Use of the polymerase chain reaction in detection of culturable and nonculturable Vibrio vulnificus cells.

Abstract:

:Vibrio vulnificus is a human pathogen associated with consumption of raw oysters. During the colder months the organism apparently enters a viable but nonculturable state and thus cannot be cultured by ordinary bacteriological methods. For this reason, another means of detecting this bacterium is necessary. In the present study we utilized the polymerase chain reaction (PCR) to detect V. vulnificus DNA, thus eliminating the problem of nonculturability. DNA from both culturable and nonculturable cells of V. vulnificus was amplified by PCR with primers flanking a 340-bp fragment of the cytotoxin-hemolysin gene. As little as 72 pg of DNA from culturable cells and 31 ng of DNA from nonculturable cells could be detected. Fifty cycles of a two-step reaction (30 s [each] at 94 and 65 degrees C) were found to be optimal as well as more time efficient than the three-step PCR. The total procedure from the point of DNA extraction to observation on a gel required less than 8 h. Possible reasons for the difficulties encountered in amplifying DNA from nonculturable cells, e.g., gene rearrangement or loss of the hemolysin gene, are discussed.

journal_name

Appl Environ Microbiol

authors

Brauns LA,Hudson MC,Oliver JD

doi

10.1128/AEM.57.9.2651-2655.1991

subject

Has Abstract

pub_date

1991-09-01 00:00:00

pages

2651-5

issue

9

eissn

0099-2240

issn

1098-5336

journal_volume

57

pub_type

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