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

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^{1, 2,},
周勇^{1, 2},
和鹏^{1, 2},
陶霞^{1, 2},
王安娜^{1, 2},
胡玉山^{1, 2},
吴新伟^{1, 2},
张晶^{1, 2, ,}

1.
广州市疾病预防控制中心广州510440
2.
广州医科大学公共卫生研究院广州510440

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
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出版历程
- 收稿日期: 2021-07-09
- 录用日期: 2021-12-02
- 网络出版日期: 2022-02-28

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

Hou Shuiping^{1, 2
,},
Zhou Yong^{1, 2},
He Peng^{1, 2},
Tao Xia^{1, 2},
Wang Anna^{1, 2},
Hu Yushan^{1, 2},
Wu Xinwei^{1, 2},
Zhang Jing^{1, 2
, ,}

1.
Guangzhou Center for Disease Control and Prevention
2.
Institute of Public Health, Guangzhou Medical University & Guangzhou Center for Disease Control and Prevention, Guangzhou 510440, China

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)

More Information

Corresponding author: Zhang Jing, Email: zhangjing9468@sina.com

摘要

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。
- 胎儿弯曲菌 /
- 多重PCR /
- 检测方法 /
- 飞行时间质谱
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）. Method 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 156bp 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 μM , 0.2 μM and 0.2 μM 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.
- Campylobaccter fetus /
- multiplex PCR /
- detection method /
- MALDI-TOF MS

HTML全文

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.

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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)

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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)

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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).

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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)

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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.

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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.

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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］
胎儿弯曲菌	Cf359r	AGCCAGTAACGCATATTATAGTAG	Cst（AY158813）	359	［3］
性病亚种	ISC2mf	AACTCTTGCATAAGCTATGG	ISCfe1（AM260752）	156	［4］
性病亚种	ISC2mr	CAAACACTTAGAAACTGGGA	ISCfe1（AM260752）	156	［4］
龟亚种	CoA266f	ACATAGCCGTAGTTGGATT	CoA（CP006833）	266	本研究
龟亚种	CoA266r	TATTATGCCTAGTTGAAGCC	CoA（CP006833）	266	本研究

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