利用实时荧光定量PCR 技术检测毒素基因快速鉴定A/B型肉毒梭菌方法的建立

黄英; 史芸; 叶长芸; 徐雪芳

doi:10.3784/j.issn.1003-9961.2019.09.015

利用实时荧光定量PCR 技术检测毒素基因快速鉴定A/B型肉毒梭菌方法的建立

doi: 10.3784/j.issn.1003-9961.2019.09.015

黄英^1,,
史芸²,
叶长芸¹,
徐雪芳^1, ,

1.
中国疾病预防控制中心传染病预防控制所，传染病预防控制国家重点实验室，北京 102206
2.
北京化工大学材料科学与工程学院，北京 100029

详细信息

作者简介:
黄英，女，北京市人，副主任技师，主要从事病原生物学和形态学研究工作，Email：Huangying@icdc.cn

通讯作者:
徐雪芳，Tel：010-58900748，Email：xuxuefang@icdc.cn

中图分类号: TS252.7
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出版历程
- 收稿日期: 2018-10-08
- 网络出版日期: 2019-04-23
- 刊出日期: 2019-09-01

Establishment of real-time PCR assays for rapid detection of Clostridium botulinum type A and B

1.
State Key Laboratory of Communicable Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
2.
College of Material Science and Engineering, Beijing University of Chemical Technology, Beijing 100029, China

More Information

Corresponding author: Xu Xuefang, Email: xuxuefang@icdc.cn

摘要

摘要: 目的建立针对A/B型肉毒梭菌毒素基因的实时荧光定量PCR（real-time PCR）检测方法，构建标准曲线，评价该方法的特异性、敏感性及检测下限，以提高肉毒梭菌检测的快速性、准确性。方法根据肉毒梭菌A/B型毒素基因设计特异性引物及探针，优化反应条件，建立real-time PCR反应体系。用25种细菌评价该方法的特异性，使用含有A/B型毒素基因特异性序列的重组质粒标准品构建标准曲线，模拟粪便标本，评价建立方法的灵敏度。结果建立的real-time PCR检测方法特异性好，携带A/B型毒素基因的重组质粒均出现相应特异性扩增曲线，对其他25种细菌进行试验扩增时，均未见出现特异性扩增曲线。根据重组质粒标准品构建的标准曲线可确定其对肉毒梭菌A/B型毒素基因检测灵敏度分别为5.04×10²拷贝/μl和6.91×10²拷贝/μl。模拟粪便标本中含有目的基因重组质粒的检测下限为A型1.71×10³拷贝/μl，B型2.14×10³拷贝/μl。结论本研究设计了适合检测国内肉毒梭菌 A/B 型的引物和探针，建立 real-time PCR 快速检测体系，为我国肉毒梭菌 A/B 型菌株的快速鉴定提供一种新的技术手段。
- A/B型肉毒梭菌 /
- 实时荧光定量PCR /
- 特异性 /
- 灵敏性
Abstract: ObjectiveTo establish real-time PCR assays for the toxin gene detections of Clostridium botulinum type A and B, construct standard curves and evaluate the specificities, sensitivities and detection thresholds of the assays, and provide evidence for the rapid and accurate detection of C. botulinum.MethodsSpecific primers and probes were designed based on the sequences of toxin genes of C. botulinum type A and B. Real-time PCR assays were established with optimized reaction conditions. 25 other intestinal bacteria and common bacteria were used to test the specificities of the assays. Standard curve construction, fecal sample simulation and sensitivity measurement were achieved with recombinant plasmids containing toxin genes of C. botulinum type A and B.ResultsThe specificities of the real-time PCR assays were high. Specific amplification curves were observed in recombinant plasmids containing toxin genes of C. botulinum type A and B. No specific amplifications were found for the 25 other bacteria. The detection thresholds of toxin genes of C. botulinum type A and type B were 5.04×10² copy/μl and 6.91×10² copy/μl respectively according to the amplification curves. The detection thresholds of recombinant plasmids containing toxin genes of C. botulinum type A and B in artificial fecal samples were 1.71×10³ copy/μl and 2.14×10³ copy/μl respectively.ConclusionIn this study, real -time PCR assays for the toxin gens detections of C. botulinum type A and type B of China were established, which can be applied in the rapid detection of C. botulinum.
- Clostridium botulinum type A and B /
- Real-time PCR /
- Specificity /
- Sensitivity

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图 1 毒素A基因重组质粒标准品荧光信号图（A）及对数值-Ct值对应标准曲线（B）

　　　　　　　　　　注：2 ~ 6分别表示5.04×10² ~ 5.04×10⁶拷贝/μl重组质粒标准品对应扩增曲线

Figure 1. Fluorescent graph and standard curve of type A toxin gene carrying recombinant plasmid amplified in real-time PCR assay

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图 2 毒素B基因重组质粒标准品荧光信号图（A）及对数值-Ct值对应标准曲线（B）

　　　　　　　　　　注：2 ~ 6分别表示6.91×10²拷贝/μl ~ 6.91×10⁶拷贝/μl重组质粒标准品对应扩增曲线

Figure 2. Fluorescent graph and standard curve of type B toxin gene carrying recombinant plasmid amplified in real-time PCR assay

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表 1 实验菌株

Table 1. Strains used in this study

序号	菌名	拉丁名	菌株
1	肉毒梭菌（A、B）	Clostridium botulinum	分离菌
2	艰难梭菌	Clostridium difficile	分离菌
3	第三梭菌	Clostridium tertium	分离菌
4	产气荚膜梭菌	Clostridium perfringens	分离菌
5	索氏梭菌	Clostridium sordellii	分离菌
6	蜡样芽孢杆菌	Bacillus cereus	分离菌
7	阴沟肠杆菌	Enterobacter cloacae	分离菌
8	猪链球菌	Streptococcus suis	分离菌
9	小肠结肠耶尔森菌	Yersinia enterocxolitica	ATCC23715
10	粪肠球菌	Enterococcus faecalis	ATCC35667
11	屎肠球菌	Enterococcus faecium	分离菌
12	单增李斯特菌	Listeria monocytogenes	分离菌
13	肠出血性大肠埃希菌	Enterohemorrhagic E. coli	EDL933
14	肠致病性大肠埃希菌	Enteropathogenic E. coli	分离菌
15	肠聚集性大肠埃希菌	Enteroaggregative E. coli	分离菌
16	肠产毒性大肠埃希菌	Enterotoxigenic E. coli	分离菌
17	肠侵袭性大肠埃希菌	Enteroinvasive E. coli	分离菌
18	空肠弯曲	Campylobacter jejuni	ATCC33291
19	霍乱弧菌	Vibrio cholerae	分离菌
20	伤寒沙门菌	Salmonella typhi	H98125
21	猪霍乱沙门菌	Salmonella choleraesuis	ATCC14028
22	弗劳地枸橼酸杆菌	Citrobacter freundii	分离菌
23	阪崎肠杆菌	Enterobacter sakazakii	ATCC51329
24	福氏志贺菌	Shigella flexneri	分离菌
25	宋内志贺菌	Shigella sonnei	ATCC25931
26	金黄色葡萄球菌	Staphylococcus aureus	ATCC6538

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表 2 引物和探针序列

Table 2. Primers and probes sequences

扩增基因	引物/探针名称	碱基序列（5′ ~ 3′）	扩增片段长度（bp）
A	F	TAA TAA AAT ATG GGT TAT TCC AGA AAG AG	111
	R	TGT TGA ATC ATA ATA TGA AAC TGG AAC T
	P	FAM-TCCTGAAGAAGGAGATTTAAATCCACCACCAG-BHQ1
B	F	CACAAACATTGCTAGTGTAACTGTTAATAA	130
	R	CTATAGTCTCATTTTCATTTAAAACTGGC
	P	JOE-CAGTAATCCAGGAGAAGTGGAGCGAAAAAAGG-BHQ2
注：F.正向引物；R. 反向引物；P. 探针

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