侯水平, 周勇, 和鹏, 陶霞, 王安娜, 胡玉山, 吴新伟, 张晶. 检测胎儿弯曲菌三种亚种的多重PCR方法建立及初步应用[J]. 疾病监测, 2022, 37(8): 1093-1099. DOI: 10.3784/jbjc.202107090392
引用本文: 侯水平, 周勇, 和鹏, 陶霞, 王安娜, 胡玉山, 吴新伟, 张晶. 检测胎儿弯曲菌三种亚种的多重PCR方法建立及初步应用[J]. 疾病监测, 2022, 37(8): 1093-1099. DOI: 10.3784/jbjc.202107090392
Hou Shuiping, Zhou Yong, He Peng, Tao Xia, Wang Anna, Hu Yushan, Wu Xinwei, Zhang Jing. Application of a multiplex PCR assay for identification of Campylobacter fetus and subspecies differentiation in fecal samples from reptile animals[J]. Disease Surveillance, 2022, 37(8): 1093-1099. DOI: 10.3784/jbjc.202107090392
Citation: Hou Shuiping, Zhou Yong, He Peng, Tao Xia, Wang Anna, Hu Yushan, Wu Xinwei, Zhang Jing. Application of a multiplex PCR assay for identification of Campylobacter fetus and subspecies differentiation in fecal samples from reptile animals[J]. Disease Surveillance, 2022, 37(8): 1093-1099. DOI: 10.3784/jbjc.202107090392

检测胎儿弯曲菌三种亚种的多重PCR方法建立及初步应用

Application of a multiplex PCR assay for identification of Campylobacter fetus and subspecies differentiation in fecal samples from reptile animals

  • 摘要:
      目的  建立一种检测胎儿弯曲菌胎儿亚种、性病亚种和龟亚种的多重PCR检测方法。
      方法  使用针对胎儿弯曲菌、性病亚种和龟亚种的特异性引物,优化反应体系及反应条件,使用38株菌株(18株胎儿弯曲菌和20株非胎儿弯曲菌)验证其特异性,并将该方法应用于53份爬行动物粪便筛查。
      结果  针对3种亚种均可扩增相应的片段,胎儿亚种只有1条359 bp大小的条带、性病亚种有2条分别为359 bp和156 bp大小的条带,龟亚种有2条分别为359 bp和266 bp大小的条带。 优化后的最佳扩增条件:退火温度58 ℃,引物MG3f和Cf359r浓度为0.1 μmol/L,ISC2mf和ISC2mr、CoA266f和CoA266r的浓度0.2 μmol/L。 对胎儿亚种、性病亚种和龟亚种的检出限分别为0.40 ng/μL、0.39 ng/μL和0.47 ng/μL。 使用18株胎儿弯曲菌和20株非胎儿弯曲菌其特异性为100%。 对53份爬行动物粪便进行筛查中有1份龟亚种阳性并分离出了相应的菌株。
      结论  本研究建立的多重PCR方法能利用1个反应体系同时鉴别3种亚种,从而为菌株快速鉴定、流行病学调查和溯源研究提供技术支持。

     

    Abstract:
      Objective   To establish a multiplex polymerase chain reaction (PCR) method to differentiate C fetus subsp fetus cff ,C fetus subsp venerealis cfv and C fetus subsp testudinum cft.
      Methods  The specific primers targetting C fetus,cfv and cft were designed and the reaction conditions were optimized. The multiplex PCR system was also evaluated by using 38 strains (including 18 Campylobacter fetus strains and 20 non-Campylobacter fetus strains) and further applied to 53 fecal samples from reptile animals.
      Results  The multiplex PCR assay with 3 primer pairs could yield specific fragments: only one fragment of 359 bp for cff, two fragments of 359 bp and 156 bp for cfv, two fragments of 359 bp and 266 bp for cft. The amplification conditions were also optimized and the extending temperature was 58 ℃. The optimal concentration of primers “MG3f and Cf359r”, “ISC2mf and ISC2mr”,“CoA266f and CoA266r” were 0.1 μmol/L , 0.2 μmol/L and 0.2 μmol/L respectively. The sensitivity of the assay for detecting cff, cfv and cft was 0.40 ng/μL、0.39 ng/μL and 0.47 ng/μL respectively. The analytical specificity was 100% by examination of 18 Campylobacter fetus strains and 20 non-Campylobacter fetus strains. In 53 fecal samples from reptile animals, one was PCR postive for cft and the suspective colonies were also identified as cft.
      Conclusion  The established multiplex PCR assay can differentiate three subspecies simutaneously and provide technical support for rapid identification, epidemiological investigation and tracing infection source.

     

/

返回文章
返回