Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-07-02
2001-08-14
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C536S023100, C536S024300
Reexamination Certificate
active
06274316
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to probe sequences and methods for detecting target nucleic acid molecules, and more specifically, to probes for detecting antibiotic vancomycin resistant enterococci (“VRE”) and methods thereof.
BACKGROUND OF THE INVENTION
Vancomycin resistant enterococci (VRE) represents a serious problem for healthcare worldwide. For example, the Center for Disease Control (CDC) has released data for antibiotic resistance associated with hospital-caused infections from January 1989 to March 1993, showing a 20-fold increase in the percentage of enterococci that were resistant to the antibiotic vancomycin (MMMWR 42:597-599, 1993) during this period. Both vanA and vanB genes of enterococci have been found to be associated with the increased resistance.
Transfer of the vanA and vanB antibiotic resistance genes to non-enterococcal species is also a growing concern. The vanA gene has been found in Corynebacterium, Arcanobacterium and Lactococcus species (Power et al.
J. Antimicrobiol. Chemother.
36:595-606, 1995). Recently, Poyart et al. (
Antimicrobiol. Agents Chemotherap.
41:24-29, 1997), reported an occurrence of a Streptococcus bovis clinical isolate with a VanB resistance phenotype. The gene was shown to be highly homologous to the prototype vanB gene from Enterococcus.
Increased use of antibiotics has resulted in the emergence of vancomycin-resistant microorganisms such as Enterococcus spp. and Staphylococcus spp. (Dutka-Malen et al.,
Antimicrobiol. Agents Chemother.
34:1875-1879, 1990). Vancomycin-resistant
S. aureus
(VISA) is certain to emerge in hospitals with high rates of methicillin resistant
Staphylococcus aureus
(MRSA) and the use of vancomycin (Edmond et al.,
Ann. Intern. Med.
124:329-334, 1996). Recently, VISA isolates have been reported in Latin America (Navarro Marin,
International Journal of Antimicrobial Agents
7:293-294, 1996).
Briefly, there are four phenotypes of enterococci that can be separated based on expression of constitutive and inducible resistance of the glycopeptides, vancomycin and teicoplanin (Leclercq and Courvalin,
Clin. Infec. Dis.
24:545-556, 1997). Inducible resistance to high levels of vancomycin (MIC≧64 mg/l) and teicoplanin (MIC≧16 mg/l) is characteristic of the VanA phenotype. This type of resistance is plasmid mediated. The vanA gene has recently been found on mobile elements that can direct their own transfer from the chromosome of one Enterococcus strain to another. The VanB phenotype is described as inducible resistant to vancomycin with MIC of 4 mg/l to ≧1,000 mg/l but displaying susceptibility to teicoplanin. The vanB gene is transferable by conjugation in certain strains. The genes in the VanC phenotype produce constitutive resistance and occur in
E. gallinarum
and
E. casseliflavis
and
E. flavenscens
(Leclercq and Courvalin Supra; Navarro & Courvalin,
Antimirobiol. Agents Chemother.
38:1788-1793, 1994). Recently, VanD phenotype has been reported and is characterized by moderate levels of vancomycin resistance and low level resistance to teicoplanin (cited in Leclercq and Courvalin Supra).
The majority of conventional methods for detection of glycopeptide resistant enterococci have drawbacks related to time, lack of specificity and sensitivity of detection. For example, detection of the glycopeptide resistant enterococci can be carried out by conventional susceptibility testing (broth and agar methods), but these techniques are slow, and automated detection is not recommended due to poor performance (Aarestrup et al.,
Antimicrob. Agents Chemother.
40:1938-1940, 1996). Although the above methods can be used to detect VRE, there is an urgent need for a rapid, user friendly and reliable method for detecting the vanA gene and vanB genes from VRE, both in the hospital and community settings. The present invention provides probes and methods for detecting the vanA and vanB genes rapidly. Further, the present invention provides other related advantages.
SUMMARY OF THE INVENTION
Briefly stated, the present invention provides compositions and methods for detecting vanA and vanB genes from enterococci.
Within one aspect of the present invention, methods are provided for determining the presence of vancomycin antibiotic resistant gene(s) of enterococci in a biological sample, comprising the steps of (a) treating cells contained within the biological sample to expose single stranded-target nucleic acid molecules; (b) reacting said target single-stranded cellular nucleic acids with one or more scissile-link containing nucleic acid probe(s) which are complementary to one or more portion(s) of the antibiotic vancomycin resistant gene, and with an enzyme molecule, under conditions, which allow the target and probe to hybridize to each other and form one or more double-stranded, target-probe complex(es), said enzyme molecule being capable of cleaving said scissile link of said target-probe complex(es) such that one or more fragments of the nucleic acid probe is released from said complex(es); and (c) determining whether cleaved portions of the nucleic acid probe are produced, and thereby detecting the presence of a vancomycin antibiotic resistant gene. Within various embodiments, determination of whether cleaved probe is produced can be accomplished by directly detecting cleaved portions of the nucleic acid probe, and/or detecting a decrease in the amount of uncleaved probe.
Within various embodiments, the scissile-link containing nucleic acid probe is complementary to a vancomycin resistant gene selected from the group consisting of vanA, vanB, vanB2, vanC1, vanC2, vanC3, vanD, or variants thereof. Within further embodiments, more than one probe may be utilized in order to multiplex, or detect more than one gene per reaction. Representative examples of suitable probes include: TTAATAACCC aaaaGGCGGG AGTAGCT (SEQ ID NO:1); TACATTCTTA CaaaaAATGC GGGCATC (SEQ ID NO:3); GAGGAACgaa aTCGGGTGCA (SEQ ID NO:7); and GCCGACAGTC TccccGCCA TACTCTCC (SEQ ID NO:9).
Within further embodiments, a single probe may be utilized in order to detect multiple genes (e.g., any one of the vanA, van B or vanB2) per reaction. Representative examples of suitable probes include CN
1
CAN
2
CCG ACCTCacagC CCGAAA (SEQ ID NO:17) and modifications thereof, wherein N
1
and N
2
can be a combination of bases typical of vanA, vanB and vanB2, abasic sites or universal bases.
Within other related aspects of the present invention, probes for detecting the presence of a vancomycin antibiotic resistant gene in a biological sample are provided, wherein said probe comprises at least a portion of a sequence which specifically recognizes a vancomycin resistant gene (e.g., Sequence ID Nos.1, 3, 9 or 17). Also provided are kits which comprise such probes, along with an enzyme (e.g., RNase H) which cleaves scissile links.
Also provided by the present invention are kits for detecting the presence of a vancomycin-resistant gene in a biological sample, comprising (a) one or more scissile-link containing nucleic acid probes which bind to a vancomycin-resistant gene, and (b) an enzyme capable of cleaving the scissile link when the probe is bound to the target. Within a further embodiment, the gene is selected from group consisting of vanA, vanB, vanB2, vanC1, vanC2, vanC3, vanD, and variants thereof. Within a related embodiment, more than one vancomycin antibiotic resistant gene is detected simultaneously. With further embodiments, the enzyme is RNase H.
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Patel et al “Multiplex PCR detection . . .
ID Biomedical Corporation
Jones W. Gary
Seed Intellectual Property Law Group PLLC
Souaya Jehanne
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