Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1997-10-23
2000-12-05
Myers, Carla J.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 912, 435 9121, 435 915, 435 9151, 536 237, 536 2433, 536 2432, C12Q 168, C12P 1934, C07H 2104
Patent
active
061565075
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
This invention relates to a diagnostic method for performing a genetic diagnosis of a resistant bacterium, especially for specifically detecting at high sensitivity methicillin-resistant Staphylococcus aureus (hereinafter referred to as "MRSA") and methicillin-resistant coagulase-negative staphylococci (hereinafter referred to as "MRC-NS"). Namely the present invention is concerned with a diagnostic method which can promptly detect and identify MRSA and MRC-NS.
BACKGROUND ART
MRSA and MRC-NS, including Staphylococcus haemolyticus and Staphylococcus epidermidis, are principal pathogenic bacteria of nosocomial infection at hospitals in all the countries of the world, and have become a serious clinical problem due to the limited availability of effective antibiotics. In clinical activities, their accurate and speedy identification has become an important theme for the diagnosis and treatment of infected patients.
MRSA is Staphylococcus aureus which produces PBP(penicillin-binding protein)2' (or PBP2a), that is, a cell-wall synthesizing enzyme PBP having low affinity to all .beta.-lactam antibiotics developed to date led by methicillin. Because of the production of this PBP2', MRSA exhibits resistance to all the conventional .beta.-lactam antibiotics. Since the report of its first clinical strain in England in 1961, it has spread around the whole world and at present, it has become, as a nosocomial infectious bacterium, a serious problem for the present-day medical treatments at hospitals in all the countries of the world.
MRSA produces PBP2' in addition to four PBPs which Staphylococcus aureus inherently have. In 1986, the mecA gene encoding this PBP was cloned by Matsuhashi et al. and its entire base sequence was determined by them. The mecA gene exists on chromosomes of MRSA and MRC-NS, but is not found on methicillin-susceptible Staphylococcus aureus (MSSA). Accordingly, the mecA gene is considered to be a gene adventitiously acquired on the chromosomes of Staphylococcus aureus. Detection of this mecA gene on the chromosomal DNA of Staphylococcus aureus, generally by PCR (polymerase chain reaction) or hybridization, makes it possible to identify it as MRSA or MRC-NS.
In Japan, a mecA identification kit by ED-PCR (enzyme detection PCR) was developed, and subsequent to its approval and the setting of its health insurance price by the Ministry of Health and Welfare, is now frequently used for clinical diagnoses (Ubukata, K., et al. J. Clin. Microbiol. 30, 1728-1733, 1992). However, the identification of MRSA by this method involves at least the following two problems.
1) It can be used only after a bacterium from a patient's sample has been cultured and procedures have then been conducted beforehand for the strain identification of Staphylococcus aureus. The above method therefore has a drawback of lack of promptness and creates various problems.
Namely, the mecA gene is also distributed widely in other strains of the genus of Staphylococcus (S.) epidermidis, S. haemolyticus, S. saprophyticus, S. capitis, S. warneri, S. sciuri and S. caprae (Eiko Suzuki et al. Antimicrb. Agents Chemother. 37, 1219-1226, 1993). In a patient's sample, these mecA-containing Staphylococcus strains are detected at the same time in many instances. Accordingly, direct detection of the mecA gene from a sample cannot be taken as a proof of the existence of MRSA. This has led to a limitation of the detection method of the mecA gene having to be used after a strain has been cultured from a patient's sample and has then been confirmed to be Staphylococcus aureus by a conventional strain identification method. Accordingly, there has been no choice other than to rely upon an empiric therapy until an infected strain is identified. Further, administration of vancomycin has been indispensable even if the administration is eventually found to have been unnecessary.
2) Lack of internal control in PCR. Described specifically, false positive or false negative may be determined in PCR operations of nowadays, depending on the wo
REFERENCES:
patent: 5702895 (1997-12-01), Mtsunaga et al.
Hiramatsu et al. FEBS. 298:133-136, Feb. 1992.
Archer et al. Antimicrobial Agents and Chemotherapy. 38:447-454, Mar. 1994.
Barberis-Maino et al. Gene. 59:107-113, 1987.
Awaya Akira
Hayashi Tsukasa
Hiramatsu Keiichi
Ito Teruyo
Ohno Hiroie
Kainos Laboratories, Inc.
Myers Carla J.
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