Probes for detecting and identifying Helicobacter pylori

Details Probes for detecting and identifying Helicobacter pylori Probes for detecting and identifying Helicobacter pylori

1999-07-12

2001-01-09

Fredman, Jeffrey (Department: 1655)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S023100, C536S024330, C435S006120

Reexamination Certificate

active

06172215

ABSTRACT:

TECHNICAL FIELD
The present invention relates to probes useful for detecting and identifying
Helicobacter pylori
, the causative bacteria of the digestive diseases including gastritis, gastric ulcer, duodenal ulcer or the like.
BACKGROUND ART
Since Warren's group reported the existence of
Helicobacter pylori
[Former Name:
Campylobacter pylori
] in the human gastric mucosal (Warren J. R. et al., “Unidentified curved bacilli on gastric epithelium in active chronic gastritis”, Lancet 1: 1273-1275 (1983)), numerous researches have been performed with regard to the biochemical properties thereof, in particular, the correlation between
Helicobacter pylori
and digestive diseases including gastric ulcer, duodenal ulcer or the like.
In view of the focus that
Helicobacter pylori
have been separated/detected at a high rate from the human gastric mucosa of the gastritis patients or the gastriculcer patients [e.g., there are reports that they were detected at a rate of 50~80% in the cases of chronic gastritis, superficial gastritis, atrophic gastritis, erosive gastritis or the like], and the symptom of these digestive diseases are alleviated with sterilization by administration of drugs, accordingly, the correlation between
Helicobacter pylori
and the digestive diseases were suggested.
Helicobacter pylori
would not make an invasion upon mucosal cells, but stay on the epithelial mucosa surface and/or the intercellular space and grow (proliferate) thereat. Then, it is thought that PAS (Periodic Acid-Sciff) reaction positive layer in the gastric mucosa are thinned through growth of
Helicobacter pylori
, thereby, effects of mucin which protects mucosa are declined, and potency on defense factor of the gastric mucosa are also declined (T. Ito, “Recent findings on
Helicobacter pylori
”, Medical Technology, 19 (10), pp. 892-893 (September 1991)).
Then, the mechanism was also reported that
Helicobacter pylori
arrive and stay in gastric mucosal epithelium, then ammonia were produced through degradation of urea in the stomach by urease from
Helicobacter pylori
, the ammonia so produced damage gastric mucosa and generate reverse diffusion of the hydrogen ion, and, tumors are thereby formed (Tsujii, M. et al., “Mechanism of gastric mucosal damage Induced by ammonia”, Gastroenterology 107: pp. 1881-1888 (1992)).
In general, conventional diagnosis of
Helicobacter pylori
, which is correlative to the human digestive diseases, includes:
(1) Direct proof on presence of
Helicobacter pylori
in a part of mucosa [Smear, Tissue-Microscopy, Cultivation],
(2) Detection utilizing character of
Helicobacter pylori
including urease activities, and
(3) Seroimmunodiagnosis (T. Shirai et al., “Diagnosis on Presence of
Campylobacter pylori
”, Saishin-Igaku, 44 (2), 284-288 (1989)).
Of the methods aforenoted, a method (cultivation method) for detecting
Helicobacter pylori
through micro-aerobic cultivation of biopsy sample on gastric mucosa is the most reliable and accurate method. But, this method usually needs about one hour for a cultivation to grow the bacteria, the longer time would therefore be necessary to obtain the test results.
Then, in consideration of urease productivity by
Helicobacter pylori
, a method for directly detecting urease in samples of gastric biopsy have also been utilized (T. Ito, “Special diagnosis for bacterial infection/4.
Campylobacter pylori
”, Rinnshoui, 15 (supplement), pp. 367-369 (1989). However, since these are methods to test the biopsy samples with an endoscope, the methods need the skilled operation and the patients will suffer from the unbearable pain.
Further, since urea in the stomach are degraded into ammonia and
14
CO
2
, there is a diagnosis including evaluation of such
14
CO
2
by a scintillation counter. But, this method also needs a skilled work to handle radioisotope.
DISCLOSURE OF INVENTION
In view of the aforenoted problems in the art, the present invention was established to develop technology for easily, specifically and effectively detect
Helicobacter pylori
without introducing into patient's body any medical instrument like an endoscope.
Then, the merit of the invention is directed to probes having specific reactivities to DNAs or RNAs of
Helicobacter pylori
which is the causative bacteria of the digestive diseases including gastritis, gastric ulcer, duodenal ulcer or the like, and to analysis of base sequences in the probes concerning the characteristic gene portion of
Helicobacter pylori.
That is to say, DNAs of the subject bacteria,
Helicobacter pylori
, can significantly be detected by the specificity between the probes of the present invention and the bacteria DNAs, thereby,
Helicobacter pylori
can be detected/identified rapidly and exactly without cultivating/growing
Helicobacter pylori.
Then, if primers are designed based on base-sequence information of these probes,
Helicobacter pylori
can be identified by amplifying DNAs with PCR techniques without performing any hybridization procedure.
When non-radioactive probes, for example, biotinylated probes are employed for hybridization, since such probes can be detected with an optical microscope in a conventional laboratory without facilities to handle radioisotype, thereby, a process for detecting bacteria can perform rapidly and simply.


REFERENCES:
Database GenBank Rel. 100, National Center for Biotechnology Information, Accession U75328, Jan. 2, 1997.
Database GenBank Rel. 100. National Center for Biotechnology Information, Accession U86610, Feb. 13, 1997.
Desai et al., “Genetic diversity ofHelicobacter pyloriindexed with respect to clinical symptomology, using a 16S rRNA and a species-specific DNA probe”J. Applied Bacteriologyvol. 75, pp. 574-582 (1993).
Li et al., “A Highly Specific and Sensitive DNA Probe Derived from Chromosomal DNA ofHelicobacter pyloriIs Useful for TypingH. pyloriIsolates”J. Clinical Microbiologyvol. 31, pp. 2157-2162 (Aug. 1993).

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