Detection of atx gene in Clostridium botulinum using TaqMan probe FQ-PCR

ObjectiveTo detect the atx gene of Clostridium botulinum(C.botulinum)type A neurotoxin(bont/A) using TaqMan probe fluorescence quantitative polymerase chain reaction(FQ-PCR).MethodsBy targeting the gene atx of bont/A,designing primer and TaqMan probe,optimizing the reaction conditions,making quantitative standard curve,and verifying sensitivity,specificity and repeatability of the method,we established a TaqMan FQ-PCR method to detect bont/A.ResultsPlasmid pUC57-△atx was constructed successfully.The standard curve established had good linearity when gene quantity was between 10³-10⁷ copies,and the coefficient of correlation was 0.997.The limit of detection for FQ-PCR was 22 copies.The sensitivity was about 100 times higher than that of ordinary PCR.The FQ-PCR method could selectively detect C.botulinum and there were no cross reactions with other five food-borne pathogen samples,consisting with the results of ordinary PCR.Repeatability tests showed that the coefficient of variation of 15 parallel samples in same concentration was only 1.0%.ConclusionA TaqMan probe FQ-PCR method with high sensitivity and specificity,and good repeatability was established for rapid,accurate and quantitative detectoin of C.botulinum type A.