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Chinese Agricultural Science Bulletin ›› 2018, Vol. 34 ›› Issue (12): 146-150.doi: 10.11924/j.issn.1000-6850.casb17030100

Special Issue: 畜牧兽医

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Development of a loop-mediated isothermal amplification assay for visual detection of Pasteurella multocida

许培文   

  • Received:2017-03-16 Revised:2017-07-20 Accepted:2017-08-22 Online:2018-05-03 Published:2018-05-03

Abstract: A rapid detection method of Pasteurella multocida (Pm) was established by loop-mediated isothermal amplification assay(LAMP) in this study. According to the published outer membrane proteins H(ompH) gene sequences of Pm in GenBank, multiple pairs of primers were designed targeting the conserved region of ompH gene. The specificity and sensitivity of the LAMP were evaluated. Meanwhile, the amplified products were colored by SYBR Green Ⅰ after completion of the reaction, so that the amplification can be observed with naked eyes. LAMP showed a highly efficient amplification for Pm genomic DNA which performed at 56℃ for 1 h. The results detected with naked eyes by the addition of SYBR Green Ⅰ at the end of reaction was observed. The LAMP showed a strong specificity that the results were all negative towards other swine pathogens detection; and it also showed a high sensitivity with a detectin limit of 0.05pg DNA, which was 100-fold higher than that of PCR. The results of 5 clinical positive samples detected by LAMP developped in this study were consistent with the ones tested before by PCR test. The results revealed a visualized LAMP method which can detect Pm was established and the results can be read with naked eyes. And the method was easy to operation, rapid, specific and sensitive, which was suitable for rapid detection of Pm.