U.S. patent application number 17/029575 was filed with the patent office on 2021-06-24 for pna probes, probe sets, methods and kits pertaining to the detection of candida.
The applicant listed for this patent is APPLIED BIOSYSTEMS, LLC. Invention is credited to Jens J. HYLDIG-NIELSEN, Kenneth M. OLIVEIRA, Susan RIGBY, Henrik STENDER.
Application Number | 20210189511 17/029575 |
Document ID | / |
Family ID | 1000005436226 |
Filed Date | 2021-06-24 |
United States Patent
Application |
20210189511 |
Kind Code |
A1 |
HYLDIG-NIELSEN; Jens J. ; et
al. |
June 24, 2021 |
PNA PROBES, PROBE SETS, METHODS AND KITS PERTAINING TO THE
DETECTION OF CANDIDA
Abstract
This invention is related to novel PNA probes, probe sets,
methods and kits pertaining to the detection of one or more species
of Candida yeast. Non-limiting examples of probing nucleobase
sequences that can be used for the probes of this invention can be
selected from the group consisting of: AGA-GAG-CAG-CAT-GCA (Seq.
Id. No. 1), AGA-GAG-CAA-CAT-GCA (Seq. Id. No. 2),
ACA-GCA-GAA-GCC-GTG (Seq. Id. No. 3), CAT-AAA-TGG-CTA-CCA-GA (Seq.
Id. No. 4), CAT-AAA-TGG-CTA-CCC-AG (Seq. Id. No. 5),
ACT-TGG-AGT-CGA-TAG (Seq. Id. No. 6), CCA-ACG-CTT-ATA-CTC-GC (Seq.
Id. No. 7), CCC-CTG-AAT-CGG-GAT (Seq. Id. No. 8),
GAC-GCC-AAA-GAC-GCC (Seq. Id. No. 9), ATC-GTC-AGA-GGC-TAT-AA (Seq.
Id. No. 10), TAG-CCA-GAA-GAA-AGG (Seq. Id. No. 11),
CAT-AAA-TGG-CTA-GCC-AG (Seq. Id. No. 12), CTC-CGA-TGT-GAC-TGC-G
(Seq. Id. No. 13), TCC-CAG-ACT-GCT-CGG (Seq. Id. No. 14),
TCC-AAG-AGG-TCG-AGA (Seq. Id. No. 15), GCC-AAG-CCA-CAA-GGA (Seq.
Id. No. 16), GCC-GCC-AAG-CCA-CA (Seq. Id. No. 17),
GGA-CTT-GGG-GTT-AG (Seq. Id. No. 18), CCG-GGT-GCA-TTC-CA (Seq. Id.
No. 19), ATG-TAG-AAC-GGA-ACT-A (Seq. Id. No. 20),
GAT-TCT-CGG-CCC-CAT-G (Seq. Id. No. 21), CTG-GTT-CGC-CAA-AAA-G
(Seq. Id. No. 22) and AGT-ACG-CAT-CAG-AAA (Seq. Id. No. 23).
Inventors: |
HYLDIG-NIELSEN; Jens J.;
(Hillerod, DK) ; STENDER; Henrik; (Gentofte,
DK) ; OLIVEIRA; Kenneth M.; (Sudbury, MA) ;
RIGBY; Susan; (Acton, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
APPLIED BIOSYSTEMS, LLC |
Carlsbad |
CA |
US |
|
|
Family ID: |
1000005436226 |
Appl. No.: |
17/029575 |
Filed: |
September 23, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15688485 |
Aug 28, 2017 |
10808288 |
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17029575 |
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14571080 |
Dec 15, 2014 |
9765405 |
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15688485 |
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13207283 |
Aug 10, 2011 |
8912312 |
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14571080 |
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10150045 |
May 17, 2002 |
8026051 |
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13207283 |
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60292147 |
May 18, 2001 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 2525/107 20130101;
C07K 14/003 20130101; C12Q 1/689 20130101; C12Q 1/6895
20130101 |
International
Class: |
C12Q 1/6895 20060101
C12Q001/6895; C07K 14/00 20060101 C07K014/00; C12Q 1/689 20060101
C12Q001/689 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OF DEVELOPMENT
[0002] The US Government has certain rights in this invention as
provided for by the terms of the Cooperative Research and
Development Agreement (CRADA) No. 58-3K95-8-631 by and between
Boston Probes, Inc. and the United States Department of
Agriculture.
Claims
1. A PNA probe comprising a probing nucleobase sequence suitable
for detecting, identifying and/or quantitating one or more species
of Candida yeast in a sample.
2. The PNA probe of claim 1, wherein at least a portion of the
probing nucleobase sequence is at least ninety percent homologous
to the nucleobase sequences, or their complements, selected from
the group consisting of: TABLE-US-00010 (Seq. Id. No. 1)
AGA-GAG-CAG-CAT-GCA, (Seq. Id. No. 2) AGA-GAG-CAA-CAT-GCA, (Seq.
Id. No. 3) ACA-GCA-GAA-GCC-GTG, (Seq. Id. No. 4)
CAT-AAA-TGG-CTA-CCA-GA, (Seq. Id. No. 5) CAT-AAA-TGG-CTA-CCC-AG,
(Seq. Id. No. 6) ACT-TGG-AGT-CGA-TAG, (Seq. Id. No. 7)
CCA-AGG-CTT-ATA-CTC-GC, (Seq. Id. No. 8) CCC-CTG-AAT-CGG-GAT, (Seq.
Id. No. 9) GAC-GCC-AAA-GAC-GCC, (Seq. Id. No. 10)
ATC-GTC-AGA-GGC-TAT-AA, (Seq. Id. No. 11) TAG-CCA-GAA-GAA-AGG,
(Seq. Id. No. 12) CAT-AAA-TGG-CTA-GCC-AG, (Seq. Id. No. 13)
CTC-CGA-TGT-GAC-TGC-G, (Seq. Id. No. 14) TCC-CAG-ACT-GCT-CGG, (Seq.
Id. No. 15) TCC-AAG-AGG-TCG-AGA, (Seq. Id. No. 16)
GCC-AAG-CCA-CAA-GGA, (Seq. Id. No. 17) GCC-GCC-AAG-CCA-CA, (Seq.
Id. No. 18) GGA-CTT-GGG-GTT-AG, (Seq. Id. No. 19)
CCG-GGT-GCA-TTC-CA, (Seq. Id. No. 20) ATG-TAG-AAC-GGA-ACT-A, (Seq.
Id. No. 21) GAT-TCT-CGG-CCC-CAT-G, (Seq. Id. No. 22)
CTG-GTT-CGC-CAA-AAA-G, and (Seq. Id. No. 23)
AGT-ACG-CAT-CAG-AAA.
3. The PNA probe of claim 1, wherein the probe is unlabeled.
4. The PNA probe of claim 1, wherein the probe is labeled with at
least one detectable moiety.
5.-7. (canceled)
8. The PNA probe of claim 1, wherein the probe is support
bound.
9. The PNA probe of claim 1, wherein the probe is
self-indicating.
10. The PNA probe of claim 1, wherein in situ hybridization is used
to detect, identify or quantitate one or more 11. A PNA probe set
suitable for detecting, identifying or quantitating one or more
species of Candida yeast in a sample wherein at least one species
of interest is selected from the group consisting of: C. albicans,
C. dubliniensis, C. krusei, C. glabrata, C. parapsilosis and C.
tropicalis.
11. A PNA probe set suitable for detecting, identifying or
quantitating one or more species of Candida yeast in a sample
wherein at least one species of interest is selected from the group
consisting of: C. cilbicans, C. dtthliniensis, C. krusei, C.
glabrata, C. parapsilosis and C. tropicalis.
12. The probe set of claim 11, wherein at least one PNA probe of
the set comprises a probing nucleobase sequence wherein at least a
portion is at least ninety percent homologous to the nucleobase
sequences, or their complements, selected from the group consisting
of: AGA-GAG-CAG-CAT-GCA (Seq. Id. No. 1), AGA-GAG-CAA-CAT-GCA (Seq.
Id. No. 2), ACA-GCA-GAA-GCC-GTG (Seq. Id. No. 3),
CAT-AAA-TGG-CTA-CCA-GA (Seq. Id. No. 4), CAT-AAA-TGG-CTA-CCC-AG
(Seq. Id. No. 5), ACT-TGG-AGT-CGA-TAG (Seq. Id. No. 6),
CCA-AGG-CTT-ATA-CTC-GC (Seq. Id. No. 7), CCC-CTG-AAT-CGG-GAT (Seq.
Id. No. 8), GAC-GCC-AAA-GAC-GCC (Seq. Id. No. 9),
ATC-GTC-AGA-GGC-TAT-AA (Seq. Id. No. 10), TAG-CCA-GAA-GAA-AGG (Seq.
Id. No. 11), CAT-AAA-TGG-CTA-GCC-AG (Seq. Id. No. 12),
CTC-CGA-TGT-GAC-TGC-G (Seq. Id. No. 13), TCC-CAG-ACT-GCT-CGG (Seq.
Id. No. 14), TCC-AAG-AGG-TCG-AGA (Seq. Id. No. 15),
GCC-AAG-CCA-CAA-GGA (Seq. Id. No. 16), GCC-GCC-AAG-CCA-CA (Seq. Id.
No. 17), GGA-CTT-GGG-GTT-AG (Seq. Id. No. 18), CCG-GGT-GCA-TTC-CA
(Seq. Id. No. 19), ATG-TAG-AAC-GGA-ACT-A (Seq. Id. No. 20),
GAT-TCT-CGG-CCC-CAT-G (Seq. Id. No. 21), CTG-GTT-CGC-CAA-AAA-G
(Seq. Id. No. 22) and AGT-ACG-CAT-CAG-AAA (Seq. Id. No. 23).
13. The probe set of claim 11, wherein a PNA probe for C. albicans
is selected from the group consisting of: AGA-GAG-CAG-CAT-GCA (Seq.
Id. No. 1), AGA-GAG-CAA-CAT-GCA (Seq. Id. No. 2),
ACA-GCA-GAA-GCC-GTG (Seq. Id. No. 3), CAT-AAA-TGG-CTA-CCA-GA (Seq.
Id. No. 4), CAT-AAA-TGG-CTA-CCC-AG (Seq. Id. No. 5),
ACT-TGG-AGT-CGA-TAG (Seq. Id. No. 6), CCA-AGG-CTT-ATA-CTC-GC (Seq.
Id. No. 7) and CCC-CTG-AAT-CGG-GAT (Seq. Id. No. 8).
14. The probe set of claim 11, wherein a PNA probe for C.
dubliniesis is selected from the group consisting of:
TAG-CCA-GAA-GAA-AGG (Seq. Id. No. 11), CAT-AAA-TGG-CTA-GCC-AG (Seq.
Id. No. 12), CTC-CGA-TGT-GAC-TGC-G (Seq. Id. No. 13) and
TCC-CAG-ACT-GCT-CGG (Seq. Id. No. 14).
15. The probe set of claim 11, wherein a PNA probe for C. glabrata
is selected from the group consisting of: TCC-AAG-AGG-TCG-AGA (Seq.
Id. No. 15), GCC-AAG-CCA-CAA-GGA (Seq. Id. No. 16),
GCC-GCC-AAG-CCA-CA (Seq. Id. No. 17), GGA-CTT-GGG-GTT-AG (Seq. Id.
No. 18) and CCG-GGT-GCA-TTC-CA (Seq. Id. No. 19).
16. The probe set of claim 11, wherein a PNA probe for C. krusei is
selected from the group consisting of: ATG-TAG-AAC-GGA-ACT-A (Seq.
Id. No. 20) and GAT-TCT-CGG-CCC-CAT-G (Seq. Id. No. 21).
17. The probe set of claim 11, wherein at least one probe of the
set is unlabeled.
18.-26. (canceled)
27. The probe set of claim 11, wherein at least one probe of the
set is support bound.
28. A method for detecting, identifying and/or quantitating one or
more species of Candida yeast in a sample; said method comprising
the steps of: a) contacting the sample with one or more PNA probes,
wherein the one or more PNA probes have a probing nucleobase
sequence that is at least ninety percent homologous to the
nucleobase sequences, or their complements, selected from the group
consisting of: AGA-GAG-CAG-CAT-GCA, (Seq. Id. No. 1)
AGA-GAG-CAA-CAT-GCA, (Seq. Id. No. 2) ACA-GCA-GAA-GCC-GTG, (Seq.
Id. No. 3) CAT-AAA-TGG-CTA-CCA-GA, (Seq. Id. No. 4)
CAT-AAA-TGG-CTA-CCC-AG, (Seq. Id. No. 5) ACT-TGG-AGT-CGA-TAG, (Seq.
Id. No. 6) CCA-AGG-CTT-ATA-CTC-GC, (Seq. Id. No. 7)
CCC-CTG-AAT-CGG-GAT, (Seq. Id. No. 8) GAC-GCC-AAA-GAC-GCC, (Seq.
Id. No. 9) ATC-GTC-AGA-GGC-TAT-AA, (Seq. Id. No. 10)
TAG-CCA-GAA-GAA-AGG, (Seq. Id. No. 11) CAT-AAA-TGG-CTA-GCC-AG,
(Seq. Id. No. 12) CTC-CGA-TGT-GAC-TGC-G, (Seq. Id. No. 13)
TCC-CAG-ACT-GCT-CGG, (Seq. Id. No. 14) TCC-AAG-AGG-TCG-AGA, (Seq.
Id. No. 15) GCC-AAG-CCA-CAA-GGA, (Seq. Id. No. 16)
GCC-GCC-AAG-CCA-CA, (Seq. Id. No. 17) GGA-CTT-GGG-GTT-AG, (Seq. Id.
No. 18) CCG-GGT-GCA-TTC-CA, (Seq. Id. No. 19)
ATG-TAG-AAC-GGA-ACT-A, (Seq. Id. No. 20) GAT-TCT-CGG-CCC-CAT-G,
(Seq. Id. No. 21) CTG-GTT-CGC-CAA-AAA-G (Seq. Id. No. 22) and
AGT-ACG-CAT-CAG-AAA; (Seq. Id. No. 23) and b) detecting, identify
and/or quantitating hybridization of the probing nucleobase
sequence of a PNA probe to the target sequence of an organism of
interest in the sample, under suitable hybridization conditions or
suitable in-situ hybridization conditions, and correlating the
result with the presence, absence or quantity of Candida yeast in
the sample.
29. The method of claim 28, wherein at least one of the PNA probes
is unlabeled.
30.-41. (canceled)
42. The method of claim 29, wherein at least one PNA probe is
support bound.
43.-56. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/292,147 filed on May 18, 2001.
BACKGROUND OF THE INVENTION
1. Field of the Invention
[0003] This invention is related to the field of probe-based
detection, analysis and/or quantitation of microorganisms. More
specifically, this invention relates to novel PNA probes, probe
sets, methods and kits pertaining for the detection, identification
and/or enumeration of organisms of the various species of the
Candida genus.
2. Description of the Related Art
[0004] Nucleic acid hybridization is a fundamental process in
molecular biology. Probe-based assays are useful in the detection,
quantitation and/or analysis of nucleic acids. Nucleic acid probes
have long been used to analyze samples for the presence of nucleic
acid from bacteria, fungi, virus or other organisms and are also
useful in examining genetically-based disease states or clinical
conditions of interest. Nonetheless, probe-based assays have been
slow to achieve commercial success. This lack of commercial success
is, at least partially, the result of difficulties associated with
specificity, sensitivity and reliability.
[0005] Despite its name, Peptide Nucleic Acid (PNA) is neither a
peptide, a nucleic acid nor is it an acid. Peptide Nucleic Acid
(PNA) is a non-naturally occurring polyamide that can hybridize to
nucleic add (DNA and RNA) with sequence specificity (See: U.S. Pat.
No. 5,539,082 and Egholm et al., Nature 365: 566-568 (1993)). Being
a non-naturally occurring molecule, unmodified PNA is not known to
be a substrate for the enzymes that are known to degrade peptides
or nucleic acids. Therefore, PNA should be stable in biological
samples, as well as have a long shelf-life. Unlike nucleic add
hybridization, which is very dependent on ionic strength, the
hybridization of a PNA with a nucleic acid is fairly independent of
ionic strength and is favored at low ionic strength, conditions
that strongly disfavor the hybridization of nucleic acid to nucleic
acid (Egholm et al., Nature, at p. 567). The effect of ionic
strength on the stability and conformation of PNA complexes has
been extensively investigated (Tomac et al., J. Am. Chem. Soc.
118:55 44-5552 (1996)). Sequence discrimination is more efficient
for PNA recognizing DNA than for DNA recognizing DNA (Egholm et
al., Nature, at p. 566). However, the advantages in point mutation
discrimination with PNA probes, as compared with DNA probes, in a
hybridization assay, appears to be somewhat sequence dependent
(Nielsen et al., Anti-Cancer Drug Design 8:53-65, (1993) and Weiler
et al., Nucl. Acids Res. 25: 2792-2799 (1997)).
[0006] Though they hybridize to nucleic add with sequence
specificity (See: Egholm et al., Nature, at p. 567), PNAs have been
slow to achieve commercial success at least partially due to cost,
sequence specific properties/problems associated with solubility
and self-aggregation (See: Bergman, F., Bannwarth, W. and Tam, S.,
Tett. Lett. 36:6823-6826 (1995), Haaima, G., Lohse, A., Buchardt,
O. and Nielsen, P. E., Angew. Chem. Int., Ed. Engl. 35:1939-4942
(1996) and Lesnik, E., Hassman, F., Barbeau, J., Teng, K. and
Weiler, K., Nucleosides & Nucleotides 16:1775-1779 (1997) at p
433, col. 1, ln. 28 through col. 2, ln. 3) as well as the
uncertainty pertaining to non-specific interactions that might
occur in complex-systems such as a cell (See: Good, L. et al.,
Antisense & Nucleic Acid Drug Development 7:431-437 (1997)).
However, problems associated with solubility and self-aggregation
have been reduced or eliminated (See: Gildea et al., Tett. Lett.
39: 7255-7258 (1998)). Nevertheless, their unique properties
clearly demonstrate that PNA is not the equivalent of a nucleic
acid in either structure or function. Consequently, PNA probes
should be evaluated for performance and optimization to thereby
confirm whether they can be used to specifically and reliably
detect a particular nucleic acid target sequence, particularly when
the target sequence exists in a complex sample such as a cell,
tissue or organism.
SUMMARY OF THE INVENTION
[0007] This invention is directed to PNA probes, probe sets,
methods and kits useful for detecting, identifying and/or
quantitating Candida yeast in a sample. The PNA probes, probe sets,
methods and kits of this invention are suitable for the analysis of
nucleic acid, whether or not it is present within an organism of
interest. Accordingly, this invention can be used for both the
analysis of organisms or for the analysis of nucleic acid extracted
from or derived from an organism of interest.
[0008] Generally, this invention can be particularly useful for the
determination of particular species of the Candida genus. The PNA
probes and probe sets of this invention comprise probing nucleobase
sequences that are particularly useful for the specific detection
of certain species of Candida, including C. albicans (also
comprising C. stellatoidea, a biovar of C. albicans), C.
dubliniensis, C. krusei, C. glabrata, C. parapsilosis and C.
tropicalis. A particularly useful probing nucleobase sequence is
Seq. Id. No. 1 (See: Table 1) because it can be highly specific for
C. albicans, a pathogen that is particularly important to determine
as early as possible in the area of blood culture analysis.
Exemplary probing nucleobase sequences for the probes of this
invention are listed in Table 1, below. The species of Candida for
which each probe is intended to determine is also listed in the
Table.
[0009] This invention is further directed to a method suitable for
detecting, identifying and/or quantitating a species of Candida in
a sample. For example, the method can be directed to the detection
of a particular species of Candida wherein the species is selected
from the group consisting of: C. albicans, C. dubliniensis, C.
krusei, C. glabrata, C. parapsilosis and C. tropicalis.
[0010] The method can comprise contacting the sample with one or
more PNA probes, wherein suitable probes are described herein.
According to the method, the presence, absence and/or number of the
one or more species of Candida in the sample is then detected,
identified and/or quantitated. Detection, identification and/or
quantitation is made possible by correlating the hybridization,
under suitable hybridization conditions or suitable in-situ
hybridization conditions, of the probing nucleobase sequence of a
PNA probe to the target sequence with the presence, absence and/or
quantity of target organism in the sample. This correlation is made
possible by direct or indirect determination of the probe/target
sequence hybrid.
[0011] In yet another embodiment, this invention is directed to
kits suitable for performing an assay that determines the presence,
absence and/or quantity of a species of Candida in a sample. The
kits of this invention comprise one or more PNA probes and other
reagents or compositions that are selected to perform an assay or
otherwise simplify the performance of an assay.
[0012] The PNA probes, probe sets, methods and kits of this
invention have been demonstrated to be relatively specific for the
species of Candida for which they are intended to determine.
Moreover, the assays described herein are rapid (2-3 hours or
less), sensitive, reliable and capable, in a single assay, of
identification as well as detection and/or enumeration of the
organisms listed in Table 1.
[0013] The PNA probes, probe sets, methods and kits of this
invention can be particularly useful for the determination of
Candida in food, beverages, water, pharmaceutical products,
personal care products, dairy products and/or environmental
samples. The analysis of beverages includes soda, bottled water,
fruit juice, beer, wine or liquor products. Suitable PNA probes,
probe sets, methods and kits can be particularly useful for the
analysis of raw materials, equipment, products or processes used to
manufacture or store food, beverages, water, pharmaceutical
products, personal care products dairy products or for the analysis
of environmental samples.
[0014] Additionally, the PNA probes, probe sets, methods and kits
of this invention can be particularly useful for the detection of
Candida species in clinical samples and clinical environments. By
way of a non-limiting example, the PNA probes, probe sets, methods
and kits of this invention can be particularly useful in the
analysis of blood cultures or in samples derived therefrom. (e.g.
subcultures). Other non-limiting examples of clinical samples
include: sputum, laryngeal swabs, gastric lavage, bronchial
washings, biopsies, aspirates, expectorates, body fluids (e.g.
spinal, pleural, pericardial, synovial, blood, pus, amniotic, and
urine), bone marrow and tissue sections (including cultures and
subcultures derived therefrom). Suitable PNA probes, probe sets,
methods and kits will also be particularly useful for the analysis
of clinical specimens, equipment, fixtures or products used to
treat humans or animals.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIGS. 1 comprises 5 images that were obtained using a
fluorescent microscope using a FITC/Texas red filter for five
culture samples, each culture sample representing a different
species of Candida, wherein all samples have been treated with the
PNA probe Can26S03 having sequence Id No. 1 as the probing
nucleobase sequence. Only the sample containing C. albicans is
positive thereby demonstrating that the probe is highly specific
for C. albicans and can be used to distinguish numerous other
Candida species, including the difficult to distinguish, C.
dubliniensis.
DETAILED DESCRIPTION OF THE INVENTION
1. Definitions:
[0016] a. As used herein, "nucleobase" means those naturally
occurring and those non-naturally occurring heterocyclic moieties
commonly known to those who utilize nucleic acid technology or
utilize peptide nucleic acid technology to thereby generate
polymers that can sequence specifically bind to nucleic acids.
Non-limiting examples of suitable nucleobases include: adenine,
cytosine, guanine, thymine, uracil, 5-propynyl-uracil,
2-thio-5-propynyl-uracil, 5-methylcytosine, pseudoisocytosine,
2-thiouracil and 2-thiothymine, 2-aminopurine,
N9-(2-amino-6-chloropurine), N9-(2,6-diaminopurine), hypoxanthine,
N9-(7-deaza-guanine), N9-(7-deaza-8-aza-guanine) and
N8-(7-deaza-8-aza-adenine). Other non-limiting examples of suitable
nucleobase include those nucleobases illustrated in FIGS. 2(A) and
2(B) of Buchardt et al. of U.S. Pat. No. 6,357;163 (incorporated
herein by reference). [0017] b. As used herein, "nucleobase
sequence" means any segment, or aggregate of two or more segments
of a polymer that comprises nucleobase-containing subunits.
Non-limiting examples of suitable polymers include
oligodeoxynucleotides (e,g. DNA), oligoribonucleotides (e.g. RNA),
peptide nucleic acids (PNA), PNA chimeras, PNA oligomers, nucleic
acid analogs and/or nucleic acid mimics. [0018] c. As used herein,
"target sequence" is: a nucleobase sequence of a polynucleobase
strand sought to be determined. The target sequence can be a
subsequence of the rRNA of Candida yeast. [0019] d. As used herein,
"polynucleobase strand" means a complete single polymer strand
comprising nucleobase subunits, [0020] e. As used herein, "nucleic
acid" is a nucleobase sequence-containing polymer, or
polynucleobase strand, having a backbone formed from nucleotides,
or analogs thereof. Preferred nucleic acids are DNA and RNA. For
the avoidance of any doubt, PNA is a nucleic acid mimic and not a
nucleic acid analog. [0021] f. As used herein, "peptide nucleic
acid" or "PNA" means any oligomer or polymer segment comprising two
or more PNA subunits (residues), including, but not limited to, any
of the oligomer or polymer segments referred to or claimed as
peptide nucleic acids in U.S. Pat. Nos. 5,539,082, 5,527,675,
5,623,049, 5,714,331, 5,718,262, 5,736,336, 5,773,571, 5,766,855,
5,786,461, 5,837,459, 5,891,625, 5,972,610, 5,986,053, 6,107,470
and 6,357,163; all of which are herein incorporated by reference.
The term "peptide nucleic acid" or "PNA" shall also apply to any
oligomer or polymersegment comprising two or more subunits of those
nucleic acid mimics described in the following publications:
Lagriffoul et al., Bioorganic & Medicinal Chemistry Letters, 4:
1081-1082 (1994); Petersen et al., Bioorganic & Medicinal
Chemistry Letters, 6: 793-796 (1996); Diderichsen et al., Tett,
Lett. 37: 475-478 (1996); Fujii et at., Bioorg. Med. Chem, Lett. 7:
637-627 (1997); Jordan et. at., Bioorg. Med. Chem. Lett. 7: 687-690
(1997); Krotz et al., Tett. Lett. 36: 6941-6944 (1995); Lagriffoul
et al., Bioorg. Med. Chem. Lett. 4: 1081-1082 (1994); Diederichsen,
U., Bioorganic & Medicinal Chemistry Letters, 7: 1743-1746
(1997); Lowe et al., J Chem. Soc. Perkin Trans. 1, (1997) 1:
539-546; Lowe et al., J. Chem. Soc. Perkin Trans. 11: 547-554.
(1997); Lowe et al., J. Chem. Soc. Perkin. Trans. 1 1:5 55-560
(1997); Howarth et al., J. Org. Chem. 62: 5441-5450 (1997);
Altmann, K-H et al., Bioorganic & Medicinal Chemistry Letters,
7: 1119-1122 (1997); Diederichsen, U., Bioorganic & Med. Chem.
Lett., 8: 165-168 (1998); Diederichsen et al., Angew. Chem. Int.
Ed. 37: 302-305 (1998); Cantin et al., Tett. Lett., 38: 4211-4214
(1997); Ciapetti et al., Tetrahedron, 53: 1167-1176 (1997);
Lagriffoule et al., Chem. Eur. J., 3: 912-919 (1997); Kumar et al.,
Organic Letters 3(9): 1269-1272 (2001); and the Peptide-Based
Nucleic Acid Mimics (PENAMs) of Shah et al. as disclosed in
WO96/04000.
[0022] In certain embodiments, a "peptide nucleic acid" or "PNA" is
an oligomer or polymer segment comprising two or more covalently
linked subunits of the formula:
##STR00001##
wherein, each J is the same or different and is selected from the
group consisting of H, R.sup.1, OR.sup.1, SR.sup.1, NHR.sup.1,
NR.sup.1.sub.2, F, Cl, Br and I. Each K is the same or different
and is selected from the group consisting of O, S, NH and NR.sup.1.
Each R.sup.1 is the same or different and is an alkyl group having
one to five carbon atoms that may optionally contain a heteroatom
or a substituted or unsubstituted aryl group. Each A is selected
from the group consisting of a single bond, a group of the formula;
--(CJ.sub.2).sub.3-- and a group of the formula;
--(CJ.sub.2).sub.3C(O)--, wherein, J is defined above and each s is
a whole number from one to five. Each t is 1 or 2 and each u is 1
or 2. Each L is the same or different and is independently selected
from: adenine, cytosine, guanine, thymine, uracil,
5-propynyl-uracil, 2-thio-5-propynyl-uracil, 5-methylcytosine,
pseudoisocytosine, 2-thiouracil and 2-thiothymine, 2-aminopurine,
N9-(2-amino-6chloropurine), N9-(2,6-diaminopurine), hypoxanthine,
N9-(7-deaza-guanine), N9-(7-deaza-8-aza-guanine) and
N8-(7-deaza-8-aza-adenine), other naturally occurring nucleobase
analogs or other non-naturally occurring nucleobases.
[0023] In certain other embodiments, a FNA subunit consists of a
naturally occurring or non-naturally occurring nucleobase attached
to the N-.alpha.-glycine nitrogen of the N-[2-(aminoethyl)]glycine
backbone through a methylene carbonyl linkage; this currently being
the most commonly used form of a peptide nucleic acid subunit.
[0024] g. As used herein, the terms "label", "reporter moiety" or
"detectable moiety" are interchangeable and refer to moieties that
can be attached to PNA oligomer or antibody, or otherwise be used
in a reporter system, to thereby render the oligomer or antibody
detectable by an instrument or method. For example, a label can be
any moiety that: (i) provides a detectable signal; (ii) interacts
with a second label to modify the detectable signal provided by the
first or second label; or (iii) confers a capture function, i.e.
hydrophobic affinity, antibody/antigen, ionic complexation. [0025]
h. As used herein, "sequence specifically" means hybridization by
base pairing through hydrogen bonding. Non-limiting examples of
standard base pairing includes adenine base pairing with thymine or
uracil and guanine base pairing with cytosine. Other non-limiting
examples of base-pairing motifs include, but are not limited to:
adenine base pairing with any of: 5-propynyl-uracil,
2-thio-5-propynyl-uracil, 2-thiouracil or 2-thiothymine; guanine
base pairing with any of: 5-methylcytosine or pseudoisocytosine;
cytosine base pairing with any of: hypoxanthine,
N9-(7-deaza-guanine) or N9-(7-deaza-8-aza-guanine); thymine or
uracil base pairing with any of: 2-aminopurine,
N9-(2-amino-6-chloropurine) or N9-(2,6-diaminopurine); and
N8-(7-deaza-8-aza-adenine), being a universal base, base pairing
with any other nucleobase, such as for example any of: adenine,
cytosine, guanine, thymine, uracil, 5-propynyl-uracil,
2-thio-5-propynyl-uracil, 5-methylcytosine, pseudoisocytosine,
2-thiouracil and 2-thiothymine, 2-aminopurine,
N9-(2-amino-6-chloropurine), N9-(2,6-diaminopurine), hypoxanthine,
N9-(7-deaza-guanine) or N9-(7-deaza-8-aza-guanine) (See: Seela et
al., Nucl. Acids, Res.: 28(17): 3224-3232 (2000)). [0026] i. As
used herein, the term "chimera" or "chimeric oligomer" means an
oligomer comprising two or more linked subunits that are selected
from different classes of subunits. For example, a PNA/DNA chimera
would comprise at least two PNA subunits linked to at least one
2'-deoxyribonucleic acid subunit (For exemplary methods and
compositions related to PNA/DNA chimera preparation See:
WO96/40709). Exemplary component subunits of the chimera are
selected from the group consisting of PNA subunits, naturally
occurring amino acid subunits, DNA subunits, RNA subunits and
subunits of analogues or mimics of nucleic acids. [0027] j. As used
herein, the term "linked polymer" means a polymer comprising two or
more polymer segments which are linked by a linker. The polymer
segments that are linked to form the linked polymer are selected
from the group consisting of an oligodeoxynucleotide, an
oligoribonucleotide, a peptide, a polyamide, a peptide nucleic acid
(PNA) and a chimera. [0028] k. As used herein "solid support" or
"solid carrier" means any solid phase material upon which a
oligomer is synthesized, attached, ligated or otherwise
immobilized. Solid support encompasses terms such as "resin",
"solid phase", "surface" and "support". A solid support may be
composed of organic polymers such as polystyrene, polyethylene,
polypropylene, polyfluoroethylene, polyethyleneoxy, and
polyacrylamide, as well as co-polymers and grafts thereof. A solid
support may also be inorganic, such as glass, silica,
controlled-pore-glass (CPG), or reverse-phase silica. The
configuration of a solid support may be in the form of beads,
spheres, particles, granules, a gel, or a surface. Surfaces may be
planar, substantially planar, or non-planar. Solid supports may be
porous or non-porous, and may have swelling or non-swelling
characteristics. A solid support may be configured in the form of a
well, depression or other container, vessel, feature or location. A
plurality of solid supports may be configured in an array at
various locations, addressable for robotic delivery of reagents, or
by detection means including scanning by laser illumination and
confocal or deflective light gathering. [0029] l. As used herein,
"support bound" means immobilized on or to a solid support. It is
understood that immobilization can occur by any means, including
for example; by covalent attachment, by electrostatic
immobilization, by attachment through a ligand/ligand interaction,
by contact or by depositing on the surface.
2. Description
I. General
PNA Synthesis:
[0030] Methods for the chemical assembly of PNAs are well known
(See: U.S. Pat. Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331,
5,718,262, 5,736,336, 5,773,571, 5,766,855, 5,786,461, 5,837,459,
5,891,625, 5,972,610, 5,986,053 an 6,107,470; all of which are
herein incorporated by reference (Also see: PerSeptive Biosystems
Product Literature)). As a general reference for PNA synthesis
methodology also please see: Nielsen et al., Peptide Nucleic Acids;
Protocols and Applications, Horizon Scientific Press, Norfolk
England (1999).
[0031] Chemicals and instrumentation for the support bound
automated chemical assembly of peptide nucleic acids are now
commercially available, Both labeled and unlabeled PNA oligomers
are likewise available from commercial vendors of custom PNA
oligomers. Chemical assembly of a PNA is analogous to solid phase
peptide synthesis, wherein at each cycle of assembly the oligomer
possesses a reactive alkyl amino terminus that can be condensed
with the next synthon to be added to the growing polymer. Because
standard peptide chemistry is utilized, natural and non-natural
amino acids can be routinely incorporated into a PNA oligomer.
Because a PNA is a polyamide, it has a C-terminus (carboxyl
terminus) and an N-terminus (amino terminus). For the purposes of
the design of a hybridization probe suitable for antiparallel
binding to the target sequence (the preferred orientation), the
N-terminus of the probing nucleobase sequence of the PNA probe is
the equivalent of the 5'-hydroxyl terminus of an equivalent DNA or
RNA oligonucleotide.
PNA Labeling:
[0032] Non-limiting methods for labeling PNAs are described in.
U.S. Pat. No. 6,110,676, U.S. Pat. No. 6,361,942, U.S. Pat. No.
6,355,421 (all incorporated herein by reference), WO99/21881, the
examples section of this specification or are otherwise well known
in the art of PNA synthesis. Other non-limiting examples for
labeling PNAs are also discussed in Nielsen et al., Peptide Nucleic
Acids; Protocols and Applications, Horizon Scientific Press,
Norfolk England (1999).
Labels:
[0033] Non-limiting examples of detectable moieties (labels) that
can be used to label PNA probes or antibodies used in the practice
of this invention can include a dextran conjugate, a branched
nucleic acid detection system, a chromophore, a fluorophore, a spin
label, a radioisotope, an enzyme, a hapten, an acridinium ester or
a chemiluminescent compound. Other suitable labeling reagents and
preferred methods of attachment would be recognized by those of
ordinary skill in the art of PNA, peptide or nucleic acid
synthesis.
[0034] Non-limiting examples of haptens include
5(6)-carboxyfluorescein, 2,4-dinitrophenyl, digoxigenin, and
biotin.
[0035] Non-limiting examples of fluorochromes (fluorophores)
include 5(6)-carboxyfluorescein (Flu),
6-((7-amino-4-methylcournarin-3-acetyl)amino)hexanoic acid (Cou),
5(and 6)-carboxy-X-rhodamine (Rox), Cyanine 2 (Cy2) Dye, Cyanine 3
(Cy3) Dye, Cyanine 3.5 (Cy3.5) Dye, Cyanine 5 (Cy5) Dye, Cyanine
5.5 (Cy5,5) Dye Cyanine 7 (Cy7) Dye, Cyanine 9 (Cy9) Dye (Cyanine
dyes 2, 3, 3.5, 5 and 5.5 are available as NHS esters from
Amersham, Arlington Heights, Ill.) or the Alexa dye series
(Molecular Probes, Eugene, Oreg.).
[0036] Non-limiting examples of enzymes include polymerases (e.g.
Taq polymerase, Klenow PNA polymerase, T7 DNA polymerase,
Sequenase, DNA polymerase 1 and phi29 polymerase), alkaline
phosphatase (AP), horseradish peroxidase (HRP), soy bean peroxidase
(SBP)), ribonuclease and protease.
Energy Transfer
[0037] In one embodiment, PNA oligomers can be labeled with an
energy transfer set. For energy transfer to be useful in
determining hybridization, there should be an energy transfer set
comprising at least one energy transfer donor and at least one
energy transfer acceptor moiety. Often, the energy transfer set
will include a single donor moiety and a single acceptor moiety,
but this is not a limitation. An energy transfer set may contain
more than one donor moiety and/or more than one acceptor moiety.
The donor and acceptor moieties operate such that one or more
acceptor moieties accept energy transferred from the one or more
donor moieties or otherwise quench the signal from the donor moiety
or moieties. Thus, in one embodiment, both the donor moiety(ies)
and acceptor moiety(ies) are fluorophores. Though the previously
listed fluorophores (with suitable spectral properties) might also
operate as energy transfer acceptors, the acceptor moiety cart also
be a non-fluorescent quencher moiety such as
4-((-4-(dimethylamino)phenyl)azo) benzoic acid (dabcyl). The labels
of the energy transfer set can be linked at the oligomer termini or
linked at a site within the oligomer. For example, each of two
labels of an energy transfer set can be linked at the distal-most
termini of the oligomer.
[0038] Transfer of energy between donor and acceptor moieties may
occur through any energy transfer process, such as through the
collision of the closely associated moieties of an energy transfer
set(s) or through a non-radiative process such as fluorescence
resonance energy transfer (FRET). For FRET to occur, transfer of
energy between donor and acceptor moieties of a energy transfer set
requires that the moieties be close in space and that the emission
spectrum of a donor(s) have substantial overlap with the absorption
spectrum of the acceptor(s) (See: Yaron et al. Analytical
Biochemistry, 95: 228-235 (1979) and particularly page 232, col. 1
through page 234, col. 1). Alternatively, collision mediated
(radiationless) energy transfer may occur between very closely
associated donor and acceptor moieties whether or not the emission
spectrum of a donor moiety(ies) has a substantial overlap with the
absorption spectrum of the acceptor moiety(ies) (See: Yaron et al.,
Analytical Biochemistry, 95: 228-235 (1979) and particularly page
229, col. 1 through page 232, col. 1). This process is referred to
as intramolecular collision since it is believed that quenching is
caused by the direct contact of the donor and acceptor moieties
(See: Yaron et al). It is to be understood that any reference to
energy transfer in the instant application encompasses all of these
mechanistically distinct phenomena. It is also to be understood
that energy transfer can occur though more than one energy transfer
process simultaneously and that the change in detectable signal can
be a measure of the activity of two or more energy transfer
processes. Accordingly, the mechanism of energy transfer is not a
limitation of this invention.
Detecting Energy Transfer In A Self-Indicating PNA Oligomer:
[0039] When labeled with an energy transfer set, we refer to the
PNA oligomer as being self-indicating. In one embodiment, a
self-indicating PNA oligomer can be labeled in a manner that is
described in co-pending and commonly owned patent application U.S.
Ser. No. 09/179,162 (now allowed), entitled: "Methods, Kits And
Compositions Pertaining To Linear Beacons" and the related PCT
application which has also now published as WO99/21881, both of
which are hereby incorporated by reference,
[0040] Hybrid formation between a self-indicating oligomer and a
target sequence can be monitored by measuring at least one physical
property of at least one member of the energy transfer set that is
detectably different when the hybridization complex is formed as
compared with when the oligomer exists in a non-hybridized state.
We refer to this phenomenon as the self-indicating property of the
oligomer. This change in detectable signal results from the change
in efficiency of energy transfer between donor and acceptor
moieties caused by hybridization of the oligomer to the target
sequence.
[0041] For example, the means of detection can involve measuring
fluorescence of a donor or acceptor fluorophore of an energy
transfer set. In one embodiment, the energy transfer set may
comprise at least one donor fluorophore and at least one acceptor
(fluorescent or non-fluorescent) quencher such that the measure of
fluorescence of the donor fluorophore can be used to detect,
identify or quantitate hybridization of the oligomer to the target
sequence. For example, there may be a measurable increase in
fluorescence of the donor fluorophore upon the hybridization of the
oligomer to a target sequence.
[0042] In another embodiment, the energy transfer set comprises at
least one donor fluorophore and at least one acceptor fluorophore
such that the measure of fluorescence of either, or both, of at
least one donor moiety or one acceptor moiety can be used to can be
used to detect, identify or quantitate hybridization of the
oligomer to the target sequence.
[0043] Self-indicating PNA oligomers can be used in in-situ
hybridization assays. However, certain self-indicating PNA
oligomers are particularly well suited for the analysis of nucleic
acid amplification reactions (e.g. PCR) either in real-time or at
the end point (See: WO99/21881).
Determining Energy Transfer In A Detection Complex:
[0044] In another embodiment, the PNA oligomers of the present
invention are labeled solely with a quencher moiety and can be used
as a component oligomer in a Detection Complex as more fully
explained in U.S. Pat. No. 6,361,942, entitled: "Methods, Kits And
Compositions Pertaining To Detection Complexes", herein
incorporated by reference. When the Detection Complex is formed, at
least one donor moiety of one component polymer is brought
sufficiently close in space to at least one acceptor moiety of a
second component polymer. Since the donor and acceptor moieties of
the set are closely situated in space, transfer of energy occurs
between moieties of the energy transfer set. When the Detection
Complex dissociates, as for example when one of the component
polymers of the Detection Complex hybridizes to a target sequence,
the donor and acceptor moieties do not interact sufficiently to
cause substantial transfer of energy from the donor and acceptor
moieties of the energy transfer set and there is a correlating
change in detectable signal from the donor and/or acceptor moieties
of the energy transfer set. Consequently, Detection Complex
formation/dissociation can be determined by measuring at least one
physical property of at least one member of the energy transfer set
that is detectably different when the complex is formed as compared
with when the component polymers of the Detection Complex exist
independently and unassociated.
Detectable and Independently Detectable Moieties/Multiplex
Analysis:
[0045] A multiplex hybridization assay can be performed in
accordance with this invention. In a multiplex assay, numerous
conditions of interest cart be simultaneously examined. Multiplex
analysis relies on the ability to sort sample components or the
data associated therewith, during or after the assay is completed.
In preferred embodiments of the invention, one or more distinct
independently detectable moieties can be used to label two or more
different probes used in an assay. The ability to differentiate
between and/or quantitate each of the independently detectable
moieties provides the means to multiplex a hybridization assay
because the data that correlates with the hybridization of each of
the distinctly (independently) labeled probe to a particular
nucleic acid sequence can be correlated with the presence, absence
or quantity of each organism sought to be detected in the sample.
Consequently, the multiplex assays of this invention can be used to
simultaneously detect the presence, absence or quantity of two or
more different organisms (e.g. species of Candida) in the same
sample and in the same assay. For example, a multiplex assay may
utilize two or more PNA probes, each being labeled with an
independently detectable fluorophore, or a set of independently
detectable fluorophores.
Spacer/Linker Moieties:
[0046] Generally, spacers are used to minimize the adverse effects
that bulky labeling reagents might have on hybridization properties
of probes. Linkers typically induce flexibility and randomness into
the probe or otherwise link two or more nucleobase sequences of a
probe or component polymer, Preferred spacer/linker moieties for
the nucleobase polymers of this invention consist of one or more
aminoalkyl carboxylic acids (e.g. aminocaproic acid) the side chain
of an amino acid (e.g. the side chain of lysine or ornithine),
natural amino adds (e.g. glycine), aminooxyalkylacids (e.g.
8-amino-3,6-dioxaoctanoic acid), alkyl diacids (e.g. succinic
acid), alkyloxy diacids (e.g. diglycolic acid) or alkyldiamines
(e.g. 1,8-diamino-3,6-dioxaoctane). Spacer/linker moieties may also
incidentally or intentionally be constructed to improve the water
solubility of the probe (For example see: Gildea et al., Tett.
Left. 39: 7255-7258 (1998)).
[0047] For example, a spacer/tinker moiety can comprise one or more
linked compounds having the formula:
--Y--(O.sub.m--(CW.sub.2).sub.n).sub.o--Z--. The group Y is
selected from the group consisting of: a single bond,
--(CW.sub.2).sub.p--, --C(O)(CW.sub.2).sub.p--,
--C(S)(CW.sub.2).sub.p-- and --S(O.sub.2)(CW.sub.2).sub.p--. The
group Z has the formula NH, NR.sup.2, S or O. Each W is
independently H, R.sup.2, --OR.sup.2, F, Cl, Br or I; wherein, each
R .sup.2 is independently selected from the group consisting of:
--CX.sub.3, --CX.sub.2CX.sub.3, --CX.sub.2CX.sub.2CX.sub.3,
--CX.sub.2CX(CX.sub.3).sub.2, and--C(CX.sub.3).sub.3. Each X is
independently H, F, Cl, Br or I. Each m is independently 0 or 1.
Each n, o and p are independently integers from 0 to 10.
Hybridization Conditions/Stringency:
[0048] Those of ordinary skill in the art of nucleic acid
hybridization will recognize that factors commonly used to impose
or control stringency of hybridization include formamide
concentration (or other chemical denaturant reagent), salt
concentration (i.e., ionic strength hybridization temperature,
detergent concentration, pH and the presence or absence of
chaotropes. Optimal stringency for a probe/target combination can
often be found by the well known technique of fixing several of the
aforementioned stringency factors and then determining the effect
of varying a single stringency factor. The same stringency factors
can be modulated to thereby control the stringency of hybridization
of a PNA to a nucleic acid, except that the hybridization of a PNA
is fairly independent of ionic strength. Optimal stringency for an
assay may be experimentally determined by examination of each
stringency factor until the desired degree of discrimination is
achieved.
Suitable Hybridization Conditions:
[0049] Generally, the more closely related the background causing
nucleic acid contaminates are to the target sequence, the more
careful stringency must be controlled. Blocking probes may also be
used as a means to improve discrimination beyond the limits
possible by mere optimization of stringency factors. Suitable
hybridization conditions will thus comprise conditions under which
the desired degree of discrimination is achieved such that an assay
generates an accurate (within the tolerance desired for the assay)
and reproducible result. Aided by no more than routine
experimentation and the disclosure provided herein, those of skill
in the art will easily be able to determine suitable, hybridization
conditions for performing assays utilizing the methods, kits and
compositions described herein. Suitable in-situ hybridization
conditions comprise conditions suitable for performing an in-situ
hybridization procedure. Thus, suitable hybridization or suitable
in-situ hybridization conditions will become apparent using the
disclosure provided herein; with or without additional routine
experimentation.
Blocking Probes:
[0050] Blocking probes are nucleic acid or non-nucleic acid probes
(e.g. PNA probes) that can be used to suppress the binding of the
probing nucleobase sequence of the probing polymer to a non-target
sequence. Preferred blocking probes are PNA probes (See: Coull et
al., WIPO publication No. WO98/24933 as well as U.S. Pat. No.
6,110,676). Typically, blocking probes are closely related to the
probing nucleobase sequence and preferably they comprise a point
mutation as compared with the probing nucleobase sequence. It is
believed that blocking probes operate by hybridization to the
non-target sequence to thereby form a more thermodynamically stable
complex than is formed by hybridization between the probing
nucleobase sequence and the non-target sequence. Formation of the
more stable and preferred complex blocks formation of the less
stable non-preferred complex between the probing nucleobase
sequence and the non-target sequence. Thus, blocking probes can be
used with the methods, kits and compositions of this invention to
suppress the binding of the PNA probe to a non-target sequence that
might be present and interfere with the performance of the assay.
Blocking probes are particularly advantageous in single point
mutation discrimination.
Probing Nucleobase Sequence:
[0051] The probing nucleobase sequence of a PNA probe is the
specific sequence recognition portion of the construct. Therefore,
the probing nucleobase sequence is a sequence of PNA subunits
designed to sequence specifically hybridize to a target sequence
wherein the presence, absence and/or amount of target sequence can
be used to detect the presence, absence and/or number of organisms
of interest in a sample. Consequently, with due consideration of
the requirements of a PNA probe for the assay format chosen, the
length of the probing nucleobase sequence of the PNA probe will
generally be chosen such that a stable complex is formed with the
target sequence under suitable hybridization conditions or suitable
in-situ hybridization conditions.
[0052] The probing nucleobase sequence suitable for detecting the
target organism listed in Table 1, will generally, but not
necessarily, have a length of 18 or fewer PNA subunits wherein the
exact nucleobase sequence can be at least 90% homologous to the
probing nucleobase sequences listed in Table 1, or their
complements. The PNA probes can be 100% homologous to said
sequences or can comprise the exact nucleobase sequences appearing
the Table 1. Complements of the probing nucleobase sequence are
included since it is possible to prepare or amplify copies of the
target sequence wherein the copies are complements of the target
sequence and thus, will bind to the complement of the probing
nucleobase sequences listed in Table 1. Useful probing nucleobase
sequences are listed in Table 1. These probing nucleobase sequences
have been shown to be relatively or highly specific for the target
organism indicated in the presence of other organisms, including
the other species of Candida (See information listed in Table 1 and
the Examples, below).
[0053] A PNA probe of this invention will generally have a probing
nucleobase sequence that is complementary to the target sequence.
Alternatively, a substantially complementary probing nucleobase
sequence might be used since it has been demonstrated that greater
sequence discrimination can be obtained when utilizing probes
wherein there exists one or more point mutations (base mismatch)
between the probe and the target sequence (See: Guo et al., Nature
Biotechnology 15:331-335 (1997)).
[0054] This invention contemplates that variations in the probing
nucleobase sequences listed in Table 1 shall provide PNA probes
that are suitable for the specific detection of the organisms
listed. Common variations include, deletions, insertions and frame
shifts. Variation of the probing nucleobase sequences within the
parameters described herein are considered to be an embodiment of
this invention.
Probe Complexes:
[0055] In still another embodiment, two probes are designed to
hybridize to the target sequence sought to be detected to thereby
generate a detectable signal whereby the probing nucleobase
sequence of each probe comprises half or approximately half of the
nucleobase sequence required for hybridization to the complete
target sequence of the organism sought to be detected in the assay
such that the aggregate nucleobase sequence of the two probes forms
the probing nucleobase sequence that hybridizes to the target
sequence. As a non-limiting example, the probing nucleobase
sequences of the two probes might be designed using the assay as
described in U.S. Pat. No. 6,027,893, entitled: "Method of
identifying a nucleic acid using triple helix formation of
adjacently annealed probes" by H. Orum et al., herein incorporated
by reference. Using this methodology, the probes that hybridize to
the target sequence may or may not be labeled. However, it is the
probe complex formed by the annealing of the adjacent probes that
is detected. Similar compositions comprised solely of PNA probes
have been described in U.S. Pat. No. 6,287,772, herein incorporated
by reference.
II. Preferred Embodiments of the Invention
[0056] a. PNA Probes:
[0057] In one embodiment, this invention is directed to PNA probes.
The PNA probes of this invention are suitable for detecting,
identifying and/or quantitating one or more species of Candida in a
sample. The PNA probes, probe sets, methods and kits of this
invention are suitable for the analysis of nucleic acid, whether or
not it is present within an organism of interest. Accordingly, this
invention can be used for both the analysis of organisms or for the
analysis of nucleic acid extracted from or derived from an organism
of interest.
[0058] With the exception of Seq. Id. Nos. 9 and 10 (See: Table 1
for a list of target organisms), generally the PNA probes
comprising the identified probing nucleobase sequences that are
specific for a certain species of Candida. General characteristics
(e.g. length, labels, linkers etc) of PNA probes suitable for the
detection, identification or quantitation of these specific
organisms have been previously described herein. Non-limiting
examples of probing nucleobase sequences of PNA probes of this
invention are listed in Table 1, below. The species of Candida that
the probing nucleobase sequence is designed to determine has also
been identified in. Table 1 as the target organism.
[0059] The PNA probes of this invention may comprise only a probing
nucleobase sequence (as previously described herein) or may
comprise additional moieties. Non-limiting examples of additional
moieties include detectable moieties (labels), linkers, spacers,
natural or non-natural amino acids, peptides, enzymes and/or other
subunits of PNA, DNA or RNA. Additional moieties may be functional
or non-functional in an assay. Generally however, additional
moieties will be selected to be functional within the design of the
assay in which the PNA probe is to be used. For example, the PNA
probes of this invention can be labeled with one or more detectable
moieties or labeled with two or more independently detectable
moieties. The independently detectable moieties can be
independently detectable fluorophores.
TABLE-US-00001 TABLE 1 Seq. ID. Target No. Organism Probing
Nucleobase Sequence 1 C. albicans AGA - GAG - CAG - CAT - GCA 2 C.
albicans AGA - GAG - CAA - CAT - GCA 3 C. albicans ACA - GCA - GAA
- GCC - GTG 4 C. albicans CAT - AAA - TGG - CTA - CCA - GA 5 C.
albicans CAT - AAA - TGG - CTA - CCC - AG 6 C. albicans ACT - TGG -
AGT - CGA - TAG 7 C. albicans CCA - AGG - CTT - ATA - CTC - GC 8 C.
albicans CCC - CTG - AAT - CGG - GAT 9 C. albicans & GAC - GCC
- AAA - GAC - GCC C. dubliniesis 10 C. albicans & ATC - GTC -
AGA - GGC - TAT - C. dubliniesis AA 11 C. dubliniesis TAG - CCA -
GAA - GAA - AGG 12 C. dubliniesis CAT - AAA - TGG - CTA - GCC - AG
13 C. dubliniesis CTC - CGA - TGT - GAC - TGC - G 14 C. dubliniesis
TCC - CAG - ACT - GCT - CGG 15 C. glabrata TCC - AAG - AGG - TCG -
AGA 16 C. glabrata GCC - AAG - CCA - CAA - GGA 17 C. glabrata GCC -
GCC - AAG - CCA - CA 18 C. glabrata GGA - CTT - GGG - GTT - AG 19
C. glabrata CCG - GGT - GCA - TTC - CA 20 C. krusei ATG - TAG - AAC
- GGA - ACT - A 21 C. krusei GAT - TCT - CGG - CCC - CAT - G 22 C.
parapsilosis CTG - GTT - CGC - CAA - AAA - G 23 C. tropicalis AGT -
ACG - CAT - CAG - AAA
[0060] The probes of this invention can be used in in-situ
hybridization (ISH) and fluorescence in-situ hybridization (FISH)
assays. Excess probe used in an ISH or FISH assay often will be
removed so that the detectable moiety of specifically bound probes
can be detected above the background signal that results from still
present but unhybridized probe. Generally, the excess probe can be
washed away after the sample has been incubated with probe fora
period of time. However, because certain types of self-indicating
probes can generate little or no detectable background, they can be
used to eliminate the requirement that excess probe be completely
removed (washed away) from the sample.
Unlabeled Non-Nucleic Acid Probes:
[0061] The probes of this invention need not be labeled with a
detectable moiety to be operable within the scope of this
invention. When using the probes of this invention it is possible
to detect the probe/target sequence complex formed by hybridization
of the probing nucleobase sequence of the probe to the target
sequence. For example, a PNA/nucleic acid complex formed by the
hybridization of a PNA probing nucleobase sequence to the target
sequence could be detected using an antibody that specifically
interacts with the complex under antibody binding conditions.
Suitable antibodies to PNA/nucleic acid complexes and methods for
their preparation and use are described in WIPO Patent Application
WO95/17430 and U.S. Pat. No. 5,412,458, herein incorporated by
reference.
[0062] The antibody/PNA/nucleic acid complex formed by interaction
of the .alpha.-PNA/nucleic acid antibody with the PNA/nucleic acid
complex can be detected by several methods. For example, the
.alpha.-PNA/nucleic acid antibody could be labeled with a
detectable moiety. Suitable detectable moieties have been
previously described herein. Thus, the presence, absence and/or
quantity of the detectable moiety can be correlated with the
presence, absence and/or quantity of the antibody/PNA/nucleic acid
complex and the species of Candida to be identified by the probing
nucleobase sequence of the PNA probe, Alternatively, the
antibody/PNA/nucleic acid complex can be detected using a secondary
antibody that is labeled with a detectable moiety. Typically the
secondary antibody specifically binds to the .alpha.-PNA/nucleic
acid antibody under antibody binding conditions. Thus, the
presence, absence and/or quantity of the detectable moiety can be
correlated with the presence, absence and/or quantity of the
antibody/antibody/PNA/nucleic acid complex and the species of
Candida to be identified by the probing nucleobase sequence of the
probe. As used herein, the term antibody includes antibody
fragments that specifically bind to other antibodies or other
antibody fragments.
Immobilization of Probes To A Surface:
[0063] One or more of the PNA probes of this invention may
optionally be immobilized to a surface for the detection of the
target sequence of a target organism of interest. PNA probes can be
immobilized to the surface using the well known process of
UV-crosslinking. A PNA probe can be synthesized on the surface in a
manner suitable for deprotection but not cleavage from the
synthesis support (See: Weiler, J. et al, Hybridization based DNA
screening on peptide nucleic acid (PNA) oligomer arrays., Nucl.
Acids Res., 25, 14:2792-2799 (July 1997)). In still another
embodiment, PNA probes can be covalently linked to a surface by the
reaction of a suitable functional group on the probe with a
functional group of the surface (See: Lester, A. et al, "PNA Array
Technology": Presented at Biochip Technologies Conference in
Annapolis (October 1997)). This method is most advantageous since
the PNA probes on the surface will typically be highly purified and
attached using a defined chemistry, thereby minimizing or
eliminating non-specific interactions.
[0064] Methods for the chemical attachment of probes to surfaces
generally involve the reaction of a nucleophilic group, (e.g. an
amine or thiol) of the probe to be immobilized, with an
electrophilic group on the support to be modified. Alternatively,
the nucleophile can be present on the support and the electrophile
(e.g. activated carboxylic acid) present on the probe. Because
native PNA possesses an amino terminus, a PNA will not necessarily
require modification to thereby immobilize it to a surface (See:
Lester et al., Poster entitled "PNA Array Technology").
[0065] Conditions suitable for the immobilization of a PNA probe to
a surface will generally be similar to those conditions suitable
for the labeling of the polymer. The immobilization reaction is
essentially the equivalent of labeling whereby the label is
substituted with the surface to which the polymer is to be
linked.
[0066] Numerous types of surfaces derivatized with amino groups,
carboxylic acid groups, isocyantes, isothiocyanates and malimide
groups are commercially available. Non-limiting examples of
suitable surfaces include membranes, chips (e.g. silicone chips),
glass, controlled pore glass, polystyrene particles (beads), silica
and gold nanoparticles.
Arrays of PNA Probes or Probe Sets:
[0067] Arrays are surfaces to which two or more probes have been
immobilized each at a specified position. The probing nucleobase
sequence of the immobilized probes can be judiciously chosen to
interrogate a sample that may contain nucleic acid from one or more
target organisms. Because the location and composition of each
immobilized probe is known, arrays can be useful for the
simultaneous detection, identification and/or quantitation of
nucleic acid from two or more target organisms that may be present
in the sample. Moreover, arrays of PNA probes can be regenerated by
stripping away any of the hybridized nucleic acid after each assay,
thereby providing a means to repetitively analyze numerous samples
using the same array. Thus, arrays of PNA probes or PNA probe sets
may be useful for repetitive screening of samples for target
organisms of interest. The arrays of this invention comprise at
least one PNA probe (as described herein) suitable for the
detection, identification and/or quantitation of at least one
species of Candida. Exemplary probing nucleobase sequences for the
immobilized PNA probes are listed in Table 1. [0068] b. PNA Probe
Sets:
[0069] In another embodiment, this invention is directed to a PNA
probe set suitable for detecting, identifying and/or quantitating
one or more species of Candida yeast in a sample of interest
wherein at least one of the species sought to be detected with the
probe set is selected from the group consisting of: C. albicans, C.
dubliniensis, C. krusei, C. glabrata, C. parapsilosis and C.
tropicalis. The general and preferred characteristics of PNA probes
suitable for the detection, identification and/or quantitation of
these specific yeast species have been previously described herein.
Preferred probing nucleobase sequences for the target species are
listed in Table 1. The grouping of PNA probes within sets
characterized for specific groups of species can be a very useful
embodiment of this invention. The PNA probes of this invention can
be combined with probes for other yeast or even for organisms other
than yeast such as been described in U.S. Pat. No. 6,280,946,
herein incorporated by reference, wherein a multiplex assay for
both yeast and bacteria has been described using a PNA probe
set.
[0070] Probe sets of this invention comprise at least one PNA probe
but need not comprise only PNA probes. For example, probe sets of
this invention may comprise mixtures of PNA probes and nucleic acid
probes, provided however that a set comprises at least one PNA
probe as described herein. In one embodiment, some of the probes of
the set can be blocking probes composed of PNA or nucleic acid. In
other embodiments, the probe set can be used to determine organisms
other than species of Candida in addition to the determination of
at least one species of Candida.
[0071] Table 1 lists several species of Candida for which two or
more probing nucleobase sequences are identified as being suitable
for detecting the identified target organism. Where alternative
probing nucleobase sequences exist, it can be advantageous to use a
probe set containing the two or more PNA probes to thereby increase
the detectable signal in the assay.
[0072] One exemplary probe set would comprise probes suitable for
determining C. albicans wherein two or more of the probes of the
set comprise a probing nucleobase sequence selected from the group
consisting of: AGA-GAG-CAG-CAT-GCA (Seq. Id. No, 1),
AGA-GAG-CAA-CAT-GCA (Seq. Id. No. 2), ACA-GCA-GAA-GCC-GTG (Seq. Id.
No. 3), CAT-AAA-TGG-CTA-CCA-GA (Seq. Id. No. 4),
CAT-AAA-TGG-CTA-CCC-AG (Seq. Id. No. 5), ACT-TGG-AGT-CGA-TAG (Seq.
Id. No. 6), CCA-AGG-CTT-ATA-CTC-GC (Seq. Id. No. 7) and
CCC-CTG-AAT-CGG-GAT (Seq. Id. No. 8). A second exemplary probe set
can comprise probes suitable for determining C. albicans and C.
dubliniensis wherein the probes of the set comprise a probing
nucleobase sequence selected from the group consisting of:
GAC-GCC-AAA-GAC-GCC (Seq. Id. No. 9) and ATC-GTC-AGA-GGC-TAT-AA
(Seq, Id No. 10). Still a third exemplary probe set can comprise
probes suitable for determining only C. dubliniensis wherein at
least two the probes of the set comprise a probing nucleobase
sequence selected from the group consisting of: TAG-CCA-GAA-GAA-AGG
(Seq. Id. No. 11), CAT-AAA-TGG-CTA-GCC-AG (Seq. Id. No. 12),
CTC-CGA-TGT-GAC-TGC-G (Seq. Id. No. 13) and TCC-CAG-ACT-GCT-CGG
(Seq. Id. No. 14). Yet a fourth exemplary probe set can comprise
probes suitable for determining only C. glabrata wherein at least
two the probes of the set comprise a probing nucleobase sequence
selected from the group consisting of: TCC-AAG-AGG-TCG-AGA (Seq.
Id. No. 15), GCC-AAG-CCA-CAA-GGA (Seq. Id. No. 16),
GCC-GCC-AAG-CCA-CA (Seq. Id. No. 17), GGA-CTT-GGG-GTT-AG (Seq. Id.
No. 18) and CCG-GGT-GCA-TTC-CA (Seq. Id. No. 19). Still a fifth
exemplary probe set can comprise probes wherein the probing
nucleobase sequence is selected from the group consisting of:
ATG-TAG-AAC-GGA-ACT-A (Seq. Id. No. 20) and GAT-TCT-CGG-CCC-CAT-G
(Seq. Id. No. 21).
[0073] In other embodiments, the probe set can comprise two or more
probes such that two or more of the species of Candida identified
herein are detected, identified and/or quantitated. Preferably, the
set comprises two or more independently detectable PNA probes
wherein each independently detectable probe is suitable for
detecting, identifying and/or quantitating a particular species of
Candida. For example, a PNA probe set comprising Seq. Id. Nos: 1
and 11, wherein each probe is labeled with an independently
detectable moiety and can be used in two independent assays, or a
single multiplex assay, for the independent determination of both
C. albicans and C. dubliniensis (See: Example 2 for a non-multiplex
assay demonstrates that these species can be independently
determined using the probing nucleobase sequences identified
herein). [0074] c. Methods:
[0075] In another embodiment, this invention is directed to a
method suitable for detecting, identifying and/or quantitating one
or more species of Candida in a sample of interest. According to
the method, one or more individual species of Candida can be
determined. The general and preferred characteristics of PNA probes
suitable for the detection, identification and/or quantitation of
these target organisms have been previously described herein.
Exemplary probing nucleobase sequences are listed in Table 1.
[0076] In one embodiment, the method can comprise contacting the
sample with one or more PNA probes, wherein suitable probes have
been previously described herein. According to the method, the
presence, absence and/or number of the one or more species of
Candida in the sample can be detected, identified and/or
quantitated by correlating hybridization of the probing nucleobase
sequence of one or more PNA probes to the target sequence of a
target organism of interest under suitable hybridization conditions
or suitable in-situ hybridization conditions. The grouping of PNA
probes within probe sets to be used with methods for detecting
specific organisms or groups of organisms can also be done.
Exemplary probes and probe sets suitable for the practice of this
method have been previously described herein. Preferred methods for
the determination of yeast, with or without the simultaneous
detection of bacteria, have been previously described in U.S. Pat.
No. 6,280,946, incorporated herein by reference. Examples 1 and 2,
below, provide further methods for the determination of the
specific yeast identified herein.
Exemplary Assay Formats:
[0077] The probes, probe sets, methods and lefts of this invention
can be used for the detection, identification and/or quantitation
of Candida yeast. In-situ hybridization (ISH) or fluorescent
in-situ hybridization (FISH) can be used as the assay format for
detecting, identifying and/or quantitating target organisms.
Specific PNA-RSH methods used to experimentally test specific PNA
probes can be found in Examples 1 and 2 of this specification. The
examples contained herein demonstrate that labeled PNA probes
comprising the probing nucleobase sequences listed in Table 1 are
specific for determining target organisms even when other organisms
listed in the Table are present in the assay. The experimental
conditions used in the Examples yield results within approximately
1-4 hours. The identical experimental protocol was found to be
specific, reliable and generally applicable regardless of the
nature or sequence of the PNA probes used.
[0078] Organisms that have been treated with the PNA probes or
probe'sets or kits described herein can be determined by several
exemplary methods. The cells can be fixed on slides and visualized
with a film, camera, slide scanner or microscope. Alternatively,
the cells can be fixed and then analyzed in a flow cytometer. Slide
scanners and flow cytometers are particularly useful for rapidly
quantitating the number of target organisms present in a sample of
interest. [0079] d. Kits:
[0080] In yet another embodiment, this invention is directed to
kits suitable for performing an assay that detects the presence,
absence and/or quantity of Candida yeast in a sample. The general
and preferred characteristics of PNA probes suitable for the
detection, identification and/or quantitation of Candida have been
previously described herein. Exemplary probing nucleobase sequences
are listed in Table 1. Furthermore, methods suitable for using the
PNA probes or PNA probe sets of a kit suitable to detect, identify
and/or quantitate target organisms in a sample of interest have
been previously described herein.
[0081] The kits of this invention comprise one or more PNA probes
and other reagents or compositions that are selected to perform an
assay or otherwise simplify the performance of an assay. The kits
can, for example, comprise buffers and/or other reagents useful for
performing a PNA-ISH or PNA-FISH assay. In other embodiments, the
buffers and/or other reagents can be useful for performing a
nucleic acid amplification reaction such as a PCR reaction.
[0082] In kits that contain sets of probes, wherein each of at
least two probes of the set are used to detect different species of
Candida, the probes of the set can be labeled with one or more
independently detectable moieties so that each specific target
organism can be individually detected, identified and/or
quantitated in a single assay (e.g. a multiplex assay). [0083] e.
Exemplary Applications For Using The Invention:
[0084] Whether support bound or in solution, the PNA probes, probe
sets, methods and kits of this invention can be useful for the
rapid, sensitive and reliable detection of Candida yeast in food,
beverages, water, pharmaceutical products, personal care products,
dairy products or for the analysis of environmental samples. The
analysis of beverages can include soda, bottled water, fruit juice,
beer, wine or liquor products. Suitable PNA probes, probe sets,
methods and kits of this invention can be particularly useful for
the analysis of raw materials, equipment, products or processes
used to manufacture or store food, beverages, water, pharmaceutical
products, personal care products, dairy products or for the
analysis of environmental samples.
[0085] Whether support bound or in solution, the PNA probes, probe
sets, methods and kits of this invention are can be useful for the
detection of Candida yeast in clinical samples and clinical
environments. By way of a non-limiting example, the PNA probes,
probe sets, methods and kits of this invention can be particularly
useful in the analysis of blood culture samples. Other non-limiting
examples of clinical samples include: sputum, laryngeal swabs,
gastric lavage, bronchial washings, biopsies, aspirates,
expectorates, body fluids (e.g. spinal, pleural, pericardial,
synovial, blood, pus, amniotic, and urine), bone marrow and tissue
sections. Suitable PNA probes, probe sets, methods and kits can
also be particularly useful for the analysis of clinical specimens,
equipment, fixtures or products used to treat humans or
animals.
EXAMPLE
[0086] This invention as now illustrated by the following examples
that are not intended to be limiting in any way.
[0087] All PNA oligomers were prepared using conventional synthesis
and purification procedures.
Example 1
Analysis of a PNA Probe Specific to Candida albicans
[0088] PNA Probe Sequence
TABLE-US-00002 Can26S03/Flu Flu-O-AGAGAGCACCATGCA-NH.sub.2 Note:
Flu = 5(6)-carboxy-fluorescein; O = 8-amino-3,6-dioxaoctanoic
acid
[0089] Reference strains and clinical isolates. Fourteen C.
albicans reference strains and nineteen other reference strains
representing phylogenetically related Candida species and
Lodderomyces elongisporus, mainly within the C. albicans clade,
were selected from the Agricultural Research Service Culture
Collection (NRRL) Peoria, Ill. One Saccharomyces cerevisiae strain
was obtained, from the American Type Culture Collection (ATCC),
Manassas, Va. Fifty-eight C. dubliniensis and thirty-nine C.
albicans clinical isolates were collected at the Institute of
Medical Microbiology, University Hospital, Aachen, Germany. The C.
dubliniensis isolates were mainly from HIV-positive patients and
from respiratory specimens of patients with cystic fibrosis.
Clinical isolates of C. albicans were from various clinical
specimens, including blood cultures, and chosen to represent
different strains, i.e. serotype A, the biovar stellatoidea as well
as phenotypically aberrant isolates such as red pigment strain and
strains that failed to assimilate glucosamine and
N-acetylglucosamine. All strains and isolates were identified by
D1/D2 26S rDNA sequence analysis. For PNA FISH analysis, reference
strains and clinical isolates were inoculated into yeast-malt (YM)
broth (Difco Laboratories, Detroit, Mich.) and incubated overnight
at 35.degree. C. Furthermore, thirty-three C. albicans isolates and
eighteen other isolates representing clinically significant yeast
species obtained from various clinical specimens, including blood
cultures (Clinical Microbiology Laboratory, Cleveland Clinic
Foundation, Ohio), were spiked into FAN BacT/Alert medium (Organon
Teknika, Durham, N.C.) and incubated in the BacT/Alert Microbial
detection system (Organon Teknika) until they were detected as
positive by the system. The eighteen non-C. albicans isolates
comprised C. glabrata (n=5), C. tropicalis (n=3), C. krusei (n=2),
C. parapsilosis (n=4), C. lusitaniae (n=3), and C. zeylanoides
(n=1).
[0090] Clinical specimens. A total of thirty-three yeast-positive
blood culture bottles (FAN BacT/Alert, Organon Teknika) from
routine testing at the Clinical Microbiology Laboratory, Cleveland
Clinic Foundation, OH were included in this study. In addition,
twenty-five simulated yeast-positive blood culture bottles (FAN
BacT/Alert, Organon Teknika) were made by inoculating routine blood
culture bottles that were negative following 7 days incubation. The
inoculation was done with just a few colony-forming units of
strains representing clinically significant non-C. albicans species
and comprised C. glabrata (n=2), C. lusitaniae (n=4), C. tropicalis
(n=4), C. guilliermormondii (n=1) C. krusei (n=3), C. parapsilosis
(n=4), famata (n=2), C. norvegensis (n=4), and C. neoformans (n=1).
These strains included reference strains from ATCC and the German
Collection of Microorganisms and Cell Cultures, Braunschweig,
Germany (DSM) as well as recent clinical isolates from the
Institute of Medical Microbiology, University Hospital RWTH Aachen,
Aachen, Germany. The blood culture bottles were re-incubated in the
BacT/Alert Microbial detection system (Organon Teknika) until they
were detected as positive by the system.
[0091] Preparation of smears. One drop of phosphate-buffered saline
(PBS) was placed in the well of a Teflon-coated microscope slide
(Clear Coat, Erie Scientific, Portsmouth, N.H.) and 10-25 .mu.L of
culture or a small drop was added, mixed and spread throughout the
well. The smear was fixed by either placing the slide on an
80.degree. C. slide warmer for 2 hours or at 60.degree. C. for 20
minutes. The slide was subsequently immersed into 95% ethanol for
1-2 minutes and allowed to air-dry.
[0092] Fluorescence in situ hybridization using PNA probes (PNA
FISH). Smears were covered with approximately 20 .mu.L of
hybridization solution containing 10% (w/v) dextran sulfate (Sigma
Chemical Co., St. Louis, Mo.), 10 mM NaCl, 30% (v/v) formamide
(Sigma), 0.1% (w/v) sodium pyrophosphate (Sigma), 0.2% (w/v)
polyvinylpyrrolidone (Sigma), 0.2% (w/v) ficoll (Sigma), 5 mM
Na.sub.2EDTA (Sigma), 0.1% (v/v) Triton X-100 (Aldrich), 50 mM
Tris/HCl pH 7.5 and 250 nM fluorescein-labeled PNA probe targeting
C. albicans. Coverslips were placed on the smears to ensure even
coverage with hybridization solution, and the slides were placed on
a slide warmer with a humidity chamber (Slidemoat, Boeckel,
Germany) and incubated for 90 min at 55.degree. C.
[0093] Following hybridization, the coverslips were removed by
submerging the slides into approximately 20 ml/slide pre-warmed 5
mM Tris, pH 10, 15 mM NaCl (J. T. Baker), 0.1% (v/v) Triton X-100
(Aldrich) in a water bath at 55.degree. C. and washed for 30 min.
The slides were then air-dried. Each smear was finally mounted
using one drop of IMAGEN Mounting Fluid (DAKO, Ely, UK) and covered
with a coverslip. Microscopic examination was conducted using a
fluorescence microscope (Optiphot, Nikon Corporation, Tokyo, Japan)
equipped with a 60.times./1.4 oil objective (Nikon), an HBO 100 W
mercury lamp, and a FITC/Texas Red dual band filter set (Chroma
Technology Corp., Brattleboro, Vt.).
[0094] Results
[0095] The probe was tested on a panel of reference strains
representing C. albicans and other Candida species, including
phylogentically closely related Candida species, clinically
relevant Candida species and other yeast species. The results are
summarized in Table 2 and show that the probe is highly
specific.
TABLE-US-00003 TABLE 2 Results for reference strains analyzed by
PNA FISH with C. albicans-specific PNA probe Yeast species Strain
ID Can26S03/Flu Candida albicans NRRL Y-107 + Candida albicans NRRL
Y-12983 + Candida albicans NRRL Y-17967 + Candida albicans NRRL
Y-17968 + Candida albicans NRRL Y-17974 + Candida albicans NRRL
Y-17976 + Candida albicans NRRL Y-302 + Candida albicans NRRL Y-477
+ Candida albicans NRRL Y-6359 + Candida albicans NRRL Y-6943 +
Candida albicans NRRL Y-79 + Candida albicans NRRL Y-81 + Candida
albicans NRRL Y-82 + Candida albicans NRRL YB-3898 + Candida
dubliniensis NRRL Y-17841 - Candida dubliniensis NRRL Y-17512 -
Candida dubliniensis NRRL Y-17969 - Candida dubliniensis NRRL
Y-17971 - Candida dubliniensis NRRL Y-17972 - Candida dubliniensis
NRRL Y-17973 - Candida glabrata NRRL Y 65 - Candida maltosa NRRL
Y-17677 - Candida tropicalis NRRL Y-12968 - Candida tropicalis NRRL
Y-1552 - Candida tropicalis NRRL Y-5716 - Candida viswanathii NRRL
Y-6660 - Candida lodderae NRRL Y-17317 +/- Candida parapsilosis
NRRL Y-12969 - Candida parapsilosis NRRL Y-543 - Candida sojae NRRL
Y-17909 - Lodderomyces elongisporus NRRL YB-4239 - Lodderomyces
elongisporus NRRL Y-7681 - S. cerevisiae ATCC 4098 -
[0096] This is to our knowledge the first probe targeting rRNA that
has been shown not to react with C. dubliniensis, a recently
discovered Candida species that is often misidentified as C.
albicans using standard methods, such as germ tube analysis and
carbon assimilation methods.
[0097] The sensitivity and specificity of the C. albicans PNA FISH
were further examined using one hundred forty-eight clinical
isolates representing C. albicans and other clinically relevant
yeast species. The results are summarized in Table 3. The assay
correctly identified all C. albicans isolates and gave negative
results with all other isolates.
TABLE-US-00004 TABLE 3 Reaction of C. albicans PNA FISH with 148
isolates representing clinically relevant yeast species. C.
albicans PNA FISH Positive Negative Species (n) (n) Candida
albicans 72 0 Candida dubliniensis 0 58 Candida glabrata 0 5
Candida tropicalis 0 3 Candida krusei 0 2 Candida parapsilosis 0 4
Candida lusitaniae 0 3 Candida zeylanoides 0 1
[0098] The diagnostic performance of C. albicans PNA FISH was
evaluated directly on 33 yeast-positive blood culture bottles as
compared to results obtained by standard methods. These comprised
nine C albicans cultures and twenty-four non-C. albicans cultures
representing six to seven different species. The specificity was
furthermore tested with twenty-five simulated blood culture bottles
representing nine different species. The results are summarized in
Table 4 and show 100% agreement with standard methods supporting a
100% diagnostic sensitivity and 100% diagnostic specificity.
TABLE-US-00005 TABLE 4 Reaction of C. albicans PNA FISH with
yeast-positive blood cultures comprising 33 real blood culture
bottles and 25 artificially spiked blood culture bottles. C.
albicans PNA FISH Positive Negative Identification (n) (n) Candida
albicans 9 0 Candida glabrata 0 11 Candida parapsilosis 0 10
Candida tropicalis 0 5 Candida krusei 0 4 Candida lusitaniae 0 9
Candida famata 0 2 Candida norvegensis 0 4 Candida guilliermondii 0
1 Cryptococcus neoformans 0 2 Other yeast, not identified 0 1
[0099] Summary
[0100] PNA FISH using PNA probes targeting rRNA of C. albicans was
used for identification of C. albicans in yeast positive blood
culture bottles in a time frame not possible using conventional
methods. The test was performed on smears made directly from the
blood culture bottles and interpretation of results was conducted
by microscopy, such that the PNA FISH procedure simply added the
high specificity of PNA probes to standard microbiological
procedures smear preparation and microscopy) to provide definitive
identification.
[0101] The C. albicans specific PNA probe showed a very high degree
of specificity, not only when tested with clinical isolates, but
also when further challenged with strains from the C. albicans
clade. These findings are ascribed to the high specificity of PNA
probes combined with the use of the D1/D2 region of 265 rRNA as
target; a region that has been used for systematic studies of
yeasts. These data are yet another example of how molecular
diagnostic methods using rRNA sequences as target can replace
classic phenotypically based microbiological identification
methods. In fact, several atypical C. albicans strains were
correctly identified by C. albicans PNA FISH. C. albicans PNA FISH
provides rapid and specific identification of C. albicans. No
confirmation testing is required for identification, thus allowing
the appropriate patient treatment to be administered more quickly,
i.e. immediate treatment with fluconazole since almost all isolates
of C. albicans are susceptible to this drug.
Example 2
PNA Probes That Distinguish Between C. dubliniensis and C.
albicans
[0102] Probe Sequence
##STR00002##
[0103] Reference strains. Reference strains were obtained from
Agricultural Research Service Culture Collection (NTRRL) Peoria,
Ill. The strains were propagated in yeast and mold broth (Difco
Laboratories, Detroit, Mich.) at 30-35.degree. C.
[0104] Preparation of smears. One drop of phosphate-buffered saline
(PBS) was placed in the well of a Teflon-coated microscope slide
(Clear Coat, Erie Scientific, Portsmouth, N.H.) and 10 .mu.L of
culture was added, mixed and spread throughout the well. The smear
was fixed by either placing the slide on an 80.degree. C. slide
warmer for 2 hours or by flame-fixation by passing the slides
through the blue cone of a Bunsen burner. The slide was
subsequently immersed into 95% ethanol for 1-2 minutes and allowed
to air-dry.
[0105] Fluorescence in situ hybridization using PNA probes (PNA
FISH). Smears were covered with approximately 10 .mu.L of
hybridization solution containing 10% (w/v) dextran sulfate (Sigma
Chemical Co. St. Louis, Mo.), 10 mM NaCl, 30% (v/v) formamide
(Sigma), 0.1% (w/v) sodium pyrophosphate (Sigma), 0.2% (w/v)
polyvinylpyrrolidone (Sigma), 0.2% (w/v) ficoll (Sigma), 5 mM
Na.sub.2EDTA (Sigma), 0.1% (v/v) Triton X400 (Aldrich), 50 mM
Tris/HCl pH 7.5 and 100 nM fluorescein-labeled PNA probe targeting
C. albicans or 500 nM fluorescein-labeled PNA probe targeting C.
dubliniensis. Coverslips were placed on the smears to ensure even
coverage with hybridization solution, and the slides were placed on
a slide warmer with a humidity chamber (Slidernoat, Boeckel,
Germany) and incubated for 30 min at 50.degree. C. Following
hybridization, the coverslips were removed by submerging the slides
into approximately 20 mL/slide pre-warmed 5 mM Tris, pH 10, 15 mM
NaCl (J. T. Baker), 0.1% (v/v) Triton X-100 (Aldrich) in a water
bath at 50.degree. C. and washed for 30 min, The slides were then
air-dried. Each smear was finally mounted using one drop of IMAGEN
Mounting Fluid (DAKO, Ely, UK) and covered with a coverslip.
Microscopic examination was conducted using a fluorescence
microscope (Optiphot, Nikon Corporation, Tokyo, Japan) equipped
with a 60.times./1.4 oil objective (Nikon), an HBO 100 W mercury
lamp, and a FITC/Texas Red dual band filter set (Chroma Technology
Corp., Brattleboro, Vt.).
[0106] Results
[0107] Fifteen C. albicans and six C. dubliniensis reference strain
were tested with the PNA FISH assay The results are summarized in
Table 5. Of the fifteen C. albicans strains, 15 (100%) produced a
positive result with the C. albicans PNA probe and a negative
result with the C. dubliniensis PNA probe. Of the six C.
dubliniensis strains, six (100%) produced a negative result with
the C. albicans PNA probe and a positive result with the C.
dubliniensis PNA probe.
TABLE-US-00006 TABLE 5 Results for reference strains analyzed by
PNA FISH with C. albicans and C. dubliniensis PNA probes Organism
Strain ID Can18S05/Flu Can23S09/Flu C. albicans NRRL Y-12983 + - C.
albicans NRRL Y-79 + - C. albicans NRRL Y-81 + - C. albicans NRRL
Y-82 + - C. albicans NRRL Y-107 + - C. albicans NRRL Y-302 + - C.
albicans NRRL Y-477 + - C. albicans NRRL Y-6359 + - C. albicans
NRRL Y-6943 + - C. albicans NRRL Y-17967 + - C. albicans NRRL
Y-17968 + - C. albicans NRRL Y-17974 + - C. albicans NRRL Y-17975 +
- C. albicans NRRL Y-17976 + - C. albicans NRRL YB-3898 + - C.
dubliniensis NRRL Y-17841 - + C. dubliniensis NRRL Y-17512 - + C.
dubliniensis NRRL Y-17969 - + C. dubliniensis NRRL Y-17971 - + C.
dubliniensis NRRL Y-17972 - + C. dubliniensis NRRL Y-17973 - +
[0108] Summary
[0109] C. dubliniensis shares many phenotypical characteristics
with C. albicans and is therefore incorrectly identified as C.
albicans by current standard methods, such as germ tube analysis
and commercially available carbon assimilation tests. We have here
shown that PNA FISH using PNA probes targeting rRNA of C.
dubliniensis and C. albicans is a 100% accurate method for the
differentiation of C. albicans and C. dubliniensis.
Example 3
Analysis of PNA Probe Specific to Candida glabrata
[0110] PNA Probe Sequence
TABLE-US-00007 Can18S11/Flu Flu-O-TCCAAGAGGTCGAGA-NH.sub.2
EuUni/Cy3 Cy3-OO-ACC-AGA-CTT-GCC-CTC-NH.sub.2 Note: Cy3 = the
cyanine 3 dye available from Amersham Pharmacia Biotech.
[0111] Reference strains. Reference strains were obtained from
Agricultural Research Service Culture Collection (NRRL) Peoria,
Ill. and American Type Culture Collection ATCC), Manassas, Va. The
strains were propagated in yeast and mold broth (Difco
Laboratories, Detroit, Mich.) at 30-35.degree. C.
[0112] Clinical specimens. A total of seventeen yeast-positive
blood culture bottles (FAN BacT/Alert, Organon Teknika) from
routine testing at the Clinical Microbiology Laboratory, Cleveland
Clinic Foundation, OH were included in this study.
[0113] Preparation of smears. One drop of phosphate-buffered saline
(PBS) was placed in the well of a Teflon-coated microscope slide
(Clear Coat, Erie Scientific, Portsmouth, N.H.) and 10 .mu.L of
culture was added, mixed and spread throughout the well. The smear
was fixed by either placing the slide on an 80.degree. C. slide
warmer for 2 hours or at 60.degree. C. for 60 minutes. The slide
was subsequently immersed into 95% ethanol for 1-2 minutes and
allowed to air-dry.
[0114] Fluorescence in situ hybridization using PNA probes (PNA
FISH). Smears were covered with approximately 5-10 .mu.L of
hybridization solution containing 10% (w/v) dextran sulfate (Sigma
Chemical Co., St. Louis, Mo.), 10 mM NaCl, 30% (v/v) formamide
(Sigma), 0.1% (w/v) sodium pyrophosphate (Sigma), 0.2% (w/v)
polyvinylpyrrolidone (Sigma), 0.2% (w/v) ficoll (Sigma), 5 mM
Na.sub.2EDTA (Sigma), 0.1% (v/v) Triton X-100 (Aldrich), 50 mM
Tris/HCl pH 7.5 and 250 nM fluorescein-labeled PNA probe targeting
C. glabrata and 1 nM Cy3-labeled PNA probe targeting eukarya.
EuUni/Cy3 was added in low conc. to give non-detected cells a
reddish appearance. Coverslips were placed on the smears to ensure
even coverage with hybridization solution, and the slides were
placed on a slide warmer with a humidity chamber (Slidemoat,
Boeckel, Germany) and incubated for 90 min at 55.degree. C.
Following hybridization, the coverslips were removed by submerging
the slides into approximately 20 ML/slide pre-warmed 5 mM Tris, pH
10, 15 mM NaCl (J. T. Baker), 0.1% (v/v) Triton X-100 (Aldrich) in
a water bath at 55.degree. C. and washed for 30 min. The slides
were then air-dried. Each smear was finally mounted using one drop
of IMAGEN Mounting Fluid (DAKO, Ely, UK) and covered with a
coverslip. Microscopic examination was conducted using a
fluorescence microscope (Optiphot, Nikon Corporation, Tokyo, Japan)
equipped with a 60.times./1.4 oil objective (Nikon), an HBO 100 W
mercury lamp, and a FITC/Texas Red dual band filter set (Chrome
Technology Corp., Brattleboro, Vt.).
[0115] Results
[0116] The probe was tested on a panel of reference strains
representing C. glabrata and other Candida species, including
phylogentically closely related Candida species, clinically
relevant Candida species and other yeast species. The results are
summarized in Table 6 and show that the probe is highly
specific.
TABLE-US-00008 TABLE 6 Results for reference strains analyzed by
PNA FISH with C. glabrata-specific PNA probe Yeast species Strain
ID Can18S11/Flu Candida glabrata NRRL Y 65 + Candida glabrata NRRL
Y-2242 + Candida glabrata NRRL YB-3659 + Candida glabrata NRRL
YB-3660 + Candida glabrata NRRL YB-4389 + Candida glabrata NRRL
YB-4319 + Candida glabrata NRRL YB-1333 + Candida glabrata NRRL
Y-1418 + Candida glabrata NRRL YB-4018 + Candida albicans NRRL
Y-17968 - Candida albicans NRRL Y-17976 - Candida albicans NRRL
Y-302 - Candida albicans NRRL Y-79 - Candida albicans NRRL Y-81 -
K. delphensis NRRL Y-2379 - K. bacillisporus NRRL Y-17846 - S.
cerevisiae ATCC 4098 -
[0117] The diagnostic performance of C. glabrata PNA FISH was
evaluated directly on seventeen yeast-positive blood culture
bottles as compared to results obtained by standard methods. These
comprised four C. glabrata cultures and thirteen non-C. glabrata
cultures representing four to five different species. The results
are summarized in Table 7 and show 100% agreement with standard
methods supporting a 100% diagnostic sensitivity and 100%
diagnostic specificity.
TABLE-US-00009 TABLE 7 Reaction of C. glabrata PNA FISH with
seventeen routine yeast-positive blood cultures. C. glabrata PNA
FISH Positive Negative Identification (n) (n) Candida albicans 0 8
Candida glabrata 4 0 Candida parapsilosis 0 2 Candida tropicalis 0
1 Candida lusitaniae 0 1 Other yeast, not identified 0 1
[0118] Having described preferred embodiments of the invention, it
will now become apparent to one of skill in the art that other
embodiments incorporating the concepts may be used. It is felt;
therefore, that these embodiments should not be limited to
disclosed embodiments but rather should be limited only by the
spirit and scope of the invention.
Sequence CWU 1
1
23115DNACandidaDescription of Combined DNA/RNA MoleculeProbing
Nucleobase Sequence of PNA Probe 1agagagcagc atgca
15215DNACandidaDescription of Combined DNA/RNA MoleculeProbing
Nucleobase Sequence of PNA Probe 2agagagcaac atgca
15315DNACandidaDescription of Combined DNA/RNA MoleculeProbing
Nucleobase Sequence of PNA Probe 3acagcagaag ccgtg
15417DNACandidaDescription of Combined DNA/RNA MoleculeProbing
Nucleobase Sequence of PNA Probe 4cataaatggc taccaga
17517DNACandidaDescription of Combined DNA/RNA MoleculeProbing
Nucleobase Sequence 5cataaatggc tacccag 17615DNACandidaDescription
of Combined DNA/RNA MoleculeProbing Nucleobase Sequence of PNA
Probe 6acttggagtc gatag 15717DNACandidaDescription of Combined
DNA/RNA MoleculeProbing Nucleobase Sequence of PNA Probe
7ccaaggctta tactcgc 17815DNACandidaDescription of Combined DNA/RNA
MoleculeProbing Nucleobase Sequence of PNA Probe 8cccctgaatc gggat
15915DNACandidaDescription of Combined DNA/RNA MoleculeProbing
Nucleobase Sequence of PNA Probe 9gacgccaaag acgcc
151017DNACandidaDescription of Combined DNA/RNA MoleculeProbing
Nucleobase Sequence of PNA Probe 10atcgtcagag gctataa
171115DNACandidaDescription of Combined DNA/RNA MoleculeProbing
Nucleobase Sequence of PNA Probe 11tagccagaag aaagg
151217DNACandidaDescription of Combined DNA/RNA MoleculeProbing
Nucleobase Sequence of PNA Probe 12cataaatggc tagccag
171316DNACandidaDescription of Combined DNA/RNA MoleculeProbing
Nucleobase Sequence of PNA Probe 13ctccgatgtg actgcg
161415DNACandidaDescription of Combined DNA/RNA MoleculeProbing
Nucleobase Sequence of PNA Probe 14tcccagactg ctcgg
151515DNACandidaDescription of Combined DNA/RNA MoleculeProbing
Nucleobase Sequence of PNA Probe 15tccaagaggt cgaga
151615DNACandidaDescription of Combined DNA/RNA MoleculeProbing
Nucleobase Sequence of PNA Probe 16gccaagccac aagga
151714DNACandidaDescription of Combined DNA/RNA MoleculeProbing
Nucleobase Sequence of PNA Probe 17gccgccaagc caca
141814DNACandidaDescription of Combined DNA/RNA MoleculeProbing
Nucleobase Sequence of PNA Probe 18ggacttgggg ttag
141914DNACandidaDescription of Combined DNA/RNA MoleculeProbing
Nucleobase Sequence of PNA Probe 19ccgggtgcat tcca
142016DNACandidaDescription of Combined DNA/RNA MoleculeProbing
Nucleobase Sequence of PNA Probe 20atgtagaacg gaacta
162116DNACandidaDescription of Combined DNA/RNA MoleculeProbing
Nucleobase Sequence of PNA Probe 21gattctcggc cccatg
162216DNACandidaDescription of Combined DNA/RNA MoleculeProbing
Nucleobase Sequence of PNA Probe 22ctggttcgcc aaaaag
162315DNACandidaDescription of Combined DNA/RNA MoleculeProbing
Nucleobase Sequence of PNA Probe 23agtacgcatc agaaa 15
* * * * *