摘要
目的建立对高致病性猪链球菌2型毒力基因SalK、virB4-89K、cps2J同步检测的多重实时荧光定量PCR检测方法。方法根据SalK、virB4-89K、cps2J基因保守区域设计并合成3对引物及其探针;通过反应体系和扩增条件优化,建立快速鉴定高致病性猪链球菌2型的方法,并对方法的特异性、敏感性、重复性指标进行评估。结果反应体系具有良好的扩增效率,全部扩增检测可在60min内完成。SalK、virB4-89K、cps2J基因检测灵敏度分别为19cfu/ml、27cfu/ml和32cfu/ml。重复性试验中变异系数均小于3%。用该方法考核33株猪链球菌,包括1/2型、1型、3型至33型,以及9株常见对照菌株,仅猪链球菌1/2型检测到cps2J基因荧光信号增强。对疫情现场获得的SS2感染者血液进行检测,3个基因均阳性。结论建立的多重实时荧光定量PCR方法可快速检测高致病性猪链球菌2型,并能鉴定是否含有89K毒力岛基因SalK、virB4-89K,具有敏感、特异、重复性好的特点。
Objective This study sought to develop a multiplex real-time TaqManR-based polymerase chain reaction（PCR） system for rapid and specific detection of highly virulent Streptococcus suis serotype 2.Methods Three pairs of specific primers and three fluorogenic-labeled probes were designed and synthesized in accordance with the target genes,the virulence genes SalK,virB4-89K,and cps2J.A set of PCR reactions was created based on optimized parameters and conditions.The sensitivity,specificity,and reproducibility of the assay were evaluated.Results The reaction system had good amplification efficiency and was completed within 60 minutes.Sensitivity for the genes SalK,virB4-89K,and cps2J was 19,27,and 32 cfu/ml,respectively.The coefficient of variation（CV） was less than 3%.Thirty-three strains of S.suis 1/2,1,and 3 to 33 were analyzed,together with 9 strains of other bacterial species related to S.suis or isolated from pigs.The PCR amplification curve had only cps2J amplification from S.suis 1/2.Blood from individuals infected with S.suis 2 tested positive for all 3 genes.Conclusion A multiplex real-time PCR assay provided a rapid way to detect highly virulent S.suis serotype 2 and allowed detection of 89K pathogenicity islands attributed to SalK and virB4-89K.This assay has the benefit of being sensitive,specific,and reproducible.
出处
《中国病原生物学杂志》
CSCD
北大核心
2012年第5期325-328,共4页
Journal of Pathogen Biology
基金
国家自然科学基金项目(No.31170124
No.30972638
No.81071317
No.81171527
No.81172794)
江苏省自然科学基金项目(No.BK2011097
No.BK2010025
No.BK2010114
No.BK2010113)
