A real-time PCR assay for the detection and quantitation of Campylobacter jejuni using SYBR Green I and the LightCycler.
Campylobacter jejuni is recognized as a leading human food-borne pathogen. Traditional biochemical identification for C. jejuni is not reliable due to special growth requirements and the possibility that this bacterium can enter a viable but nonculturable (VNC) state. Nucleic acid-based tests have emerged as a useful alternative to traditional testing. In this article, we present fluorescent quantitative PCR assay for quantitative detection of C. jejuni, the assay was carried out using a LightCycler instrument and product formation was monitored continuously with the fluorescent double-stranded DNA binding dye SYBR Green I. When this assay was applied, the assay positive for all of the isolates of C. jejuni tested (11 isolates, including type strain ATCC33560) and negative for all other Campylobacter spp. (three isolates) and several other bacteria (five species tested). The total assay could be completed in 60 min with a detection limit of approximately 1 CFU, and a correlation coefficient was 1.000. Result indicated that fluorescent quantitative detection methods provided a special, sensitive, rapid, reproducible and accurate method for quantitative detection of C. jejuni.
Yang
Chengbo
C
College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, PR China.
Jiang
Yuan
Y
Huang
Kehe
K
Zhu
Changqing
C
Yin
Yulong
Y
Gong
Jian-Hua
JH
Yu
Hai
H
D020640:Arcobacter / Q000378:metabolism; D052160:Benzothiazoles; D002167:Campylobacter / Q000378:metabolism; D016123:Campylobacter jejuni / Q000378:metabolism*; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D004269:DNA, Bacterial; D003959:Diamines; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000378:metabolism; D005456:Fluorescent Dyes / Q000494:pharmacology; D005516:Food Microbiology; D016998:Helicobacter / Q000378:metabolism; D009930:Organic Chemicals / Q000494:pharmacology*; D011485:Protein Binding; D011804:Quinolines; D012044:Regression Analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D052160:Benzothiazoles; D017931:DNA Primers; D004269:DNA, Bacterial; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; D004247:DNA