Establishment of real-time PCR assays for rapid detection of Clostridium botulinum type A and B
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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。 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. -
Key words:
- Clostridium botulinum type A and B /
- Real-time PCR /
- Specificity /
- Sensitivity
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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 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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2018-0434 利用Real-time PCR 技术检测毒素基因快速鉴定AB型肉毒梭菌方法的建立.docx
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