›› 2012, Vol. 39 ›› Issue (10): 22-26.

Previous Articles     Next Articles

Development of Real-time Fluorescence Quantitative PCR Assay for Detection of Bovine Mycobacterium paratuberculosis

WANG Su-hua1, WANG Zhong-cai1, LI Xiao-jun2, DU Ai-fang3   

  1. 1. Wenzhou Entry-exit Inspection and Quarantine Bureau, Wenzhou 325027, China;
    2. Zhoushan Entry-exit Inspection and Quarantine Bureau, Zhoushan 316000, China;
    3. Institute of Preventive Veterinary Medicine, Zhejiang University, Hangzhou 310029, China
  • Received:2012-03-13 Online:2012-10-20 Published:2012-10-19

Abstract: According to the published sequences of bovine Mycobacterium paratuberculosis in GenBank, a pair of primers were designed and synthesized for the C-2 chromosome(ISMav2)gene of Mycobacterium paratuberculosis, and a SYBR-GreenⅠfluorescent Real-time quantitative PCR assay was developed. The positive standard plasmid pMD-ISMav2 were used as quantitative template to make the standard curves by optimizing the reaction conditions, the correlation coefficient (R2) was 0.999. By using the positive standard as template, specificity and sensitivity were tested. According to the experiment, all negative controls such as Brucella, Escherichia coli, Salmonella and Streptococcus showed negative detection. By sensitivity analysis, the Real-time PCR indicated that a minimum of 1.96×101 copies of plasmid DNA was detected. As a result of the specificity and sensitivity of the assay with a relatively rapid and simple procedure, the Real-time PCR can be used as a routine assay for the diagnosis of Mycobacterium paratuberculosis.

Key words: Mycobacterium paratuberculosis; Real-time fluorescence quantitative; detection

CLC Number: