC) 10 ECA30-356S ECA30-589A
(CGATGTGTGATGAGATAG AGC) 11 ECA30-355S ECA30-589Ba
(TGATTGGGATGGAAGTCCAATA- C) (CGATGTGTGATGAGATAG AGCG) 12 ECA30-353S
ECA30-597A (CATGATTGGGATGGAAGTCCAATAC) (ATGGCTGCCGATGTGTGA TG) 13
ECA30-278S ECA30-484A (CCAAGTGTCTCACATTTTGGTAGC)
(TGGGCCACCCATTGAGTA AC)
[0028] The predicted annealing scores and specificity values, as
compared to the p28 gene of E. chaffeensis, of these primer sets
are shown in Table 2 below.
2TABLE 2 Ranks of E. canis p30 primer sets with predicted annealing
and specificity values. Identity Scores to E. chaffeensis.sup.a
Total Primer Cluster I Cluster II Cluster III Annealing Set Forward
Reverse Forward Reverse Forward Reverse Mean.sup.b Score.sup.c
Rank.sup.d 1 0.64 0.74 0.48 0.87 0.56 0.78 0.68 52 2 2 0.59 0.45
0.55 0.65 0.55 0.60 0.56 57 1 3 0.74 0.45 0.61 0.65 0.70 0.60 0.62
58 3 4 0.81 0.55 0.86 0.75 0.71 0.70 0.73 63 5 5 0.82 0.55 0.82
0.77 0.73 0.73 0.74 64 6 6 0.91 0.55 0.86 0.75 0.77 0.70 0.76 64 7
7 0.91 0.55 0.86 0.77 0.77 0.73 0.76 65 9 8 0.68 0.45 0.60 0.68
0.68 0.64 0.62 66 4 9 0.64 0.86 0.64 0.95 0.72 0.91 0.79 67 12 10
0.91 0.62 0.86 0.81 0.77 0.71 0.78 67 10 11 0.87 0.64 0.83 0.82
0.74 0.73 0.77 68 11 12 0.88 0.45 0.84 0.65 0.80 0.70 0.72 68 8 13
0.67 0.85 0.67 0.95 0.71 0.90 0.79 68 13 .sup.aIdentity Score = No.
oligonucleotide bases identical to aligned template .div. total No.
oligonucleotide bases. .sup.bMean identity score for both primers
of each set to all three E. chaffeensis clusters. .sup.cDetermined
by GCG (version 10) Prime program. Sum of annealing scores for
primer secondary structure, non-specific primer binding to the
template sequence and self-complementarity of individual primers
and complementarity between primer pairs; the lowest scores
indicate the primer sets with least complementarity to sequences
other than the target binding site. .sup.dRank determined by
product of annealing score and mean identity score; lower values
have higher rank.
[0029] The primers in the E. chaffeensis p28 primer set are based
upon select sequences in the p28 gene of E. chaffeensis. The p28
gene encodes a major outer membrane protein of E. chaffeensis. The
sequences of the first and second primers in the p28 primer set are
distinct from sequences found in the closely related p30 gene in E.
canis. The first primer in the p28 primer set is an oligonucleotide
of from 15 to 35 nucleotides in length., preferably from 18 to 30
nucleotides in length. The second primer in the E. chaffeensis p 28
primer set is an oligonucleotide of from 15 to 35 nucleotides,
preferably from 18 to 30 nucleotides in length. The first p28
primer, i.e., the forward primer, comprises a sequence which is
substantially identical to the complement of consecutive sequence
located between nucleotides 15 and 101 of the sense strand of the
open reading frame sequence of the p28 gene of E. chaffeensis. The
sequence of the sense strand in this region is as follows: A
GTTTTCATAA CAAGTGCATT GATATCACTA ATATCTTCTC TACCTGGAGT ATCATTTTCC
GACCCAACAG GTAGTGGTAT TAACGG, SEQ ID NO. 3.
[0030] The second p28 primer, i.e., the reverse primer, comprises a
sequence which is substantially identical to and the inverse of a
consecutive sequence located between nucleotides 341 through 365 or
nucleotides 641 through 674 of the sense strand of the p28 gene of
E. chaffeensis. The sequence of the second p28 primer is
substantially identical to the complement of the inverse complement
of a consecutive sequence contained within one of the following two
sequences: CAT TTCTAGGTTT TGCAGGAGCT ATTGGCTACT CAATGGATGG
TCCAAGAATA GAGCTTGAAG TATCTTATGA, SEQ ID NO. 4, or C AAGGAAAGTT
AGGTTTAAGC TACTCTATAA GCCCAGA, SEQ ID NO. 5.
[0031] In specific embodiments, the first and second primers in the
E. chaffeensis p28 primer set comprise the sequences shown in Table
3 below. The first and second primers may also comprise sequences
which are shorter by one or two oligonucleotides than the sequences
shown in Table 3 below. The first and second primers of the E.
chaffeensis p28 primer set may also comprise a sequence which is
longer than the sequences shown in Table 3 below. Such sequences
have one or two additional nucleotides attached to the 5' end of
the above-listed sequences. The additional nucleotides are selected
from the group consisting of adenylic acid, guanylic acid, and
combinations thereof.
3TABLE 3 product: 1 [DNA] = 50.000 nM [salt] = 50.000 mM PRIMERS 5'
3' forward primer (18-mer): 84 AGGTAGTGGTATTAACGG 101 reverse
primer (20-mer): 360 AGATACTTCAAGCTCTATTC 341 forward reverse
primer % GC: 44.4 35.0 primer Tm (degrees Celsius): 44.4 43.6
PRODUCT product length: 277 product % GC: 31.0 product Tm: 70.2
degrees Celsius difference in primer Tm: 0.8 degrees Celsius
annealing score: 57 optimal annealing temperature: 47.3 degrees
Celsius Product: 2 [DNA] = 50.000 nM [salt] = 50.000 mM PRIMERS 5'
3' forward primer (18-mer): 84 AGGTAGTGGTATTAACGG 101 reverse
primer (20-mer): 365 TCATAAGATACTTCAAGCTC 346 forward reverse
primer % GC: 44.4 35.0 primer Tm (degrees Celsius): 44.4 44.0
PRODUCT product length: 282 product % GC: 30.9 product Tm: 70.2
degrees Celsius difference in primer Tm: 0.5 degrees Celsius
annealing score: 57 optimal annealing temperature: 47.4 degrees
Celsius Product: 3 [DNA] = 50.000 nM [salt] = 50.000 mM PRIMERS 5'
3' forward primer (19-mer): 50 CTTCTCTACCTGGAGTATC 68 reverse
primer (21-mer): 669 GCTTATAGAGTAGCTTAAACC 649 forward reverse
primer % GC: 47.4 38.1 primer Tm (degrees Celsius): 44.8 45.5
PRODUCT product length: 620 product % GC: 30.0 product Tm: 71.1
degrees Celsius difference in primer Tm: 0.8 degrees Celsius
annealing score: 61 optimal annealing temperature: 48.3 degrees
Celsius Product: 4 [DNA] = 50.000 nM [salt] = 50.000 mM PRIMERS 5'
3' forward primer (18-mer): 83 CAGGTAGTGGTATTAACG 100 reverse
primer (19-mer): 364 CATAAGATACTTCAAGCTC 346 forward reverse primer
% GC: 44.4 36.8 primer Tm (degrees Celsius): 43.6 42.2 PRODUCT
product length: 282 product % GC: 31.2 product Tm: 70.3 degrees
Celsius difference in primer Tm: 1.4 degrees Celsius annealing
score: 63 optimal annealing temperature: 47.0 degrees Celsius
Product: 5 [DNA] = 50.000 nM [salt] = 50.000 mM PRIMERS 5' 3'
forward primer (18-mer): 83 CAGGTAGTGGTATTAACG 100 reverse primer
(19-mer): 359 GATACTTCAAGCTCTATTC 341 forward reverse primer % GC:
44.4 36.8 primer Tm (degrees Celsius): 43.6 41.9 PRODUCT product
length: 277 product % GC: 31.4 product Tm: 70.3 degrees Celsius
difference in primer Tm: 1.7 degrees Celsius annealing score: 63
optimal annealing temperature: 46.9 degrees Celsius Product: 6
[DNA] = 50.000 nM [salt] = 50.000 mM PRIMERS 5' 3' forward primer
(19-mer): 50 CTTCTCTACCTGGAGTATC 68 reverse primer (20-mer): 669
GCTTATAGAGTAGCTTAAAC 650 forward reverse primer % GC: 47.4 35.0
primer Tm (degrees Celsius): 44.8 42.8 PRODUCT product length: 620
product % GC: 30.0 product Tm: 71.1 degrees Celsius difference in
primer Tm: 2.0 degrees Celsius annealing score: 63 optimal
annealing temperature: 47.7 degrees Celsius Product: 7 [DNA] =
50.000 nM [salt] = 50.000 mM PRIMERS 5' 3' forward primer (20-mer):
55 CTACCTGGAGTATCATTTTC 74 reverse primer (21-mer): 670
GGCTTATAGAGTAGCTTAAAC 650 forward reverse primer % GC: 40.0 38.1
primer Tm (degrees Celsius): 44.7 45.5 PRODUCT product length: 616
product % GC: 30.0 product Tm: 71.1 degrees Celsius difference in
primer Tm: 0.9 degrees Celsius annealing score: 64 optimal
annealing temperature: 48.3 degrees Celsius Product: 8 [DNA] =
50.000 nM [salt] = 50.000 mM PRIMERS 5' 3' forward primer (18-mer):
45 AATATCTTCTCTACCTGG 62 reverse primer (19-mer): 359
GATACTTCAAGCTCTATTC 341 forward reverse primer % GC: 38.9 36.8
primer Tm (degrees Celsius): 40.8 41.9 PRODUCT product length: 315
product % GC: 32.1 product Tm: 70.9 degrees Celsius difference in
primer Tm: 1.1 degrees Celsius annealing score: 65 optimal
annealing temperature: 47.0 degrees Celsius Product: 9 [DNA] =
50.000 nM [salt] = 50.000 mM PRIMERS 5' 3' forward primer (18-mer):
15 AGTTTTCATAACAAGTGC 32 reverse primer (19-mer): 364
CATAAGATACTTCAAGCTC 346 forward reverse primer % GC: 33.3 36.8
primer Tm (degrees Celsius): 42.5 42.2 PRODUCT product length: 350
product % GC: 31.4 product Tm: 70.9 degrees Celsius difference in
primer Tm: 0.3 degrees Celsius annealing score: 66 optimal
annealing temperature: 47.4 degrees Celsius Product: 10 [DNA]
50.000 nM [salt] = 50.000 mM PRIMERS 5' 3' forward primer (18-mer):
15 AGTTTTCATAACAAGTGC 32 reverse primer (19-mer): 359
GATACTTCAAGCTCTATTC 341 forward reverse primer % GC: 33.3 36.8
primer Tm (degrees Celsius): 42.5 41.9 PRODUCT product length: 345
product % GC: 31.6 product Tm: 70.9 degrees Celsius difference in
primer Tm: 0.6 degrees Celsius annealing score: 66 optimal
annealing temperature: 47.3 degrees Celsius Product: 11 [DNA] =
50.000 nM [salt] 50.000 mM PRIMERS 5' 3' forward primer (25-mer):
50 CTTCTCTACCTGGAGTATCATTTTC 74 reverse primer (26-mer): 662
GAGTAGCTTAAACCTAACTTTCCTTG 637 forward reverse primer % GC: 40.0
38.5 primer Tm (degrees Celsius): 50.9 52.1 PRODUCT product length:
613 product % GC: 30.0 product Tm: 71.1 degrees Celsius difference
in primer Tm: 1.2 degrees Celsius annealing score: 67 optimal
annealing temperature: 50.1 degrees Celsius Product: 12 [DNA] =
50.000 nM [salt] = 50.000 mM PRIMERS 5' 3' forward primer (19-mer):
50 CTTCTCTACCTGGAGTATC 68 reverse primer (22-mer): 662
GAGTAGCTTAAACCTAACTTTC 641 forward reverse primer % GC: 47.4 36.4
primer Tm (degrees Celsius): 44.8 46.6 PRODUCT product length: 613
product % GC: 30.0 product Tm: 71.1 degrees Celsius difference in
primer Tm: 1.8 degrees Celsius annealing score: 67 optimal
annealing temperature: 48.3 degrees Celsius Product: 13 [DNA] =
50.000 nM [salt] 50.000 mM PRIMERS 5' 3' forward primer (18-mer):
45 AATATCTTCTCTACCTGG 62 reverse primer (18-mer): 365
TCATAAGATACTTCAAGC 348 forward reverse primer % GC: 38.9 33.3
primer Tm (degrees Celsius): 40.8 40.2 PRODUCT product length: 321
product % GC: 31.8 product Tm: 70.8 degrees Celsius difference in
primer Tm: 0.6 degrees Celsius annealing score: 68 optimal
annealing temperature: 46.7 degrees Celsius Product: 14 [DNA] =
50.000 nM [salt] = 50.000 mM PRIMERS 5' 3' forward primer (18-mer):
45 AATATCTTCTCTACCTGG 62 reverse primer (19-mer): 364
CATAAGATACTTCAAGCTC 346 forward reverse primer % GC: 38.9 36.8
primer Tm (degrees Celsius): 40.8 42.2 PRODUCT product length: 320
product % GC: 31.9 product Tm: 70.9 degrees Celsius difference in
primer Tm: 1.4 degrees Celsius annealing score: 69 optimal
annealing temperature: 47.0 degrees Celsius Product: 15 [DNA] =
50.000 nM [salt] 50.000 mM PRIMERS 5' 3' forward primer (24-mer):
51 AATATCTTCTCTACCTGG 62 reverse primer (19-mer): 364
CATAAGATACTTCAAGCTC 645 forward reverse primer % GC: 37.5 38.5
primer Tm (degrees Celsius): 49.8 51.3 PRODUCT product length: 620
product % GC: 30.0 product Tm: 71.1 degrees Celsius difference in
primer Tm: 1.5 degrees Celsius annealing score: 69 optimal
annealing temperature: 49.8 degrees Celsius Product: 16 [DNA[ =
50.000 nM [salt] =50.000 mM PRIMERS 5' 3' forward primer (22-mer):
53 CTCTACCTGGAGTATCATTTTC 74 reverse primer (22-mer): 670
GGCTTATAGAGTAGCTTAAACC 649 forward reverse primer % GC: 40.9 40.9
primer Tm (degrees Celsius): 47.7 48.1 PRODUCT product length: 618
product % GC: 30.1 product Tm: 71.2 degrees Celsius difference in
primer Tm: 0.4 degrees Celsius annealing score: 71 optimal
annealing temperature: 49.2 degrees Celsius Product: 17 [DNA] =
50.000 nM [salt] 50.000 mM PRIMERS 5' 3' forward primer (22-mer):
53 CTCTACCTGGAGTATCATTTTC 74 reverse primer (22-mer): 662
GAGTAGCTTAAACCTAACTTTC 641 forward reverse primer % GC: 40.9 36.4
primer Tm (degrees Celsius): 47.7 46.6 PRODUCT product length: 610
product % GC: 30.0 product Tm: 71.1 degrees Celsius difference in
primer Tm: 1.1 degrees Celsius annealing score: 71 optimal
annealing temperature: 48.8 degrees Celsius Product: 18 [DNA] =
50.000 nM [salt] = 50.000 mM PRIMERS 5' 3' forward primer (18-mer):
57 ACCTGGAGTATCATTTTC 74 reverse primer (18-mer): 674
TCTGGGCTTATAGAGTAG 657 forward reverse primer % GC: 38.9 44.4
primer Tm (degrees Celsius): 42.6 43.0 PRODUCT product length: 618
product % GC: 30.1 product Tm: 71.2 degrees Celsius difference in
primer Tm: 0.4 degrees Celsius annealing score: 73 optimal
annealing temperature: 47.7 degrees Celsius Product: 19 [DNA] =
50.000 nM [salt] = 50.000 mM PRIMERS 5' 3' forward primer (19-mer):
50 CTTCTCTACCTGGAGTATC 68 reverse primer (18-mer): 673
CTGGGCTTATAGAGTAGC 656 forward reverse primer % GC: 47.4 50.0
primer Tm (degrees Celsius): 44.8 45.1 PRODUCT product length: 624
product % GC: 30.3 product Tm: 71.2 degrees Celsius difference in
primer Tm: 0.4 degrees Celsius annealing score: 73 optimal
annealing temperature: 48.4 degrees Celsius Product: 20 [DNA] =
50.000 nM [salt] = 50.000 mM PRIMERS 5' 3' forward primer (20-mer):
55 CTTCTCTACCTGGAGTATC 68 reverse primer (18-mer): 673
CTGGGCTTATAGAGTAGC 656 forward reverse primer % GC: 40.0 50.0
primer Tm (degrees Celsius): 44.7 45.1 PRODUCT product length: 619
product % GC: 30.2 product Tm: 71.2 degrees Celsius difference in
primer Tm: 0.5 degrees Celsius annealing score: 75 optimal
annealing temperature: 48.3 degrees Celsius
[0032] Sample
[0033] For the vertebrate hosts the sample is a tissue sample or
bodily fluid, such as for example, buffy coats and peripheral blood
mononuclear cells. For the invertebrate vectors which may transmit
the pathogen from one vertebrate host to another, the sample can be
dissected from ticks (e.g., midgut, salivary glands and hemolymph),
ticks can be cut into pieces, and ticks can be frozen and smashed
in preparation for PCR assay. Further preparation of tick tissues
may involve just heating the sample, digesting the samples with
proteinase or isolating pure DNA from the tick tissues.
[0034] Methods
[0035] Optionally, DNA is extracted from the sample using standard
methods. For example, DNA may be extracted from fluids using
commercially available PCR filters such as, for example, the
Isocode filters which are available from Schleicher and Schuell,
New Hampshire. Methods of extracting DNA from tissue samples, are
also described in Maniatis, T., J. Sambrook, and E. F. Fritsch.
1989. Molecular Cloning: a Laboratory manual, 2nd ed. Cold Spring
Harbor Laboratory Press, Cold Spring Harbor N. Y., which is
specifically incorporated herein by reference. An optimal method
for vertebrates involves collection of whole blood in the presence
of an anticoagulant such as heparin, collecting the buffy coat
fraction from the whole blood, removal of host hemoglobin by
hypotonic lysis of erythrocytes remaining with the buffy coat
followed by several washings of the buffy coat prior to preparation
of the template for PCR assay. For detecting the pathogens in the
invertebrate vectors, DNA is extracted from tick halves, salivary
glands and midgut by digestion with proteinase K in the presence of
nonionic detergents such as Tween-20 and NP-40 followed by heat
inactivation of the proteinase and an optional procedure to extract
proteins with phenol and chloroform followed by DNA precipitation
by salt and ethanol.
[0036] The DNA is then amplified with the one of the primer sets at
PCR conditions equivalent to or comparable to denaturation for 0.5
to 1.0 minute at 94.degree. C. in a solution, followed by annealing
at about 50-72.degree. C. for 0.5 to 1.0 minutes. As recognized in
the art, suitable stringency conditions can be attained by varying
a number of factors such as the length and nature, i.e., DNA or
RNA, of the primer; the length and nature of the target sequence;
the concentration of the salts and other components of the PCR
solution; the temperature and time of each step, e.g.,
denaturation, annealing, and elongation. All of these factors may
be varied to generate conditions of stringency which are equivalent
to the conditions listed above. Changes in stringency are
accomplished primarily through the manipulation of annealing
temperature and time.
[0037] Thereafter, the size of the PCR products is determined.
Preferably the size of the PCR products is determined using gel
electrophoresis and a plurality of DNA standards of varying sizes.
The PCR products and DNA standards that are present on the gel are
visualized using standard techniques, such as ethidium bromide
staining. Optionally, the PCR products may be separated, preferably
by gel electrophoresis and the sequence of the PCR products
determined using standard techniques.
[0038] To further enhance the sensitivity and specificity of the
method for detecting E. canis, a nested PCR assay which employs two
different E. canis primer sets is used. In such assay, the sample
is first amplified with a primer set comprising two external
primers, i. e. ECA30-351S and ECA30-591A, ECA30-350S and
ECA30-591bA, ECA30-356S and ECA30-591A, ECA30-356S and ECA30-591Ba,
ECA30-356S and ECA30-589A, ECA30-355S and ECA30-589bA, and
ECA30-353S and ECA30-597A. Thereafter, the products of the first
PCR are amplified with a second primer set that comprises internal
primers, eg. ECA30-384S and ECA30. A highly preferred primer set
for this second amplification comprises a first primer comprising
the sequence 5'-ATAAACACGCTGACTTTACTGTTCC-3' and a second primer of
comprising the sequence 5'-GTGATGAGATAGAGCGCAGTACC-3'. Following
the second amplification, the size or sequence of the PCR products
are determined to detect a PCR product which indicates that a DNA
molecule having a sequence which corresponds to the region of the
p30 gene that is located between the consecutive sequences to which
the internal primers bind is present in the sample.
[0039] To further enhance the sensitivity and specificity of the
method for detecting E. chaffeensis, a nested PCR assay which
employs two different E. chaffeensis primer sets is used. In such
assay, the sample is first amplified with a primer set that
comprises external primers, i.e. E. chaffeensis primer set 3 A
highly preferred primer set for this first amplification comprises
a first primer comprising the sequence CTTCTCTACCTGGAGTATC and a
second primer comprising the sequence GCTTATAGAGTAGCTTAAACC.
Thereafter, the products of the first PCR are amplified with a
second primer set that comprises two internal primers, e.g. E.
chaffeensis primer set 1. A highly preferred primer set for this
second amplification comprises a first primer comprising the
sequence AGGTAGTGGTATTAACGG and a second primer which comprises the
sequence AGATACTTCAAGCTCTATTC. Following the second amplification,
the size or sequence of the PCR products are determined to detect a
PCR product which indicates that a DNA molecule having a sequence
which corresponds to 277 bp of the p28 gene is present in the
sample.
EXAMPLES
[0040] The following examples are for purposes of illustration only
and are not intended to limit the scope of the invention as defined
in the claims which are appended hereto. The references cited in
this document are specifically incorporated herein by
reference.
Example 1
[0041] A. Obtaining DNA from Blood Samples of Infected Animals
[0042] Two colony-reared Beagle dogs that were seronegative for
exposure to E. canis by IFA were inoculated with heparinized blood
from a donor dog infected with E. canis (Ebony isolate).
Heparinized blood was collected from each animal and split into 0.5
ml aliquots and subjected to the following treatments: (1) DNAzol
extraction of buffy coat, (2) DNAzol-BD (Molecular Research Center)
extraction of whole blood, (3) DNAzol-BD extraction of buffy coat,
(4) spin-column purification of peripheral blood mononuclear cell
DNA with a QIAamp Kit (Qiagen) and (5) phenol/chloroform extraction
followed by ethanol precipitation of buffy coat samples. All sample
preparation methods were done in duplicate and in two trials. Buffy
coats were isolated from the heparinized whole blood after
centrifugation at 1000.times.g for 20 min, except for those to be
purified with spin-columns, which were isolated after isopycnic
centrifugation as described in Iqbal, Z., and Y. Rikihisa. 1994.
Application of the polymerase chain reaction for the detection of
Ehrlichia canis in tissues of dogs. Vet. Microbiol. 42:281-7. All
DNA isolations with commercial kits were performed in strict
accordance to the manufacturer's instructions. DNAzol and DNAzol-BD
isolations were performed in the presence of polyacryl carrier
(Molecular Research Center) as recommended by the manufacturer.
[0043] The remaining method was a modification of a protocol that
was previously described in Stich, R. Wet al. 1991. Preliminary
development of a polymerase chain reaction assay for Anaplasma
marginale in ticks. Biotechnol. Techniq. 5:269-274.; and Stich, R.
W., et al 1993. Detection of Anaplasma marginale (Rickettsiales:
Anaplasmataceae) in secretagogue-induced oral secretions of
Dermacentor andersoni (Acari: Ixodidae) with the polymerase chain
reaction. J. Med. Entomol. 30:789-94. Briefly, the buffy coats were
removed from whole blood and transferred to a 1.5 ml microfage
tube. Erythrocytes remaining in the buffy coat were lysed with two
volumes of TE (10 mM Tris.HCl, pH 8.0, 1 mM EDTA), and the
hemoglobin was removed by washing pelleted cells (13,000.times.g
for 1 min) three times in TE. The final cell suspension was in 400
.mu.l of RPMI 1640 containing 100 .mu.g/ml proteinase K and 0.45%
(v/v) NP-40 and 0.45% (v/v) Tween-20, and the protein was digested
for 1-2 hr at 55.degree. C. Digests were extracted one time each
with equal volumes of buffer-saturated phenol (pH>7.5) (Life
Technologies), phenol/chloroform/isoamyl alcohol (25:24:1), and
chloroform/isoamyl alcohol (24:1). DNA was precipitated by adding
{fraction (1/10)} volume of 3 M sodium acetate and 2.5 volumes of
absolute ethanol (-20.degree. C.) and stored overnight at
-20.degree. C. The DNA samples were centrifuged at 13,000.times.g
for 20 min at 4.degree. C., allowed to air dry in a sterile,
bleach-treated cabinet and resuspended in 25 .mu.l of HPLC grade
H.sub.2O. The samples containing E. canis DNA were diluted
empirically throughout the optimization process.
[0044] B. Nested PCR
[0045] The primers used in the first PCR reaction is set 12 shown
in Table 1 above. The primers used in the second PCT reaction is
set 1 shown in Table 1 above. PCR was performed with a Perkin-Elmer
2400 thermal cycler. Master mixes, made with the PE Biosystems
Reagents (Foster City, Calif.), were divided into 50 or 25 .mu.l
final reaction volumes containing PCR Gold buffer, 0.8 mM dNTP mix
and specified amounts of MgCl.sub.2, primers, Amplitaq-Gold DNA
Polymerase and 10% (v/v) template. The reaction profile, except for
stated exceptions, consisted of 95.degree. C. for 10 min followed
by 35 cycles of 94.degree. C. for 1.0 min, 60.degree. C. for 0.5
min, and 72.degree. C. for 0.5 min followed by a final extension at
72.degree. C. for 7.0 min.
[0046] The following PCR parameters were progressively optimized:
(1) annealing temperature (37.degree., 50.degree., 55.degree.,
60.degree., 65.degree. and 70.degree. C. with 1.5 mM MgCl.sub.2,
0.5 .mu.M primers and 0.025 U/.mu.l Amplitaq-Gold), (2) MgCl.sub.2
concentration (1.0-4.0 mM MgCl.sub.2 in 0.5 mM increments), (3)
primer concentration (0.1-1.0 .mu.M in 0.1 .mu.M increments), (4)
Amplitaq-Gold concentration (0.01-0.1 U/.mu.l in 0.01 U/.mu.l
increments) and (5) cycle number (25-65 cycles in 5 cycle
increments).
[0047] C. Analysis of PCR Products
[0048] PCR product (20 .mu.l) was added to 5 .mu.l of loading
buffer (40% (w/v) sucrose, 89 mM Tris, 89 mM boric acid, 2 mM EDTA)
and electrophoresed on a 1.5% (w/v) agarose gel with 0.5 .mu.g/ml
ethidium bromide in 1.times. TBE (89 mM Tris, 89 mM boric acid, 2
mM EDTA) at 60-80 V for 1-1.5 hr. A 100 bp ladder (Life
Technologies, Rockville, Md.) was used as the molecular weight
standard. DNA bands were visualized with ultraviolet light and
documented using an Alpha Innotech 2000 gel imaging system.
RESULTS
[0049] Amplification of the 200 bp target sequence by nested PCR of
E. canis-infected peripheral blood from dogs with acute
experimental CME.
[0050] A 200 bp band was amplified from DNA prepared from blood
samples collected from three dogs during acute or chronic
experimental CME (FIG. 1). This band was not observed from blood
collected prior to infection of the two dogs assayed during acute
CME. The DNA sequence of this amplicon was found to be identical to
the 200 bpp3O target sequence and the reverse complement of
ECAR1583A (FIG. 2).
[0051] Optimum Conditions for Isolating DNA from Blood Samples
[0052] Several approaches to blood preparation for PCR assay were
compared with duplicate samples in two trials. Venous blood was
collected with heparin from an experimentally infected E. canis
carrier, equal aliquots that were subjected to different protocols
for the preparation of template for PCR. Inhibition of PCR by
hemoglobin is well known, thus removing hemoglobin from the blood
sample is a major concern of any procedure used to assay these
samples with PCR. Commercially available chemical and spin column
kits for DNA isolation did provide some positive results with buffy
coat and whole blood samples, but these treatments were
inconsistent, failing to result in amplification of DNA in both
duplicates of both trials. However, buffy coat isolation followed
by lysing residual erythrocytes, removal of hemoglobin, protein
digestion, protein extraction and DNA precipitation was the only
procedure that resulted in amplification of the 200 bp target in
both duplicates of both trials (FIG. 5). Moreover, this method has
been used to detect E. canis in peripheral blood of a carrier dog
for over one year (data not shown).
[0053] Optimization of the p30-based PCR assay for E. canis.
Initial optimization experiments with primer set 2 resulted in the
amplification of multiple bands, thus this primer set is less
preferred. The remaining assays employed primer sets 1 and 12. For
the primary reaction with set 12, optimum PCR conditions were
determined to be 1.5 mM MgCl.sub.2, 0.2 .mu.M of each primer, 0.04
units/.mu.l of Amplitaq-Gold and, after enzyme activation at
95.degree. C. for 10 min, 55 cycles with 94, 65 and 72.degree. C.
denaturation, annealing and extension temperatures for 30 sec each.
For the secondary or nested PCR with internal primer set 1, optimum
reaction conditions were determined to be 2.5 mM MgCl.sub.2, 0.5
.mu.M of each primer, 0.03 units/.mu.l of Amplitaq-Gold, 10%
(vol/vol) of the appropriate primary reaction with primer set 12
and 40 cycles with 94, 60 and 72.degree. C. denaturation, annealing
and extension temperatures for 30 sec each.
[0054] To confirm that the 200 bp amplicon associated with E. canis
infection originated from the target DNA sequence, several
p30-based nested PCR assays of buffy coat DNA from an E.
canis-infected dog were pooled and the amplicons isolated with the
QIAquik PCR Purification Kit (Qiagen, Valencia, Calif.) according
to the manufacturer's instructions. The purified amplicon was
submitted to The Ohio State University Neurobiotechnology DNA
Sequencing Facility for cycle sequencing in the presence of the
primer, ECAF1384S.
[0055] The optimum number of cycles for both reactions,
particularly the primary reaction, were somewhat greater than
expected. One explanation for this may be the enzyme used,
Amplitaq-Gold, which requires activation at 95.degree. C. for 10
min prior to the amplification cycles. It is possible that a
portion of this enzyme remains inactive until the amplification
cycles are underway; thus additional active enzyme becomes
available in the course of the reaction, increasing the number of
cycles required for optimal PCR.
[0056] Specificity and Sensitivity.
[0057] The sensitivity of this p30-based assay was compared to that
of the previously reported 16S rDNA-based assay as described in
Wen, B., Y. Rikihisa, J. M. Mott, R. Greene, H. Y. Kim, N. Zhi, G.
C. Couto, A. Unver, and R. Bartsch. 1997. Comparison of nested PCR
with immunofluorescent-antibody assay for detection of Ehrlichia
canis infection in dogs treated with doxycycline. J. Clin.
Microbiol. 35:1852-5.The buffy coat fraction was removed from 5 ml
of heparinized blood from an experimentally infected dog during
acute CME, and subjected to DNA isolation with DNAzol (Molecular
Research Center) according to the manufacturer's instructions. A
tenfold dilution series of the buffy coat DNA was then tested with
both the p30- and the 16S rDNA-based PCR assays.
[0058] Specificity of the p30-based assay was tested by attempts to
amplify 50 ng samples of DNA isolated from E. muris, E. chaffeensis
and HGE agent in experimentally infected host cells. DNA. E.
chaffeensis and E. muris are the two known species most closely
related to E. canis (based on 16S rDNA sequence homology) Faint
bands at sizes other than that of the target sequence were
occasionally observed, indicating that similar omp-1 sequences may
be amplified under these conditions, but the robust 200 bp band was
only observed with E. canis template.
Example 2
[0059] E. chaffeensis p28 ORF sequences were compared to identify
prospective oligonucleotide primer sequences for a PCR assay.
Design of the primers for this PCR assay was similar to that
described for E. canis, except that identities of these primers to
the three clusters of p28 sequences among the E chaffeensis
isolates were also considered by dividing the product of the
annealing and E. canis identity scores by the mean of the three E.
chaffeensis cluster identity scores. Primers were designed from the
E. chaffeensis Arkansas isolate p28 sequence. These primer
sequences were aligned with the homologous portions of the E. canis
p30 geme and the three p28 clusters and the identity of these
sequences determined Total annealing scores, identity to the
different p28 clusters and identity to p30 were then used to rank
these primer sets. A second approach to primer design involved the
design of primers to the consensus sequence of all three E.
chaffeensis p28 clusters through less stringent annealing score
parameters. These primers, which are shown in Table 3 above are
expected to be universal for all five E. chaffeensis isolates that
composed these three clusters.
[0060] Primers for E. chaffeensis were designed with the Wisconsin
Package (GCG) Version 10 software suite (Madison, Wisc.). This was
accomplished with a multiple sequence alignment of the target
template from isolates representing all three clusters of E.
chaffeensis of and the E. canis p30 sequence. Initially optimal
primer sequences could not be obtained from the consensus of all
three p28 sequences, thus primers were designed from the E.
chaffeensis Arkansas isolate p28 sequence since this is the isolate
to be used in the proposed investigation. Twenty-five sets of
primers were then identified. These primer sequences were aligned
with the homologous portions of p30 and the three p28 clusters and
the identity of these sequences determined. Total annealing scores,
identity to the different p28 clusters and identity to p30 were
then used to rank these primer sets. Three primer sets were chosen
for development of a p28-based assay by virtue of their overall
rank and relative positions flanking or internal to other primer
sets for nested PCR and found to amplify E. chaffeensis DNA from
infected canine cells. Two of the E. chaffeensis Arkansas
isolate-specific primer sets were optimized and used as a nested
PCR assay to detect the pathogen in intrastadially infected male
ticks of four species known to parasitize dogs.
4TABLE 4 Ranks of E. chaffeensis p28 primer sets with predicted
annealing and specificity values..sup.a Identity Scores to E.
chaffeensis Identity Scores to Primers Anealing Cluster I Cluster
II Cluster III E. canis Set Fwd. Rev. Score Fwd. Rev. Fwd. Rev.
Fwd. Rev. Mean Fwd. Rev. Mean Rank 1.sup.b 67-85 341-320 55 1.0 1.0
0.90 0.95 1.0 1.0 0.98 0.68 0.91 0.80 8 2 58-78 321-302 58 1.0 0.95
0.95 0.95 1.0 1.0 0.98 0.57 0.90 0.74 7 3 58-78 314-294 58 1.0 0.90
0.95 0.90 1.0 1.0 0.96 0.57 0.86 0.72 5 4 61-82 339-319 58 1.0 0.90
0.91 0.95 1.0 1.0 0.96 0.64 0.86 0.75 10 5 61-82 315-294 59 1.0
0.91 0.91 0.91 1.0 1.0 0.96 0.64 0.86 0.75 15 6 61-82 320-301 59
1.0 1.0 0.91 0.95 1.0 1.0 0.98 0.64 0.90 0.77 16 7 60-80 314-294 60
1.0 0.90 0.95 0.90 1.0 1.0 0.96 0.62 0.86 0.74 13 8 59-79 314-294
60 1.0 0.90 0.95 0.90 1.0 1.0 0.96 0.62 0.86 0.74 14 9 58-79
322-302 60 1.0 0.95 0.95 0.95 1.0 1.0 0.98 0.59 0.90 0.75 11 10
58-78 320-301 60 1.0 1.0 0.95 0.95 1.0 1.0 0.98 0.57 0.90 0.74 9
*11.sup.b 58-78 341-320 61 1.0 1.0 0.95 0.95 1.0 1.0 0.98 0.57 0.91
0.74 12 12 59-79 320-301 62 1.0 1.0 0.95 0.95 1.0 1.0 0.98 0.62
0.90 0.76 19 *13.sup.b' 78-99 314-294 62 1.0 0.90 0.95 0.90 1.0 1.0
0.96 0.41 0.86 0.64 2 14 59-80 320-301 62 1.0 1.0 0.95 0.95 1.0 1.0
0.98 0.64 0.90 0.77 20 15 58-78 352-331 62 1.0 1.0 0.95 0.95 1.0
1.0 0.98 0.57 0.77 0.67 3 16 58-78 339-319 64 1.0 0.90 0.95 0.95
1.0 1.0 0.97 0.57 0.86 0.72 18 17 78-99 341-320 64 1.0 1.0 0.95
0.95 1.0 1.0 0.98 0.41 0.91 0.66 4 18 58-78 313-293 65 1.0 0.95
0.95 0.90 1.0 1.0 0.97 0.57 0.95 0.76 25 19 60-80 341-320 64 1.0
1.0 0.95 0.95 1.0 1.0 0.98 0.62 0.91 0.77 21 20 78-99 352-331 65
1.0 1.0 0.95 0.95 1.0 1.0 0.98 0.41 0.77 0.59 1 21 61-82 314-293 65
1.0 0.91 0.91 0.91 1.0 1.0 0.96 0.64 0.86 0.75 24 22 59-79 341-320
65 1.0 1.0 0.95 0.95 1.0 1.0 0.98 0.62 0.91 0.77 23 23 59-80
352-331 66 1.0 1.0 0.95 0.95 1.0 1.0 0.98 0.64 0.77 0.71 17
24.sup.b' 78-99 314-293 66 1.0 0.91 0.95 0.91 1.0 1.0 0.96 0.41
0.86 0.64 6 25 60-80 339-319 66 1.0 0.90 0.95 0.95 1.0 1.0 0.97
0.62 0.86 0.74 22 .sup.aAll values determined as described for
Table 1, except for Rank, which was determined by the product of
annealing score and mean E. canis identity score, divided by mean
E. chaffeensis annealing score; lower values have higher rank.
.sup.b,b'Nested primer sets. *Primer sets chosen for
optimization.
[0061] Primers derived from both approaches were chosen for use in
a p28-based assay by virtue of their overall rank and relative
positions flanking or internal to other primer sets for nested PCR.
These candidate primer were synthesized and they were all tested
with 50 ng of DNA from E. chaffeensis-infected DH82 cells and Life
Technologies reagents at concentrations of 1.times. PCR buffer, 1.5
mM MgCl.sub.2, 0.5 .mu.M of each primer and 0.025 U/.mu.l Platinum
Taq DNA Polymerase at an initial incubation period at 94.degree. C.
for 2 min followed by 25 cycles of 94.degree. C. for 30 sec,
50.degree. C. for 30 sec and 72.degree. C. for 1 min 7 min final
extension step at 72.degree. C. Each primer set tested amplified a
clean, robust amplicon of the expected size under unoptimized
conditions. Primer sets F58/R341 and F78/R314 were chosen first for
optimization due to the more stringent parameters used for their
selection.
[0062] Optimum parameters for primer sets F58/R341 and F78/R314
were then used to assay canine and tick host samples. Dog number
146 was inoculated iv. with DH82 cells that were infected in vitro
with E. chaffeensis (Arkansas isolate). Once the dog was
PCR-positive by the 16S rDNA-based PCR assay and seroconverted, it
was infested with 150 adult males of A. americanum, and 10 each of
A. maculatum, D. variabilis and R. sanguineus. These ticks were
allowed to simultaneously acquisition feed seven days prior to
their removal and incubation in a humidity chamber for another 10
days. The held ticks were then placed at 37.degree. C. at 100% rh
for 80 hr before they were aseptically bisected and digested with
proteinase K in the presence of nonionic detergents. These ticks
(2.5 .mu.l) were then assayed with E. chaffeensis p28-specific
nested primer sets 11.sub.A (F58/R341)and 13.sub.A (F78/R314).
[0063] Samples from each tick species fed tested PCR-positive at a
frequency of 20-40% with the nested assay. These results are the
first to our knowledge to demonstrate detection of E. chaffeensis
in experimentally infected ticks, only the second time to detect E.
chaffeensis in individual ticks and the first detection of this
pathogen in A. maculatum or R. sanguineus.
[0064] Further sequence analysis with less stringent criteria did
reveal 20 primer sequences complementary to the p28 consensus
sequence of all three clusters. Two sets of the consensus primers
(See * in Table 5 below) were also tested and found to amplify
robust amplicons from E. chaffeensis DNA.
5TABLE 5 Ranks of universal E. chaffeensis p28 primer sets with
predicted values. Identity Scores to Primers Anealing E. canis Set
Fwd. Rev. Score Fwd. Rev. Mean Rank *1 84-101 360-341 57 0.5 0.8
0.65 1 2 84-101 365-346 57 0.5 0.8 0.65 1 *3 50-68 669-649 61 0.58
0.67 0.62 3 4 83-100 364-346 63 0.44 0.79 0.62 5 5 83-100 359-341
63 0.44 0.79 0.62 5 6 50-68 669-650 63 0.58 0.65 0.61 4 7 55-74
670-650 64 0.65 0.67 0.66 7 8 45-62 359-341 65 0.61 0.79 0.70 10 9
15-32 364-346 66 0.78 0.79 0.78 19 10 15-32 359-341 66 0.78 0.79
0.78 19 11 50-74 662-637 67 0.64 0.73 0.69 11 12 50-68 662-641 67
0.58 0.68 0.63 8 13 45-62 365-348 68 0.61 0.78 0.69 13 14 45-62
364-346 69 0.61 0.79 0.70 15 15 51-74 670-645 69 0.67 0.58 0.62 9
16 53-74 670-649 71 0.69 0.64 0.66 12 17 53-74 662-641 71 0.67 0.72
0.69 17 18 57-74 674-657 73 0.67 0.72 0.69 14 19 50-68 673-656 73
0.58 0.72 0.65 14 20 55-74 673-656 75 0.65 0.72 0.69 18
[0065]
Sequence CWU 1
1
51 1 135 DNA Artificial Sequence Description of Artificial Sequence
Primer 1 ccaagtgtct cacattttgg tagcttctca gctaaagaag aaagcaaatc
aactgttgga 60 gtttttggat taaaacatga ttgggatgga agtccaatac
ttaagaataa acacgctgac 120 tttactgttc caaac 135 2 133 DNA Artificial
Sequence Description of Artificial Sequence Primer 2 gttactcaat
gggtggccca agaatagaat tcgaaatatc ttatgaagca ttcgacgtaa 60
aaagtcctaa tatcaattat caaaatgacg cgcacaggta ctgcgctcta tctcatcaca
120 catcggcagc cat 133 3 87 DNA Artificial Sequence Description of
Artificial Sequence Primer 3 agttttcata acaagtgcat tgatatcact
aatatcttct ctacctggag tatcattttc 60 cgacccaaca ggtagtggta ttaacgg
87 4 73 DNA Artificial Sequence Description of Artificial Sequence
Primer 4 catttctagg ttttgcagga gctattggct actcaatgga tggtccaaga
atagagcttg 60 aagtatctta tga 73 5 38 DNA Artificial Sequence
Description of Artificial Sequence Primer 5 caaggaaagt taggtttaag
ctactctata agcccaga 38 6 25 DNA Artificial Sequence Description of
Artificial Sequence Primer 6 ataaacacgc tgactttact gttcc 25 7 23
DNA Artificial Sequence Description of Artificial Sequence Primer 7
gtgatgagat agagcgcagt acc 23 8 22 DNA Artificial Sequence
Description of Artificial Sequence Primer 8 aacacgctga ctttactgtt
cc 22 9 20 DNA Artificial Sequence Description of Artificial
Sequence Primer 9 atggctgccg atgtgtgatg 20 10 23 DNA Artificial
Sequence Description of Artificial Sequence Primer 10 acgctgactt
tactgttcca aac 23 11 21 DNA Artificial Sequence Description of
Artificial Sequence Primer 11 aacatgattg ggatggaagt c 21 12 20 DNA
Artificial Sequence Description of Artificial Sequence Primer 12
gccgatgtgt gatgagatag 20 13 22 DNA Artificial Sequence Description
of Artificial Sequence Primer 13 aaacatgatt gggatggaag tc 22 14 22
DNA Artificial Sequence Description of Artificial Sequence Primer
14 gccgatgtgt gatgagatag ag 22 15 22 DNA Artificial Sequence
Description of Artificial Sequence Primer 15 gattgggatg gaagtccaat
ac 22 16 25 DNA Artificial Sequence Description of Artificial
Sequence Primer 16 acacgctgac tttactgttc caaac 25 17 22 DNA
Artificial Sequence Description of Artificial Sequence Primer 17
atggctgccg atgtgtgatg ag 22 18 25 DNA Artificial Sequence
Description of Artificial Sequence Primer 18 gtgtctcaca ttttggtagc
ttctc 25 19 22 DNA Artificial Sequence Description of Artificial
Sequence Primer 19 cttgggccac ccattgagta ac 22 20 21 DNA Artificial
Sequence Description of Artificial Sequence Primer 20 cgatgtgtga
tgagatagag c 21 21 23 DNA Artificial Sequence Description of
Artificial Sequence Primer 21 tgattgggat ggaagtccaa tac 23 22 22
DNA Artificial Sequence Description of Artificial Sequence Primer
22 cgatgtgtga tgagatagag cg 22 23 25 DNA Artificial Sequence
Description of Artificial Sequence Primer 23 catgattggg atggaagtcc
aatac 25 24 24 DNA Artificial Sequence Description of Artificial
Sequence Primer 24 ccaagtgtct cacattttgg tagc 24 25 20 DNA
Artificial Sequence Description of Artificial Sequence Primer 25
tgggccaccc attgagtaac 20 26 18 DNA Artificial Sequence Description
of Artificial Sequence Primer 26 aggtagtggt attaacgg 18 27 20 DNA
Artificial Sequence Description of Artificial Sequence Primer 27
agatacttca agctctattc 20 28 20 DNA Artificial Sequence Description
of Artificial Sequence Primer 28 tcataagata cttcaagctc 20 29 19 DNA
Artificial Sequence Description of Artificial Sequence Primer 29
cttctctacc tggagtatc 19 30 21 DNA Artificial Sequence Description
of Artificial Sequence Primer 30 gcttatagag tagcttaaac c 21 31 18
DNA Artificial Sequence Description of Artificial Sequence Primer
31 caggtagtgg tattaacg 18 32 19 DNA Artificial Sequence Description
of Artificial Sequence Primer 32 cataagatac ttcaagctc 19 33 19 DNA
Artificial Sequence Description of Artificial Sequence Primer 33
gatacttcaa gctctattc 19 34 20 DNA Artificial Sequence Description
of Artificial Sequence Primer 34 gcttatagag tagcttaaac 20 35 20 DNA
Artificial Sequence Description of Artificial Sequence Primer 35
ctacctggag tatcattttc 20 36 21 DNA Artificial Sequence Description
of Artificial Sequence Primer 36 ggcttataga gtagcttaaa c 21 37 18
DNA Artificial Sequence Description of Artificial Sequence Primer
37 aatatcttct ctacctgg 18 38 18 DNA Artificial Sequence Description
of Artificial Sequence Primer 38 agttttcata acaagtgc 18 39 25 DNA
Artificial Sequence Description of Artificial Sequence Primer 39
cttctctacc tggagtatca ttttc 25 40 26 DNA Artificial Sequence
Description of Artificial Sequence Primer 40 gagtagctta aacctaactt
tccttg 26 41 22 DNA Artificial Sequence Description of Artificial
Sequence Primer 41 gagtagctta aacctaactt tc 22 42 18 DNA Artificial
Sequence Description of Artificial Sequence Primer 42 tcataagata
cttcaagc 18 43 22 DNA Artificial Sequence Description of Artificial
Sequence Primer 43 ctctacctgg agtatcattt tc 22 44 18 DNA Artificial
Sequence Description of Artificial Sequence Primer 44 acctggagta
tcattttc 18 45 18 DNA Artificial Sequence Description of Artificial
Sequence Primer 45 tctgggctta tagagtag 18 46 18 DNA Artificial
Sequence Description of Artificial Sequence Primer 46 ctgggcttat
agagtagc 18 47 1607 DNA Ehrlichia canis 47 attttattta ttaccaatct
tatataatat attaaatttc tcttacaaaa atctctaatg 60 ttttatacct
aatatatata ttctggcttg tatctacttt gcacttccac tattgttaat 120
ttattttcac tattttaggt gtaatatgaa ttgcaaaaaa attcttataa caactgcatt
180 aatatcatta atgtactcta ttccaagcat atctttttct gatactatac
aagatggtaa 240 catgggtggt aacttctata ttagtggaaa gtatgtacca
agtgtctcac attttggtag 300 cttctcagct aaagaagaaa gcaaatcaac
tgttggagtt tttggattaa aacatgattg 360 ggatggaagt ccaatactta
agaataaaca cgctgacttt actgttccaa actattcgtt 420 cagatacgag
aacaatccat ttctagggtt tgcaggagct atcggttact caatgggtgg 480
cccaagaata gaattcgaaa tatcttatga agcattcgac gtaaaaagtc ctaatatcaa
540 ttatcaaaat gacgcgcaca ggtactgcgc tctatctcat cacacatcgg
cagccatgga 600 agctgataaa tttgtcttct taaaaaacga agggttaatt
gacatatcac ttgcaataaa 660 tgcatgttat gatataataa atgacaaagt
acctgtttct ccttatatat gcgcaggtat 720 tggtactgat ttgatttcta
tgtttgaagc tacaagtcct aaaatttcct accaaggaaa 780 actgggcatt
agttactcta ttaatccgga aacctctgtt ttcatcggtg ggcatttcca 840
caggatcata ggtaatgagt ttagagatat tcctgcaata gtacctagta actcaactac
900 aataagtgga ccacaatttg caacagtaac actaaatgtg tgtcactttg
gtttagaact 960 tggaggaaga tttaacttct aattttattg ttgccacata
ttaaaaatga tctaaacttg 1020 tttttawtat tgctacatac aaaaaaagaa
aaatagtggc aaaagaatgt agcaataaga 1080 gggggggggg ggaccaaatt
tatcttctat gcttcccaag ttttttcycg ctatttatga 1140 cttaaacaac
agaaggtaat atcctcacgg aaaacttatc ttcaaatatt ttatttatta 1200
ccaatcttat ataawatatt aaatttctct tacaaaaatc actagtattt tataccaaaa
1260 tatatattct gacttgcttt tcttctgcac ttctactatt tttaatttat
ttgtcactat 1320 taggttataa taatatgaat tgcmaaagat ttttcatagc
aagtgcattg atatcactaa 1380 tgtctttctt acctagcgta tctttttctg
aatcaataca tgaagataat ataaatggta 1440 acttttacat tagtgcaaag
tatatgccaa gtgcctcaca ctttggcgta ttttcagtta 1500 aagaagagaa
aaacacaaca actggagttt tcggattaaa acaagattgg gacggagcaa 1560
cactaaagga tgcaagcwgc agccacacaw tagacccaag tacaatg 1607 48 849 DNA
Ehrlichia chafeensis 48 atgaattaca aaaaagtttt cataacaagt gcattgatat
caytaatatc ttctctacct 60 ggagtatcat tttcygaccc arcaggtagt
ggtattaacg gyaatttcta yatcagtgga 120 aaatayatgc caagygcttc
gcattttggr gtrttytctg ctaaggaaga aagaartaca 180 acagytggag
trtttggayt gaagcaarat tgggayggma gygcaatayc ymacwcyhmy 240
mswrahrmtv yattyactgt ytcaaaytay tcrtttaaat atgaaaayaa yccrtttyta
300 ggwtttgcag gagctattgg ytactcaatg gatggyccaa gaatagagct
tgaagtatct 360 tatgaracat tygatgtwaa aaatcaaggt aacarytaya
agaaygaagc dcatagrtay 420 tgtgctytat cycrtmasrs ywcarbarca
rrcatgwska gtgcarrtra tamwtttgty 480 tttctaaaaa atgaaggryt
acttgacrta tcrttyatgc tgaacgcatg ctatgaygta 540 rtargygaag
gmataccttt ttctccttay atatgygyag gtatyggkac tgatttagta 600
tccatgtttg aagytacaaa ycctaaaatt tcttaccaag gaaagttagg tttaagctac
660 tctataagcc cagaarcttc tgtstttrty ggyggrcayt tycataaggt
ratrggraac 720 gaattyagag atattcctrc trtaatacct avtggatcaa
swcttgcagg aamaggraay 780 yaccctgcaa tagtaayact rgaygtatgc
cactttggwa tagarcttgg aggaagrttt 840 gctttctaa 849 49 22 DNA
Artificial Sequence Description of Artificial Sequence Primer 49
ggcttataga gtagcttaaa cc 22 50 24 DNA Artificial Sequence
Description of Artificial Sequence Primer 50 ttctctacct ggagtatcat
tttc 24 51 26 DNA Artificial Sequence Description of Artificial
Sequence Primer 51 ggcttataga gtagcttaaa cctaac 26
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