PCR for detection of Shigella spp. in mayonnaise

Abstract

The use of PCR to amplify a specific virA gene fragment serves as a highly specific and sensitive method to detect virulent bacteria of the genus Shigella and enteroinvasive Escherichia coli. Amplification of a 215-bp DNA band was obtained by using isolated genomic DNA of Shigella, individual cells of Shigella dysenteriae, and mayonnaise contaminated with S. dysenteriae. Moreover, a multiplex PCR with specific (virA) and bacterium-restricted (16S ribosomal DNA) primers generated an amplification product of approximately 755 bp for all bacteria tested and an additional 215-bp product for Shigella and enteroinvasive E. coli.

FIG. 1

PCR with virA primers and different amounts of DNA of S. dysenteriae serovar 1. (A) Twenty-five microliters of the amplification reaction solutions containing different amounts of DNA subjected to agarose gel electrophoresis; (B) same solutions as those in panel A after filter DNA hybridization against the virA Dig-labeled probe. Lane M contains a 100-bp marker, with numbers indicating the size in base pairs. Lanes 1 to 6 contain 0, 1, 5, 25, 50, and 100 fg of template DNA, respectively.

FIG. 2

Multiplex PCR with DNA from Shigella spp. or EIEC. (A) Ten microliters of amplification reaction solutions with 100 ng of DNA from various bacteria after agarose gel electrophoresis; (B) the same solutions as those in panel A after filter DNA hybridization against the virA Dig-labeled probe. Lane M, 100-bp marker, with numbers indicating the size in base pairs. Lanes 1 to 7, control without DNA, S. boydii serovar 10, S. dysenteriae serovar 1, S. flexneri serovar 2a, S. sonnei serovar a, EIEC 41, and EIEC 121, respectively.