DNA probes for campylobacter, arcobacter and helicobacter

Details DNA probes for campylobacter, arcobacter and helicobacter DNA probes for campylobacter, arcobacter and helicobacter

1999-12-07

2001-06-12

Whisenaut, Ethan (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C536S023100, C536S024300

Reexamination Certificate

active

06245516

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to nucleotide probes that are useful in detecting and identifying bacterial pathogens. More particularly, the invention relates to nucleotide probes that are useful in detecting and identifying Campylobacter, Helicobacter and Arcobacter spp. bacterial pathogens.
BACKGROUND OF THE INVENTION
Campylobacter, Helicobacter, and Arcobacter spp. are examples of common human and animal pathogens (Thomas, C. A. et al., 1966). Although the pathogenicity of such bacteria has long been known, their phylogenetic relationships, isolation, detection, identification, and classification by traditional biochemical tests, have been variable and difficult. This is largely due to their fastidious growth requirements, inability to ferment carbohydrates, and diverse growth characteristics which vary, not only between genera and species, but also within species. Thus, their large phenotypic variations have made biochemical tests unreliable as a sole method for identifying and differentiating these bacteria.
Many of the species in the genera Helicobacter and Arcobacter were once classified under the genus Campylobacter. However, the phylogenetic relationships of these bacteria have been reevaluated based on information from DNA-DNA hybridization, 23S rRNA-DNA hybridization (Vandamme et al., 1991; Vandamme et al., 1993), and partial 16S rRNA sequences (Li et al., 1993; Patton et al., 1991; Totten et al., 1987). These phylogenetic studies have led to the formation of the current classification of the Campylobacter and Vibrio organisms into Campylobacter, Helicobacter, and Arcobacter.
Other than the conventional biochemical tests, alternative methods based on molecular and genetic approaches, have been proposed to improve the identification and differentiation of these bacteria to the species level. These methods include serology (Hebert et al., 1983; Penner, J. L., 1988), enzymology (Elharrif, Z. and Megraud, F., 1986; Paster et al., 1991), cellular fatty acid compositions (Goodwin et al., 1985), electrophoretic protein patterns (Costas et al., 1987; Penner, J. L., 1988), random PCR-DNA fingerprinting (Eyers et al., 1993; Giesendorf et al., 1993; Giesendorf et al., 1994; and Vandamme et al., 1992), and DNA-DNA hybridization (Macario, A. J. L. and Macario, E. C. de. (eds.), 1990; and Penner, J. L., 1988). A highly specific DNA-DNA hybridization method is oligo hybridization. By varying hybridization conditions such as ionic concentration and temperature, oligo probes can detect single nucleotide sequence differences (Lee/Lane, 1992).
SUMMARY OF THE INVENTION
The present inventors have developed a method for preparing nucleic acid probes for identifying species of bacterial pathogens, and have deceloped nucleic acid probes for identifying species of Campylobacter, Helicobacter and Arcobacter.
In particular, the inventors have identified several probes that are specific for the Campylobacter species, including
Campylobacter jejuni
(
C.jejuni
),
Campylobacter coli
(
C.coli
),
Campylobacter lari
(
C.lari
) and
Campylobacter upsaliens
(
C.upsaliens
); the Helicobacter species, including
Helicobacter cinaedi
(
H. cinaedi
);
Helicobacter pylori
(
H.pylori
);
Helicobacter canis
(
H. canis
); and the Arcobacter species, including
Arcobacter nitrofigalis
(
A.nitrofigalis
);
Arcobacter butzleri
(
A. butzleri
) and
Arcobacter butzleri-like
(
A. butzleri-like
).
The probes are useful in detecting the presence of a bacterial pathogen and are further useful in determining the identity of the specific pathogen.
In one aspect, the present invention relates to an isolated nucleic acid probe for detecting or identifying
C.jejuni
. In one embodiment the probe is designated CJATC-1 and has the sequence 5′-TTTTC CGCAC ACTCA TGTAG TAAGC TCAAC TA-3′, and is identified as SEQ ID NO: 1. In another embodiment the probe is designated CJATC-2 and has the sequence 5′-GAAAA AGTAA GAGAA ATTGC TAAAA AAGAA-3′, and is identified as SEQ ID NO: 2.
In another aspect, the present invention relates to an isolated nucleic acid probe for detecting or identifying
C. coli
. In one embodiment the probe is designated CC-1 and has the sequence 5′-ATTTC CTCAT GCTCA TGTAG TAAGC TCTAC AA-3′, and is identified as SEQ ID NO: 3. In another embodiment the probe is designated CC-2 and has the sequence 5′-GAAAA AGTTA GGGAA ATTGC TCATA TTGTA-3′, and is identified as SEQ ID NO: 4.
In another aspect, the present invention relates to an isolated nucleic acid probe for detecting or identifying
C. lari
. In one embodiment the probe is designated CL-1 and has the sequence 5′-ATTCC CTTAT GCTCA TGTTG TAAGT TCT-3′, and is identified as SEQ ID NO:5. In another embodiment the probe is designated CL-2 and has the sequence 5′-GATAA AGTTA GAGAG ATAGC AAAAG AGATT-3′, and is identified as SEQ ID NO: 6.
In another aspect, the present invention relates to an isolated nucleic acid probe for detecting or identifying
C. upsaliens
. In one embodiment the probe is designated CU-1 and has the sequence 5′-TTTCC CTCAC GCACA CATCG TAAGC TCA-3′, and is identified as SEQ ID NO: 7. In another embodiment the probe is designated CU-2 and has the sequence 5′-GAAAA AGTAA GAGAA ATAGC ACACA TCGTT-3′, and is identified as SEQ ID NO: 8. In a further embodiment the probe is designated GlyA-CU and has the sequence 5′-GGT TAG TAG CTC GGG TAA AAT GTA TGA AAG C-3′ and is identified as SEQ ID NO: 15.
In another aspect, the present invention relates to an isolated nucleic acid probe for detecting or identifying
H. cinaedi
. In one embodiment the probe is designated HC-1 and has the sequence 5′-TGAGC GCGTG AAGCA GCTAT TTGGC TGTGC GT-3′, and is identified as SEQ ID NO:9.
In another aspect, the present invention relates to an isolated nucleic acid probe for detecting or identifying
H.pylori
. In one embodiment the probe is designated HP-1 and has the sequence 5′-AGAAA GGGCT AAAAA GCTTT TCAAT TGCCA GT-3′, and is identified as SEQ ID NO: 10.
In another aspect, the present invention relates to an isolated nucleic acid probe for detecting or identifying
H. canis
. In one embodiment the probe is designated GlyA-HC and has the sequence 5′-CAG GAT TGA TTA CGA CAA GCT ACG CCA AAG CGC GC-3′ and is identified as SEQ ID NO: 16. In another embodiment the probe is designated GlyA-HC2 and has the sequence 5′-TTC TGC CTA TAC AAG AGA GCT AGA TTT TGC CAA G-3′ and is identified as SEQ ID NO: 17.
In another aspect, the present invention relates to an isolated nucleic acid probe for detecting or identifying
A. nitrofigalis
. In one embodiment the probe is designated AN-1 and has the sequence 5′-AGATA GAGCT TGTGA AATTT TTGGT TGTAA AT-3′, and is identified as SEQ ID NO: 11.
In another aspect, the present invention relates to an isolated nucleic acid probe for detecting or identifying
A. Butzleri
. In one embodiment the probe is designated GlyA-AB and has the sequence 5′-GCT TCT GCA TAC GCA AGA GAA ATT GAT TCA AA- 3′ and is identified as SEQ ID NO: 12.
In another aspect, the present invention relates to an isolated nucleic acid probe for detecting or identifying
A. Butzleri
-like. In one embodiment the probe is designated GlyA-BL and has the sequence 5′-GCA AGT GCA TAT GCA AGA GAG ATT GAT TTT AA-3′ and is identified as SEQ ID NO: 13. In another embodiment the probe is designated GlyA-BL2 and has the sequence 5′-AAG TAA ACC AAG CTT TTC AGG GCA AAA CTA CTC T-3′ and is identified as SEQ ID NO: 14.
The nucleic acid probes of the present invention permit the detection and identification of pathogenic bacteria in various samples including biological, food, or environmental samples.
Accordingly, the invention provides a method for detecting the presence of a specific bacteria in a sample comprising contacting the nucleic acid molecules of the sample with a nucleic acid probe according to the present invention and determining if the sample hybridizes with the nucl

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