Development of a single nucleotide polymorphism-based electronic DNA microarray technique for the detection and species differentiation of viable Campylobacter.

摘要

Campylobacter jejuni, C. coli, and C. lari are the three major food-borne pathogenic Campylobacter species that cause the most frequent occurrences of acute bacterial gastroenteritis around the world. This thesis presents the successful development of a reverse transcription-polymerase chain reaction (RT-PCR) electronic DNA microarray approach for the simultaneous detection and species differentiation of viable Campylobacter. The mRNA of the 60-kDa heat shock protein gene ( hsp60) was used as the viability marker, and two closely located single nucleotide polymorphisms (SNPs) within this gene were chosen as the species marker. A 200-bp fragment amplified from the hsp60 mRNA by RT-PCR was detected with species-specific fluorescently-labeled reporters using an electronic DNA microarray technique.; This technique can detect as few as two viable Campylobacter cells, and will not detect dead cells. The evaluation of 14 blind Campylobacter samples showed 100% agreement with their identities, demonstrating its high specificity. This technique was preliminarily applied to six authentic chicken samples as well.