河弧菌Taqman实时荧光PCR检测方法建立

目的 建立以Taqman实时PCR检测河弧菌的方法。方法 根据河弧菌toxR基因的保守序列设计引物和Taqman探针,建立检测河弧菌的实时PCR方法;对引物和探针的浓度进行优化,对优化后建立的方法分别进行实验室内的灵敏度和特异性评价,并与常规PCR比较。结果 建立了检测河弧菌的实时PCR方法,经优化该方法选择引物的浓度为100 nmol/L,探针浓度为200 nmol/L;该方法对纯菌的检测下限为4.17×10²cfu/mL,比常规PCR高1000倍;针对toxR基因建立的河弧菌实时PCR方法,对8种其他弧菌和肠道细菌的染色体无扩增。结论 建立的方法能够特异和敏感地检测河弧菌,可用于河弧菌的快速筛检。

Detection of Vibrio fluvialis with Taqman real-time PCR assay

Objective To establish a real-time PCR Taqm an assay for the detection of Vibrio fluvialis.Methods The conserved region of tox Rgene of V.fluvialis was used to design primers and Taqman probe.The real-time PCR Taqm an system for V.fluvialis detection was established and the concentration of primers and probe were optimized.The sensitivity and specificity of the assay were evaluated and compared with PCR.Results The real-time PCR Taqman system for V.fluvialis detection was established.The concentration of primers and probe in the assay was 100 nmol/L and 200 nmol/L, respectively.The sensitivity of the real-time PCR Taqman system to detect pure bacterial was 4.17×10² cfu/mL and was 1000 times higher than that of PCR.No amplification of other bacterium was observed in the templates when targeting tox R gene of V.fluvialis was detected.Conclusion The established assay can detect V.fluvialis sensitively and specifically and is a sensitive and rapid way to detect V.fluvialis.