检测胎儿弯曲菌三种亚种的多重PCR方法建立及初步应用

侯水平 周勇 和鹏 陶霞 王安娜 胡玉山 吴新伟 张晶

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.
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.
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.
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.

检测胎儿弯曲菌三种亚种的多重PCR方法建立及初步应用

基金项目: 广州市医学重点学科(2021-2023-11);广州市重点实验室基础研究计划项目(202102100001)
详细信息
    作者简介:

    侯水平, 男, 广东省广州市人, 硕士, 副主任技师, 从事病原微生物检测研究工作. Email:gzcdc367@163.com

    通讯作者:

    张晶,(补充座机电话),Email:zhangjing9468@sina.com

  • 中图分类号: R211

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

Funds: This study was supported by the fund of The Key Project of Medicine Discipline of Guangzhou (NO.2021-2023-11) and The Basic Research Project of Key Laboratory of Guangzhou (202102100001)
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  • 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为:退火温度58 ℃,引物MG3f和Cf359r浓度为0.1 μM,ISC2mf和ISC2mr、CoA266f和CoA266r的浓度0.2 μM。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。
  • 图  1  PCR引物验证电泳图。(M为marker;lane1、2、3、4为以MG3f和Cf359r为引物,以胎儿亚种、性病亚种、龟亚种、阴性对照为模板的电泳图;lane5、6为以ISC2mf和ISC2mr为引物,以性病亚种、阴性对照为模板的电泳图;lane7、8为以Coa266f和Coa266r为引物,龟亚种、阴性对照为模板的电泳图)

    Figure  1.  PCR results with three pairs of primers. Lane M: marker; lane1−4: cff, cfv, cft and negative control, "MG3f and Cf359r" as primers; lane 5−6: cfv and negative control, "ISC2mf and ISC2mr" as primers; lane7−8:cft and negative control, "Coa266f and Coa266r" as primers.

    图  2  退火温度在56 ℃,58 ℃,60 ℃和62 ℃时三种亚种标准菌株的PCR产物电泳图。(M为marker;lane1,4,7,10为胎儿亚种;lane2,5,8,11为性病亚种;lane3,6,9,12为龟亚种)

    Figure  2.  Multiplex PCR results for identification of three subspecies at four different annealing temperature (56 ℃, 58 ℃, 60 ℃ and 62 ℃). (M: marker; lane1, 4, 7, 10: cff; lane2, 5, 8, 11:cfv; lane3, 6, 9, 12: cft)

    图  3  引物在不同浓度比例的PCR产物电泳图。(M为marker;lane1-3为MG3f和Cf359r浓度0.2 μM,lane4-6为MG3f和Cf359r浓度0.1μM,lane7-9为MG3f和Cf359r浓度0.05μM)

    Figure  3.  Multiplex PCR results at different relative concentration of primers. (M: marker; lane1−3: The concentration of MG3f and Cf359r is 0.2 μM, lane4−6: The concentration of MG3f and Cf359r is 0.1μM, lane7−9: The concentration of MG3f and Cf359r is 0.05 μM)

    图  4  多重PCR方法对胎儿亚种、性病亚种和龟亚种的灵敏度实验结果。(lane1-4:胎儿亚种DNA浓度为40 ng/μL、4 ng/μL、0.4 ng/μL和0.04 g/μL;lane5-8:性病亚种DNA浓度为39 ng/μL、3.9 ng/μL、0.39 ng/μL和0.04 g/μL;龟亚种DNA浓度为47 ng/μL、4.7 ng/μL、0.47 ng/μL和0.05 ng/μL)

    Figure  4.  The sensitivity of the multiplex PCR for detecting cff, cfv and cft. (lane1−4: the concentration of cff is 40 ng/μL、4 ng/μL、0.4 ng/μL and 0.04 g/μL; lane 5−8: the concentration of cfv is 39 ng/μL、3.9 ng/μL、0.39 ng/μL and 0.04 g/μL; lane 9−12: the concentration of cft is 47 ng/μL、4.7 ng/μL、0.47 ng/μL and 0.05 ng/μL).

    图  5  多重PCR特异性实验结果。(M:marker;lane1-2:胎儿弯曲菌胎儿亚种;lane3-4:胎儿弯曲菌性病亚种;lane5-18:胎儿弯曲菌龟亚种;lane19:空肠弯曲菌;lane20:大肠弯曲菌;lane21:海鸟弯曲菌;lane22:大肠埃希氏;lane23:沙门菌; lane24:志贺菌; lane25:变形杆菌; lane26:霍乱弧菌; lane27:副溶血性弧菌; lane28:创伤弧菌;lane29:铜绿假单胞菌; lane30:金黄色葡萄球菌; lane31:脑膜炎奈瑟菌; lane32:猪链球菌; lane33:溶血性链球菌; lane34:柠檬酸杆菌; lane35:粪产碱杆菌; lane36:嗜水气单胞菌; lane37:粘质沙雷菌; lane38:蜡样芽孢杆菌)

    Figure  5.  The specifity results of the multiplex PCR assay. (M:marker; lane1−2: cff; lane3−4: cfv; lane5−18: cft; lane19: Campylobacter jejuni; lane20:Campylobacter coli; lane21: Campylobacter lari; lane22:E.coli; lane23:Salmonella; lane24:Shigella; lane25:Proteus ; lane26: Vibrio cholerae; lane27:Vibrio parahaemolyticus; lane28:Vibrio vulnificus; lane29:Pseudomonas aeruginosa; lane30:Staphylococcus aureus; lane31:Neisseria meningitidis; lane32:Streptococcus suis; lane33:Hemolytic streptococci; lane34:Citrobacter; lane35: Alcaligenes faecalis; lane36: Aeromonas hydrophila; lane37:Serratia marcescens; lane38:Bacillus cereus)

    图  6  分离株juxi和弯曲菌属标准菌株的质谱图谱及聚类分析

    Figure  6.  Dendrogram of the MS peaks of juxi and most closely related species of the genus Campylobacter using Pearson correlation with neighbor-joining method.

    图  7  基于16S rRNA序列使用邻接法构建进化树。

    Figure  7.  Phylogenetic tree based on 16S rRNA gene sequences constructed by the neighbor-joining method. Bootstrap values (%) are indicated at nodes.

    表  1  多重PCR引物序列及扩增产物大小

    Table  1.   The nucleotide sequences of primer sets and the size of the multiplex PCR products

    引物名称引物序列靶基因
    (Accession NO)
    片段大小(bp)参考文献
    胎儿弯曲菌 MG3f GGTAGCCGCAGCTGCTAAGAT Cst(AY158813) 359 3
    Cf359r AGCCAGTAACGCATATTATAGTAG
    性病亚种 ISC2mf AACTCTTGCATAAGCTATGG ISCfe1(AM260752) 156 4
    ISC2mr CAAACACTTAGAAACTGGGA
    龟亚种 CoA266f ACATAGCCGTAGTTGGATT CoA(CP006833) 266 本研究
    CoA266r TATTATGCCTAGTTGAAGCC
    下载: 导出CSV
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