Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1991-02-11
1998-10-06
Marschel, Ardin H.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 693, 435 912, 4351723, 436501, 536 231, 536 241, 536 243, 536 2431, 536 2432, 536 2433, 935 9, 935 77, 935 78, C12Q 168, C07H 2104
Patent
active
058174591
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to nucleotide sequences of Actinomycetales, in particular of mycobacteria, to oligonucleotides contained within the said sequences, to their uses as primers for the synthesis of Actinomycetales DNA and as probes for the detection of DNA and/or of the transcription products of Actinomycetales, in particular of mycobacteria, to the products of expression of the said sequences, to their uses and to antibodies directed towards the said products, to a method for detecting and identifying Actinomycetales and its uses, as well as to immunogenic compositions comprising the said expression products.
Tuberculosis and leprosy are known to be major public health problems. They are currently approximately 60.times.10.sup.6 individuals suffering from tuberculosis in the world (with an annual mortality of 3.times.10.sup.6), and approximately 15.times.10.sup.6 individuals suffering from leprosy. In France, approximately 10.sup.4 new cases of tuberculosis appear every year. Vaccination with BCG (Bacillus Calmette-Guerin, an attenuated strain of M. bovis) is far from effective in all populations. This efficacy varies approximately from 80% in Western countries such as England to 0% in India (results of the latest vaccination trial in Chingleput). The appearance of strains of M. tuberculosis resistant to the usual antituberculosis agents and the existence of mycobacterioses due to other, increasingly common mycobacteria such as M. avium, especially in patients with immunosuppression (AIDS in the largest number of cases), add to the urgency of developing a rapid method of detecting and identifying mycobacteria.
The diagnosis of tuberculosis and other related mycobacterioses is difficult to carry out; in effect the microorganisms responsible for these diseases are often present in small amounts, and when the amount of them is detectable by the methods conventionally used, the disease is already progressing and the patients are contagious to those around them. As a result of the very long generation time of these bacteria (24 h for M. tuberculosis compared with 20 min for E. coli), culturing these organisms is difficult. Thus, it requires 6 to 8 weeks to identify the microorganisms, and longer to obtain an antibiogram usable for appropriate treatment of the patients. The need for a detection test not requiring culturing of the microorganisms, and directly usable with the pathological samples even when the microorganisms are present therein at low concentrations, is hence essential.
Several techniques are currently used in clinical practice for identifying a mycobacterial infection.
In the first place, direct detection of the microorganisms in the microscope should be mentioned; this technique is rapid, but does not permit identification of the mycobacterial species observed, and lacks sensitivity in as much as a large number of microorganisms have to be present in the sample (>10.sup.4 /ml) in order to permit reliable detection (RATES J., CHEST, 1979, 76, (suppl.), 757-763).
Cultures, when positive, have a specificity approaching 100%, and permit identification of the mycobacterial species isolated; nevertheless, as specified above, growth of mycobacteria in vitro can be achieved only in the space of 3 to 6 weeks, and when few mycobacteria are present at the site of infection, repeated culturing is necessary in order to ensure a positive result (BATES J., 1979 and BATES J. et al., Am. Rev. Respir. Dis., 1986, 134, 415-417).
Serological techniques can prove useful under some conditions, but their use is limited by their low sensitivity and/or their low specificity (DANIEL T. M. et al., Am. Rev. Respir. Dis., 1987, 135, 1137-1151).
The presence or absence of mycobacteria may also be determined by hybridisation with DNA or RNA using probes specific for DNA sequences (KIEHN T. E. et al., J. Clin. Microbiol., 1987, 25, 1551-1552; ROBERTS M. C. et al., J. Clin. Microbiol., 1987, 25, 1239-1243; DRAKE T. A. et al., J. Clin. Microbiol., 1987, 25, 1442-1445). However, these methods also require culturing
REFERENCES:
patent: 4358535 (1982-11-01), Falkow et al.
Tubercle, vol. 69, No. 1, Mar. 1988, Longman Group UK Ltd., (Londres, GB), C.C. Pao et al.: "The detection of mycobacterial DNA sequences in uncultured clinical specimens with cloned Mycobacterium tuberculosis DNA as probes", pp. 27-36.
Molecular and Cellular Probes, vol. 2, 1988, Academic Press Ltd, R.N. Picken et al.: "DNA probes for mycobacteria. I. Isolation of DNA probes for the identification of Mycobacterium tuberculosis complex and for mycobacteria other than Tuberculosis (MOTT)" pp. 111-124.
Molecular Microbiology, vol. 3, No. 7, 1989, (New York, US), A.J. Hance et al.: "Detection and identification of mycobacteria by amplification of mycobacterial DNA" pp. 843-849.
Abstracts of the Annual Meeting of the American Society for Microbiology, vol. 89, No. 0, 1989, (US), B.B. Plikaytis et al.: "Rapid, sensitive and specific detection of mycobacteria using gene amplification techniques", Abstract U-43.
Gicquel Brigitte
Grandchamp-Desraux Bernard
Hance Allan Johnson
Levy-Frebault Veronique
Brunelle Jan P.
Dreger Walter H.
Institut National de la Sante et de la Recherche Mediale-Inserm
Institute Pasteur
Marschel Ardin H.
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