摘要
本研究依据GB 4789.4-2016标准对沙门氏菌ACAS-PT526能力验证样品进行常规培养法检测,同时使用Taq Man实时荧光聚合酶链反应(polymerase chain reaction,PCR)技术对预增菌培养物进行快速检测和鉴定。本研究首先以沙门氏菌特异性基因hut基因为靶基因,设计合成特异性引物和探针,提取各类食源性菌种的核酸DNA进行实时荧光PCR反应,仅沙门氏菌属出现阳性扩增,非沙门氏菌属、阴性对照和空白对照均无扩增信号,验证设计合成的引物探针具有较高的特异性。其次将能力验证样品和加标样品经预增菌、增菌、分离、纯化、生化试验和血清学鉴定,同时将预增菌培养物经实时荧光PCR测定后,18-D319和加标样品有显著的S型扩增曲线,Ct值分别为24.34和26.21,为沙门氏菌阳性,18-M906无显著荧光信号,Ct值>40.00,为沙门氏菌阴性。经API20E试剂条鉴定,18-D319为猪霍乱沙门菌亚利桑那亚种,鉴定百分率为99.90%,T值为0.97,18-M906为大肠埃希氏菌,鉴定百分率为99.80%,T值为0.94。实时荧光PCR检测结果与常规培养法检测结果一致,且更为简单快速,从预增菌到结果判定仅需12h,结果准确度高,一批次可检测多个样品,可用于大量样品中沙门氏菌的快速筛查和对能力验证样品的检测验证。
According to GB 4789.4-2016 standard,the conventional culture method was used to test the ACAS-PT526 proficiency test sample for Salmonella,and TaqMan real-time fluorescence PCR was used to detect and identify rapidly the pre-enriched culture.In this study,specific primers and probes were designed and synthesized through using the Salmonella specific gene hut gene as the target gene.The nucleic acid DNA of various foodborne strains was extracted for real-time fluorescent PCR reaction.Only Salmonella showed positive amplification,while non Salmonella species,negative control and blank control had no amplification signal,confirming thee relatively high specificity of the designed and synthesized primer probes.Secondly,after pre-enrichment,enrichment,isolation,purification,biochemical tests and serological identification of the proficiency test samples and spiked samples,as well as real-time fluorescent PCR detection of pre-enriched culture,the 18-D319 and spiked samples exhibited significant S-type amplification with the Ct value being 24.34 and 26.21,respectively(which were positive for Salmonella),whilst 18-M906 had no significant fluorescence signal with the Ct value higher than 40(which was negative for Salmonella).Based on the determination using API20 E reagent strips,18-D319 was a subspecies of Salmonella cholerae in Arizona,with the percentage of identification as 99.90%and T value as 0.97.18-m906 was Escherichia coli,with the percentage of identification as 99.80%and T value as 0.94.The results of real-time fluorescence PCR were consistent with those obtained by the conventional culture method,and the PCR method was simpler and more rapid,requiring only 12 hours from pre-enrichment to result determination,and leading to results with high accuracy and ability to detect multiple samples in one batch.This method allows rapid screening of Salmonella in a large number of samples and detection and verification of proficiency testing samples.
作者
王凤军
叶素丹
WANG Feng-jun;YE Su-dan(Department of Applied Engineering,Zhejiang Institute of Economic and Trade,Hangzhou 310018,China)
出处
《现代食品科技》
EI
CAS
北大核心
2020年第12期300-306,83,共8页
Modern Food Science and Technology
基金
浙江省教育厅一般科研项目(Y201840745)
浙江省基础公益研究计划项目(LGC19C200007)
浙江经贸职业技术学院省属高校基本科研业务费专项(20SBYB03)。
关键词
沙门氏菌
能力验证
实时荧光PCR
快速检测
鉴定
Salmonella
proficiency testing
real-time fluorescent PCR
rapid detection
identification
