Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1999-01-06
2000-04-11
Myers, Carla J.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 912, 536 232, 536 237, 536 2432, 536 2433, C12Q 168, C12P 1934, C07H 2104
Patent
active
06048697&
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention relates to oligonucleotide primers for amplification of the target nucleotide sequence characteristic of Vibrio parahaemolyticus (abbreviated as "VP" somewhere hereinafter). This invention relates to the method for detecting Vibrio parahaemolyticus based on the polymerase chain reaction (PCR) using a primer specific for the DNA gyrase sub-unit B gene (Nucleotide sequence of DNA gyrase B subunit, abbreviated as "gyrB" hereinafter).
PRIOR ART
Vibrio parahaemolyticus is known to cause food poisoning in many countries. It is found not only in the intestine but also in other organs and in the postoperative wound. Vibrio parahaemolyticus is a Gram negative, polymorphic, bacilliform, halophilic, facultative anaerobe, which ferments carbohydrate to generate gas. It forms green colonies on thiosulfate-citrate-bile-sucrose (TCBS) agar.
For detection of Vibrio parahaemolyticus, is used usually a method where the specimen is cultivated in an enrichment medium followed by isolation by the selective plate culture. The conventional method of detection requires one week, and therefore a more rapid method has been desired.
The fluorescence assay based on determination of trypsin activity can detect rapidly Vibrio parahaemolyticus but cannot differentiate Vibrio parahaemolyticus from Vibrio alginolyticus or Vibrio harveyi. The conventional methods for identification of Vibrio parahaemolyticus and Vibrio alginolyticus are time-consuming because the 16S rRNA sequence reveals homology of 99.7% between Vibrio parahaemolyticus and Vibrio alginolyticus.
The genus Vibrio includes 37 different species, all of which are derived from aquatic environment. Based on the systematic data of rRNA, species known as V. angullarum, V. ordalli, and V. damsels have been newly classified as Listonella or Photobacterium. Ten species are involved in gastroenteritis, infection of the wound, and human septicemia, while 7 species are known to be pathogens for fish. Vibrio parahaemolyticus occurs usually in an environment such as river-mouth and sea, being isolated from sea water and fishes and shellfishes often in summer.
For isolation and identification of Vibrio parahaemolyticus, the specimen is inoculated into a selective medium such as the bromothymolblue-teepol agar medium or TCBS agar medium, followed by isolation of bluish green colonies and examinations for the biochemical properties of the colonies. Unfortunately many Vibrio species show the same responses, and thus more detailed biochemical examinations are required for reliable identification. Examinations for a variety of biochemical properties on many isolates are time-consuming and laborious. Serological identification of Vibrio parahaemolyticus shows a cross reaction with other Vibrio species.
A method for identification of a Vibrio species was developed which used I)NA. In this method were used DNA probes capable of amplifying the cholera toxin operon from V. cholera 01 to identify specifically the bacterium. These probes cross-react with Vibrio species other than cholera toxin-producing V. cholera. A method for identification of V. vulnificus has been developed in which hybridization is carried out on a membrane filter by using a fluorescent-labeled oligonucleotide probe (Wright, A. C. et al., Appl. Environ. Microbiol. 59: 541-546, 1993).
In addition, the oligonucleotide DNA probe was constructed from a portion of the cytolysin gene (hlyA) sequence of V. vulnificus and labeled through the covalent bond. These probes do not react with non-toxinogenic V. vulnificus and therefore do not detect all strains of V. vulnificus.
Similarly, other molecular biologic methods using the toxic factor (TDH, TRH) genes as the target can detect toxinogenic V. parahaemolyticus, though based on the toxic factor, all strains of V. parahaemolyticus cannot be detected.
DISCLOSUR OF THE INVENTION
The object of this invention is to provide a method for differentiation of Vibrio parahaemolyticus from other 36 Vibrio species.
The object of this invention is to provi
REFERENCES:
Dorsch, M. et al. Int. J. Systemic Bacteriology 42(1):58-63, Jan. 1992.
Lee, C. et al. J. Gen. Microbiology 139:3225-3231, Dec. 1993.
Jun Okuda et al., Microbial Pathogenesis 1995; 18:167-172.
Mitsuaki Nishibuchi et al., FEMS Microbiology Letters 67(1990) pp. 251-256.
Randall K. Saiki et al., Science (1988), vol. 239, No. 29, pp. 487-491.
Anita C. Wright et al., Applied and Environmental Microbiology, Feb. 1993, pp. 541-546.
Satoshi Yamamoto and Shigeaki Harayama, Applied and Environmental Microbiology, Mar. 1995, pp. 1104-1109.
Doumoto Nobuhiko
Venkateswaran Kasthuri
Johannsen Diana
Myers Carla J.
Nippon Suisan Kaisha Ltd.
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