Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-07-02
2003-01-07
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S024000, C536S024300, C536S024320, C536S023100
Reexamination Certificate
active
06503709
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to medical diagnostics, and more specifically, to methods, probes and primers for detecting the mecA gene of methicillin resistant staphylococci.
BACKGROUND OF THE INVENTION
Methicillin-resistant
Staphylococcus aureus
(MRSA) is an extremely virulent antibiotic-resistant form of
Staphylococcus aureus
which has been rapidly spreading worldwide. MRSA has been found to be common in many hospitals, with a prevalence ranging from less than 1% to greater than 80% (Pittet and Waldvogel,
Q. J. Med.
239-241, 1997). Unfortunately, MRSA is no longer considered only a hospital associated pathogen, since community-acquired MRSA is also increasing.
Resistance to methicillin by
Staphylococcus aureus
is caused by the expression of a low affinity penicillin binding protein (PBP) called PBP2a or PBP2′, in addition to the usual PBPs (Hartman and Tomasz,
J. Bacteriol.
158:513-516; 1984; Murakami and Tomasz,
J. Bacteriol.
171:874-879, 1989). The responsible mecA gene which encodes the PBP2′, mecA, is present in resistant strains but not susceptible ones (Hartman and Tomasz,
J. Bacteriol.
158:513-516, 1984; Matsuhashi et al.,
J. Bacteriol.
167: 975-980, 1986), and has only a slight sequence variation from strain to strain (Ryffel et al.,
Gene
94:137-138, 1990). MRSA strains carrying the mecA gene also tend to demonstrate resistance to other antibiotics such as aminoglycosides, macrolides, quinolones, beta-lactams, monobactams, imipenem, meropenem (Maple et al.,
Lancet
I:537-540, 1989; Kayser,
Chemotherapy
42 (
suppl
2):2-12; Chambers et al. cited in Bowler,
Q. J Med.
90:243-246, 1997).
Some
S. aureus
strains may exhibit heterogeneity of expression of the mecA gene (Hartman et al.,
Antimicrob. Agents Chemother.
29:85-92, 1986; Hiramatsu et al.,
Microbiol. Immunol.
36:445-453, 1992; Ryffel et al., Antimicrob. Agents Chemother. 38:724-728, 1994). Within these populations, although all cells carry mecA, the majority of cells are susceptible to low concentrations of the antibiotic and only a minority of the cells (1 in 105-106 ) express resistance (Hackbarth et al.,
Antimicrob. Agents Chemother.
33:991-994, 1989; Ryffel et al.,
Antimicrob. Agents Chemother.
38:724-728, 1994). Consequently, these strains exhibit low MIC and may be incorrectly characterized as sensitive to methicillin. Conversely, some
S. aureus
strains that lack the mecA gene may display borderline or low level resistance to methicillin due to alternate mechanisms of resistance (Chambers et al.,
Antimicrob. Agents Chemother.
33:424-428, 1989; Hiramatsu et al., Supra). Low level resistance to methicillin due to hyper &bgr;-lactamase production, methicillinase production, or the synthesis of modified PBP (Knapp, et al,
J. Clin. Microbiol.
34:1603-1605, 1996; McDougal, et al.,
J. Clin. Microbiol.
23:832-839, 1986; Tomasz et al.,
Antimicrob. Agents Chemother.
33:1869-1874, 1989.) may be incorrectly diagnosed as MRSA by conventional susceptibility tests.
Rapid detection, both for prevention of transmission and treatment of methicillin resistant Staphylococcus (MRS) species has become a worldwide priority. There are a number of techniques available in the diagnostic field for detecting MRS, including conventional biochemical tests and immunological tests. For example, one accepted method for detection of MRS is the screening for isolates on Mueller-Hinton agar containing 4% NaCl and 6 &mgr;g/ml of oxacillin. Many laboratories now use automated screening systems such as Microscan API (Dade) and Vitek GPS-SA card (bioMerieux, Hazlewood, Mo.) which give susceptibility results within 24 hours (Knapp et al., Supra). The BBL® Crystal™ MRSA ID test (Beckton Dickinson, Cockeysville, Md.) is a more rapid susceptibility test that permits diagnosis after 4 to 6 hours (Wallet et al.
J. Antimicrobiol. Chemother.
37:901-909; Martinez et al,
Rev. Esp. Quimioterap.
9:130-133, 1996). However, none of the susceptibility tests can reliably differentiate between heterogeneous MRSA and borderline oxacillin resistant
S. aureus
(BORSA, Knapp et al., Supra; Lencastre et al.,
Eur. J. Clin. Microbiol. Infect. Dis.
12:S13-S18).
Many of these techniques also have drawbacks related to time, lack of specificity and sensitivity of detection. To address such issues, detection of the mecA gene utilizing the polymerase chain reaction (“PCR”) has also been attempted. Briefly, PCR has been used in several studies for detecting the mecA gene, but test results using this method are expensive and can take up to 6 hours (Wallet, Supra). In addition, there are a number of problems associated with such methods, including background contamination, carry over contamination, thermocycling, special room requirements, cumbersome from the point of view of the number of primers and probes required, number of steps and time involved in processing the samples and the special training required. In spite of the wide use of PCR in the molecular biology area due to its high sensitivity and multiple applications there are only a few FDA approved PCR or other target amplification based diagnostic products. The main factor limiting the use of these technologies in clinical diagnostic setting is the inherent problem of amplicon contamination. There are several chemical or physical methods that were developed to reduce or eliminate the problem of contamination. Generally these methods add extra steps and are costly.
Although the above methods can be used to detect MRSA, there is an urgent need for a rapid, user friendly and reliable method for detecting the mecA gene in nosocomial and non-nosocomial settings. The present invention provides probes, primers and methods for detecting the mecA gene that meet these needs. Further, the present invention provides other, related advantages.
SUMMARY OF THE INVENTION
Briefly stated, the present invention provides compositions and methods for detecting the mecA gene of the methicillin resistant staphylococci.
Within one aspect of the present invention, methods are provided for determining the presence of an antibiotic resistant mecA gene in a biological sample, comprising the steps of (a) treating cells contained within a biological sample to expose target single-stranded nucleic acid molecule, (b) reacting target single-stranded nucleic acids with a scissile link-containing nucleic acid probe which is complementary to a portion of an antibiotic resistant mecA gene, and with an enzyme which cleaves double-stranded target-probe complexes, under conditions which allow the target and probe to hybridize to each other to form a double-stranded target-probe complex, the enzyme molecule being capable of cleaving the scissile link of the target-probe complex such that one or more fragments of the nucleic acid probe released from said complex, and (c) determining whether cleaved portions of the nucleic acid probe are produced, and thereby detecting the presence of an antibiotic resistant mecA 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 one embodiment, the probe comprises at least a portion of the nucleotide sequence GACGATAATA GCAATACAAT CGCACATACA TTAATAGAGA AAAAGAAAAA AGATGGCAAA GATATTCAAC TAACTATTGA TGCTAAAGTT CAAAAGAGTA TTTATAAC (SEQ ID NO:13). Within a further embodiment, the probe consists essentially of at least a portion of the nucleotide sequence GAACTTTAGC ATCAATAGTT AGTTGAATAT CTTTGCCATC TTTTTTCTTT TTCTCTATTA ATGTATGTGC GATTGTATTG CTATTATCG (SEQ ID NO:4). Other representative probes include: AATAGAGAAA AAGAAAAAAG ATGGCAAAG (SEQ ID NO:1); and AATAGAGaaaaAGAAAAA AGATGGCAAAG-3′(SEQ ID NO:5) wherein large letters represent deoxyribonucleotides and small letters represent ribonucleotides. As utilized herein, a probe should be at least 8 nucleotides in length, and may be 10, 12, 14, 15, 16, 18, 20, 30 or even 100 or more nucleotides in length.
Within
Bekkaoui Faouzi
Cloney Lynn P.
Einsmann Juliet
ID Biomedical Corporation
Jones W. Gary
Seed IP Law Group PLLC
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