Oligonucleotide for detection and identification of...

Details Oligonucleotide for detection and identification of... Oligonucleotide for detection and identification of...

2001-11-28

2003-12-30

Horlick, Kenneth R. (Department: 1637)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S007200, C435S091100, C435S091200, C536S023100, C536S023700, C536S024100, C536S024300, C536S024320, C536S024330

Reexamination Certificate

active

06670130

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to oligonucleotides that can be used for detection and identification of mycobacteria. More particularly, the present invention identifies the nucleotide sequence of ITS (Internal Transcribed Spacer region) of non-tuberculosis mycobacteria,
Mycobacterium fortuitum, Mycobacterium chelonae, Mycobacterium abscessus, Mycobacterium vaccae, Mycobacterium flavescens, Mycobacterium asiaticum, Mycobacterium porcinum, Mycobacterium acapulcensis
and
Mycobacterium diernhoferi
, and using the nucleotide sequences, it provides oligonucleotide primers or probes used for detection and identification of mycobacteria.
2. Description of the Related Art
A Even though the number of patients of tuberculosis has steadily decreased in these days, about 8 million patients have come out and about 3 million patients died of tuberculosis in a year. Moreover, in underdeveloped countries, inadequate treatment and lack of drugs for tuberculosis increase chronic carriers of drug-resistant bacteria. In 1980's, spread of AIDS has increased patients of tuberculosis even in advanced countries. In this condition, it is expected that about twelve million patents of tuberculosis would newly come out in the year 2000 (J. P. Natain, M. C. Raviglione, and A. Kochi,
Tubercle and Lung Disease
, 73: 311-321, 1992; Murray C J L. And Lopez A D. The global burden of disease.
Global burden of disease and injury series. Vol.
1. Cambridge, Mass.: Harvard University Press, 1996, p349-350; Global Tuberculosis Programme: Anti-tuberculosis drug resistance, WHO Report 1997: World Health Organization. 1997).
In 1950's, it was reported that non-tuberculosis mycobacteria (NTM) has been able to cause diseases in human. After the report that
M. avium
complex (MAC) would bring about systemic disease in the patients of AIDS in 1980, non-tuberculosis mycobacteria have taken an interest. Diseases caused by non-tuberculosis mycobacteria are similar to tuberculosis in clinical condition and general pathological view. Non-tuberculosis mycobacteria are distributed in a wide range of living environment, and it is difficult to judge whether they have pathogenicity or not in clinical test sample. Further, since they have resistance to a number of drugs for tuberculosis, the infection is hard to treat and the recurrence rate is high. The infection of non-tuberculosis mycobacteria should be treated by other means than for tuberculosis, and therefore, accurate and fast method of detecting and identifying non-tuberculosis mycobacteria is required. The accurate and fast method of detecting and identifying both TB complex and NTM is also needed for effective treatment and management of tuberculosis.
Many a method has been developed to diagnose mycobacterial infection and to detect and identify mycobacteria strains. Among them, the following methods are used at present;
The first is a microbiological method, that is, smearing, staining and culturing test. However, this method is not suitable for mycobacteria, since they have long generation term and need long culturing time. Further, such pathogenic microorganism as mycobacteria is dangerous to infect the personnel in culture room;
The second is a PCR (Polymerase Chain Reaction) method. It is highly sensitive and specific to the mycobacteria and very useful to detect mycobacteria which have a long culturing time. Especially, it does not require a culturing process but uses a small amount of DNA to be amplified, therefore, only a small amount of pathogens in test sample is enough to detect and identify mycobacteria. Many a PCR process has been introduced with different target DNAs each other, and IS6110 and 16S rRNA are often used as the target (Bauer J, Andersen A B, Kremer K, and Miorner H, Usefulness of spoligotyping to discriminate IS6110 low-copy-number
Mycobacterium tuberculosis
complex strains cultured in Denmark, 1999
, J. Clin. Microbiol
. 37: 2602-2606; Troesch, A., H. Nguyen, C. G. Miyada, S. Desvarenne, T. R. Gingeras, P. M. Kaplan, P. Cros and C. Mabilat. 1999, Mycobacterium species identification and rifampin resistance testing with high-density DNA probe arrays,
J. Clin. Microbiol
. 37: 49-55);
The third is a physico-chemical process, in which lipid component in mycobacteria has been detected by HPLC, GC or mass spectrophotometer. This method is very specific but rquires expensive equipments;
The fourth is a method of detecting mycobacteria composition by serological method. This method uses a coagulation reaction of latex particles or blood corpuscles adsorbed with antibody to mycobacterial antigen or enzyme-linked immunological method in which enzyme is linked with antibody. It is, however, very sensitive only to be proceeded within a limited place. Further, it is difficult for this method to distinguish present infection from previous infection;
The next method to detect mycobacteria consists of infecting mycobacteria with mycobacteriophage L5 inserted with luciferase gene, and inspecting luminescence by luciferin in medium (W. R. Jacobs, R. G. Barletta, R. Udani, J. Chan, G. Kalkut, G. Sosne, T. Kieser, G. J. Sarkis, G. F. Hatful, and B. R. Bloom. 1993
, Science
260: 819-822); and
The last is a method of detecting and identifying mycobacteria by hybridization of oligonucleotide (A. Troesch, H. Nguyen, C. G. Miyada, S. Desvarenne, T. R. Gingeras, P. M. Kaplan, P. Cros and C. Mabilat. 1999
. J. Clin. Microbiol
37: 49-55).
Besides
Mycobacierium avium
complex (MAC) described above,
M. Fortuitum, M. chelonae
complex,
M. terrae
and
M. vaccae
are also known as non-tuberculosis mycobacteria. Among them,
M. chelonae
complex are classified into
M. chelonae
and
M. abscessus
, and there is no means to distinguish one from the other at present.
SUMMARY OF THE INVENTION
To solve the problems in the prior method of detection and identification of mycobacteria, it is an objective of the present invention to provide specific oligonucleotides as probes or primers for PCR which can be used to detect mycobacteria, to distinguish TB complex from NTM, and to identify species of mycobacteria with an accuracy and effectiveness.
To accomplish the above objective, the present invention provides a DNA of ITS (Internal Transcribed Spacer region) of
Mycobacterium fortuitum, Mycobacterium chelonae, Mycobacterium abscessus, Mycobacterium vaccae, Mycobacterium flavescens, Mycobacterium asiaticum, Mycobacterium porcinum, Mycobacterium acapulcensis
and
Mycobacterium diernhoferi
genes set forth in SEQ ID NOs: 1 to 9.
Further, the present invention provides, as a primer for PCR or a probe for hybridization, an oligonucleotide for detection of mycobacteria set in forth in one of SEQ ID NOs: 10 to 14;
an oligonucleotide for distinction of TB complex from NTB among mycobacteria set in forth in one of SEQ ID NOs: 15 to 23;
an oligonucleotide for detection and identification of MAC (
Mycobacterium avium
and
Mycobacterium intracellulare
) set in forth in one of SEQ ID NOs: 24 to 27;
an oligonucleotide for detection and identification of
Mycobacterium fortuitum
set in forth in one of SEQ ID NOs: 28 to 38;
an oligonucleotide for detection and identification of
Mycobacterium chelonae
set in forth in one of SEQ ID NOs: 39 to 46;
an oligonucleotide for detection and identification of
Mycobacterium abscessus
set in forth in one of SEQ ID NOs: 47 to 52;
an oligonucleotide for detection and identification of
Mycobacterium vaccae
set in forth in one of SEQ ID NOs: 53 to 64;
an oligonucleotide for detection and identification of
Mycobacterium flavescens
set in forth in one of SEQ ID NOs: 65 to 72;
an oligonucleotide for detection and identification of
Mycobacterium gordonae
set in forth in one of SEQ ID NOs: 73 to 77;
an oligonucleotide for detection and identification of
Mycobacterium terrae
set in forth in one of SEQ ID NOs: 78 to 100;
an oligonucleotide for detection and identification of
Mycobacterium scrofulaceum
set in forth in one of SEQ ID NOs: 101 to 108;
an oligonucleotide for detection

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