Evaluation of a polymerase chain reaction-based test for detecting Salmonella spp. in food samples: Probelia Salmonella spp

A commercially available polymerase chain reaction (PCR) kit was evaluated for the detection of Salmonella spp. in food samples. The test combines PCR amplification and sandwich hybridization of the amplified DNA in microtiter plates. The sensitivity and specificity was evaluated with 52 Salmonella strains and 51 non-Salmonella strains and showed that the test was entirely reliable. The threshold sensitivity was 10(2) CFU/ml. The limit of detection of dead cells that determines the minimum detection level of dead cells in food samples was superior to 10(6) CFU/25 g, a level rarely achieved in naturally contaminated samples. After an 18-h pre-enrichment step, the test could detect viable Salmonella in artificially contaminated food samples, even for the lower contamination level (3 CFU/25 g). There was complete agreement between the PCR test and the ISO 6579 bacteriological reference method with artificially contaminated samples. Regarding the accuracy of the results obtained from 253 naturally or noncontaminated foods and from 32 artificially contaminated foods, the agreement percentage was 99.6%. The fidelity of the technique was evaluated in a collaborative study with eight European laboratories and showed a correlation of 98.4%.     

IQ-Check Salmonella II Kit方法对食品中沙门氏菌检测的评价

通过对标准菌株和人工污染沙门氏菌的食品样品进行检测,评价IQ-Check Salmonella Ⅱ试剂盒方法。该研究采用试剂盒方法对50株不同血清型的沙门氏菌和50株非沙门氏菌进行检测,分析该方法的灵敏性和特异性;通过对人工污染沙门氏菌食品样品(包括液体奶、婴幼儿配方乳粉、肉及肉类制品)的检测,评价试剂盒方法与GB 4789.4-2016方法的一致性。实验结果表明:当菌浓度在10³ CFU/mL及以上,试剂盒方法对50株沙门氏菌实现全部检出,其对不同血清型沙门氏菌检出限的平均值为6.98×10² CFU/mL。试剂盒方法对50株非沙门检测结果均为阴性,说明试剂盒特异性较好。试剂盒方法与GB方法对人工污染沙门氏菌的食品样品阳性检出率分别为98.77%(161/163)和96.32%(157/163);相对准确度为:96%、99%、97%,总体准确度为97.33%;相对灵敏度为:96.22%、100%、100%,总体灵敏度为98.73%;相对特异性为:95.74%、98.15%、92.86%,总体特异性为95.80%。参照ISO 16140进行方法一致性分析,结果表明两方法在统计学上无显著性差异。该研究表明该试剂盒方法具有高灵敏性和特异性强的特点,在人工染菌食品样品检测中与GB方法呈高度一致性,值得在食品沙门氏菌快速检测中推广应用。     

食品中沙门氏菌FTA膜结合跨越式滚环等温扩增检测方法的建立

为实现食品中沙门氏菌的简便和快速现场检测,本研究采用FTA膜(Flinders technology associates,FTA)结合跨越式滚环等温扩增(Saltatory rolling circle amplification,SRCA)方法(FTA-SRCA)建立一种新型的沙门氏菌检测方法。利用FTA膜快速提取模板DNA,根据沙门氏菌的inv A基因设计及筛选引物,建立FTA-SRCA反应体系。扩增反应在能够实现集约化检测的凹孔板中进行,反应结束后添加荧光染料观察结果。确定了该方法的特异性、灵敏度和人工污染样品的检出限,并对60个实际样品进行检测,评估其敏感性、特异性和符合率。结果表明:检测的17株沙门氏菌均为阳性结果,29株非沙门氏菌均为阴性结果,特异性良好。FTA-SRCA方法的灵敏度为6.81×100 CFU/m L,比PCR方法高100倍,比SRCA方法高10倍。对于人工污染的牛奶样品检测,FTA-SRCA方法的检出限为3.22×100CFU/m L,比PCR方法低1000倍,比SRCA方法低10倍。检测实际样品的敏感性、特异性和符合率分别为100.00%,94.64%,95.00%。本研究建立的FTA-SRCA方法具有操作简便快速、成本低廉、特异性强、灵敏度高、检出限低等优点,可用于食品中沙门氏菌的大批量集约化快速现场检测。     

食品中沙门氏菌恒温隔绝式PCR检测方法的建立

为弥补传统培养方法耗时长和现场检测步骤繁琐等缺陷,该研究建立了一种针对食品中沙门氏菌的恒温隔绝式PCR快速检测方法。根据沙门氏菌的inv A基因设计特异性引物和探针,通过水浴法快速提取细菌DNA,优化引物、探针以及模板用量,建立了一种基于恒温隔绝式PCR快速检测沙门氏菌的方法,并对方法的特异性和灵敏度及稳定性进行评价,最后对比建立的方法与传统PCR方法、传统分离培养法对实际食品样品中沙门氏菌污染的检测效果。建立的恒温隔绝式PCR检测方法特异性好,灵敏度高且与其他细菌无交叉反应,最低检出限可达75 CFU/mL,可在6 h内完成检测实际食品样品中污染的沙门氏菌,传统PCR方法至少需12 h才能达到与之相同的检测效果,传统培养法验证了建立方法的准确性和可靠性。本研究建立的恒温隔绝式PCR方法更快速,且操作简便,适用于现场检测食品中污染的沙门氏菌。     

Evaluation of TaqMan PCR assay for detecting Salmonella in raw meat and shrimp.

We evaluated the TaqMan Salmonella amplification/detection kit from PE Applied Biosystems, which uses a polymerase chain reaction (PCR) assay for rapid detection of Salmonella in food samples. This system uses the 5' nuclease activity of Taq DNA polymerase, which digests an internal fluorogenic probe to monitor the amplification of the target gene. The system's sensitivity and specificity were evaluated using 42 serotypes of 68 Salmonella strains isolated from fecal samples from patients in Tokyo, Japan, and 39 non-Salmonella strains in 22 genera. There were no false-negative or false-positive results. This PCR assay can detect 3 CFU per PCR tube of Salmonella in pure culture (120 CFU/ml of TSB culture). PCR signals were attenuated with artificially contaminated shrimp, but a similar detection limit was obtained. TaqMan's performance was tested with 100 meat and chicken samples purchased from stores in Tokyo. Overall, two of the DNA extraction protocols (the Chelex and EnviroAmp methods) worked equally well, with some exceptions. Of the 100 samples analyzed, 10 were positive for Salmonella with both conventional culture methods and the kit and 89 were negative with both. One sample was negative by the culture method but positive by the kit assay. These results indicate that TaqMan is a reliable and rapid method for Salmonella analysis in the food industry. With this system, food samples can be analyzed for Salmonella in less than 20 h.     

多重PCR检测4种常见食源性致病菌

目的 建立一种多重聚合酶链式反应法(multiplex polymerase chain reaction, MPCR)快速检测肉制品中金黄色葡萄球菌、沙门氏菌、志贺氏菌和单增李斯特氏菌的分析方法。方法 选取金黄色葡萄球菌nuc基因、沙门氏菌SipB基因、志贺氏菌ipaH基因、单增李斯特菌inlA基因作为目标基因, 设计4对PCR引物, 建立并优化多重PCR反应体系, 评价该体系的特异性和灵敏度, 并对人工污染的熟肉样品进行检测。结果 构建的多重PCR方法特异性强、灵敏度高, 人工污染熟肉匀浆中4种致病菌的检出限为103 CFU/mL。结论 构建的多重PCR检测方法能够快速、准确、高效地检测肉制品中金黄色葡萄球菌、沙门氏菌、志贺氏菌和单增李斯特氏菌, 为食源性疾病菌的快速检测提供参考依据。     

基于cgcA基因实时荧光PCR快速检测穆汀斯克罗诺杆菌的研究

为实现奶粉中常见污染菌穆汀斯克罗诺杆菌的快速检测与控制,本文根据cgcA基因序列设计出特异性探针和引物,建立了运用实时荧光定量PCR法检测穆汀斯克罗诺杆菌的反应体系和反应条件,并对该法的特异性、灵敏度、稳定性进行评价,并进行人工染菌样品检测实验。特异性结果表明,对穆汀斯克罗诺杆菌的荧光PCR检测结果的特异性为100%;灵敏度结果表明,穆汀斯克罗诺杆菌检出限在440 cfu/m L;稳定性结果表明,穆汀斯克罗诺杆菌组内实验CV在0.48~0.69%之间波动,而组间实验CV在0.69~0.73%之间波动;人工染菌样品试验表明,在增菌6 h后能检出阳性。本研究所建立的实时荧光定量PCR法特异性好、灵敏度高、稳定性强,有望成为快速检测食品中穆汀斯克罗诺杆菌污染的新方法,具有很好的研究价值和应用前景。     

甲型副伤寒沙门菌特异性基因筛选及PCR检测体系建立

以甲型副伤寒沙门菌为检测目标,通过比较基因组和聚合酶链式反应（polymerase chain reaction,PCR）验证方法筛选到4 个该血清型的特异性基因,其中以gene_3105作为该血清型的检测靶点设计引物PA23;并结合沙门菌属特异性引物139-141,建立一种甲型副伤寒沙门菌的PCR检测方法。优化PCR反应体系,并对该检测体系的特异性、灵敏度、抗干扰能力及人工污染样品检出限等方面进行评价。结果表明,当样品中含有甲型副伤寒沙门菌时,该体系能扩增出2?条特异性条带,含有其他血清型的沙门菌仅能扩增出284?bp条带,不含沙门菌无扩增条带产生。灵敏度评价表明,基因组DNA和纯菌菌落检出限分别为32.4?pg/μL和4.3×103?CFU/mL;抗干扰能力实验显示,当鸡肉背景菌群和猪肉背景菌群浓度在106?CFU/mL和4.87×107?CFU/mL时,检出限为6.43×104?CFU/mL。当无菌的鸡肉和猪肉样品中添加N?CFU/25?g甲型副伤寒沙门菌时,经10?h增菌,检测结果为阳性（0＜N＜10）。实验建立甲型副伤寒沙门菌PCR检测方法具有较好的特异性和灵敏度,有很好的应用价值,可在食品安全领域广泛应用。     

免疫捕捉通用引物PCR检测食品中沙门氏菌

利用免疫吸附富集结合经典PCR 技术建立免疫捕捉通用引物PCR(IC-UPPCR)检测食品中沙门氏菌。采用细菌16S rRNA 基因保守区设计特异性引物,建立通用引物PCR 技术是可行的,该IC-UPPCR 检测沙门氏菌的灵敏度最低,能检测到2 × 102CFU/ml,检测沙门氏菌属和非沙门氏菌属标准株的特异性为100%,无假阳性和假阴性出现。结果表明该方法具有简单、快速、特异性好和敏感性高等特点,并可满足大批样品沙门氏菌筛选检测的要求,适用于食品卫生监管、商品检验检疫以及临床诊断等领域。     

3种常见食源性致病菌多重重组酶聚合酶扩增检测方法研究

目的建立了食品中沙门菌、单核细胞增生李斯特菌和蜡样芽胞杆菌多重重组酶聚合酶扩增（recombinase polymerase amplification,RPA）快速检测方法。方法选择沙门菌invA基因、单增李斯特菌hlyA基因和蜡样芽孢杆菌16S RNA序列为目标基因进行扩增,建立并优化多重RPA扩增体系和扩增条件;评价反应体系的特异性和灵敏度,并对人工污染食品样品和实际样品进行检测。结果多重RPA反应体系能够在20 min完成三种目标基因的扩增,特异性强;对沙门菌、单增李斯特菌和蜡样芽孢杆菌的灵敏度分别为2.70×105、1.30×105、1.44×104 CFU/mL;能够用于人工污染样品和实际样品的检测。结论本研究建立的多重RPA等温扩增方法特异性强,操作快速、简单,为食源性致病菌的快速检测提供新方向     

Evaluation of an immunomagnetic separation-real-time PCR assay for the rapid detection of Salmonella in meat

The aim of this study was the comparison of an immunomagnetic separation (IMS)-real-time PCR assay for the detection of Salmonella with the cultural reference method according to and 35 of the German Law on Food and Commodities (LMBG, L 00.00.20:1998). The IMS-real-time PCR assay includes a nonselective preenrichment step, an IMS, DNA extraction, as well as DNA purification followed by hybridization probe-based real-time PCR analysis. An accurate comparability was achieved, because both methods analyzed the same preenrichment. The evaluation was carried out using both artificially and naturally contaminated meat samples. The IMS-real-time PCR assay provides a result after 12 to 13 h. Compared with the reference method and regarding artificially contaminated meat samples, the IMS-real-time PCR assay achieved a specificity of 80% (false-positive rate of 20%) and a sensitivity of 100% (false-negative rate of 0%). The relative accuracy was 94%. The detection limit of both methods was 10 CFU/25 g. The concordance index kappa, which defines the statistical accordance, was 0.85 and indicated the agreement of both methods on statistical criteria. Compared to the reference method and analyzing naturally contaminated meat samples (n = 491), the IMS-real-time PCR assay showed a specificity of 99.3% (false-positive rate of 0.7%) and a sensitivity of 83.7% (false-negative rate of 16.3%). The relative accuracy was 98%. The concordance index kappa had a value of 0.87 and highlighted the statistical agreement of both methods. In conclusion, the IMS-real-time PCR assay is suitable as specific, sensitive, and rapid screening method for the detection of Salmonella from meat.     

An 8-hour system for Salmonella detection with immunomagnetic separation and homogeneous time-resolved fluorescence PCR

We describe a system consisting of rapid sample enrichment and homogeneous end-point PCR analysis that enables the detection of Salmonella in various food matrices in 8 h. Sample preparation starts with 6 h enrichment step in supplemented broth, after which Salmonella cells are collected with immunomagnetic particles. The particles are washed and dispensed to ready-to-use PCR reaction vessels, which contain dried assay-specific reagents and an internal amplification control. PCR is performed with a novel instrument platform utilising the sensitive label technology of time-resolved fluorometry. Qualitative assay results are automatically interpreted and available in 45 min after sample addition. The overall accuracy, sensitivity and specificity of the Magda CA Salmonella system were 99.1%, 98.4% and 100.0%, respectively, based on the evaluation of 107 samples (beef, pork, poultry and ready-to-eat meals) artificially contaminated with sub-lethally injured Salmonella cells.     

恒温隔绝式聚合酶链式反应检测食品中金黄色葡萄球菌

建立一种恒温隔绝式聚合酶链式反应(insulated isothermal polymerase chain reaction,iiPCR)检测食品中金黄色葡萄球菌的方法.根据nuc基因设计特异性引物、探针,并探索针对食品样品快速提取金黄色葡萄球菌模板DNA的方法;通过优化引物、探针及模板的用量,对iiPCR的特异性、...     

可视化环介导恒温扩增法检测蔬菜产地环境灌溉水源中沙门氏菌

目的建立可视化环介导恒温扩增体系(loop-mediated isothermal amplification, LAMP)检测灌溉水源中沙门氏菌的分析方法。方法以沙门氏菌侵袭蛋白A(invA)基因序列设计特异性LAMP检测引物,优化LAMP反应体系和反应条件后,通过特异性实验、灵敏度实验对LAMP检测方法进行测试,并与国标检测方法对比检测人工污染的灌溉水源样品,验证该方法的准确性。结果灵敏度实验结果显示本LAMP检测方法最低检出限为7 CFU/25μL,特异性实验结果显示本LAMP检测方法具有良好的特异性,干扰菌株对本LAMP检测方法无影响。该方法在与国家标准检测方法对比,检测人工污染的灌溉水源样品时具有相同的准确性,检测灵敏度为1×10^2 CFU/mL。结论本研究建立了快速、准确、灵敏的恒温扩增体系,可以应用于灌溉水源中沙门氏菌的快速检测。     

环介导等温扩增方法在食品中沙门菌检测的应用和评价

将环介导等温扩增检测方法应用于食品中沙门菌的检验,并在检测方法特异性、灵敏度等方面与实时荧光PCR和传统检测方法进行比较。方法 针对沙门菌属高度保守的fimY基因设计环介导等温扩增检测引物并优化反应体系,在特异性、灵敏度和实际样品检测等方面与实时荧光PCR及传统检测方法比对。结果 本研究建立的LAMP方法检测沙门菌93株和非目标菌31株,具有良好的特异性。在纯培养、无需增菌情况下,其检测灵敏度为6.4×10²cfu/ml,与实时荧光PCR方法相当。食品基质添加试验中,环介导等温扩增方法检测低限为2cfu/25g样品;对45份实际食品样品检测结果表明,该方法实际样品检出率为11.1%,与实时荧光PCR及传统方法检测结果一致。结论 本研究建立的沙门菌环介导等温扩增检测方法具有良好的特异性,检测灵敏度与实时荧光PCR相当,适用于沙门菌的快速筛选。     

原位荧光LAMP技术检测食源性沙门氏菌

以沙门氏杆菌为研究对象,探讨了将原位荧光LAMP技术应用于检测沙门氏菌所需要的具体实验方法与数据,并成功将原位荧光LAMP技术应用于检测人工污染的食品中的沙门氏菌。实验选取invA基因作为靶序列设计了2对引物。结果证明,与传统沙门氏菌检测方法和PCR方法相比,原位荧光LAMP无需破碎细胞提DNA,反应方便易行,耗时少;反应条件恒定温和,能够减少细胞损伤;单基因检测水平证明反应的高灵敏度;良好的基因定位以及清晰的背景条件使反应结果更加直观,易于观察。     

An immunoconcentration-PCR assay to detect Salmonella in the environment of poultry houses.

An immunoconcentration-PCR assay was developed for the rapid and specific detection of Salmonella. This assay was evaluated against a conventional bacteriological method for the detection of Salmonella from environmental swabs of poultry houses. The 120 samples investigated were pre-enriched in phosphate buffered peptone water and Salmonella was separated by an immunoconcentration process using an automated system (VIDAS bioMérieux, Marcy l'Etoile, France) prior to PCR. The specificity of the assay was high as no false-positives were found. The sensitivity of the assay was 70%. The correlation between the ICS-PCR assay and the bacteriological method was 84%.     

多重PCR检测婴幼儿配方奶粉中3种食源性致病菌

为建立一种能够同时检测婴幼儿配方奶粉(Powdered Infant Formula,PIF)中克罗诺杆菌、沙门氏菌和金黄色葡萄球菌3种食源性致病菌的多重PCR检测方法。通过单重PCR验证了9对引物的特异性,筛选出其中3对特异性好的引物建立了多重PCR体系,对其特异性和灵敏度进行评价,并将其应用于人工污染PIF中3种食源性致病菌的检测。结果表明,针对克罗诺杆菌、沙门氏菌和金黄色葡萄球菌等3种食源性致病菌所筛选的3对引物分别能扩增出469、638和796 bp的目的条带,具有高度特异性。多重PCR检测体系的最佳退火温度为55℃,最佳Mg2+浓度为2.00 mmol/L,最佳引物浓度为400 nmol/L。在此条件下3种目的菌在102 CFU/mL均可同时扩增出较清晰条带。将建立的多重PCR检测体系应用于人工污染PIF中3种食源性致病菌的检测,其检出限达到103 CFU/g。本研究初步建立了一种能准确、快速、特异性的检测PIF中克罗诺杆菌、沙门氏菌和金黄色葡萄球菌的多重PCR方法,适用于PIF中3种常见的食源性致病菌的快速检测。     

Validation of a Diagnostic PCR Method for Routine Analysis of Salmonella spp. in Animal Feed Samples

As a part of a validation study, a comparative study of a PCR method and the standard culture-based method NMKL-71, for detection of Salmonella, was performed according to the validation protocol from the Nordic validation organ for validation of alternative microbiological methods (NordVal) on 250 artificially or naturally contaminated animal feed samples. The PCR method is based on culture enrichment in buffered peptone water followed by PCR using the DNA polymerase Tth and an internal amplification control. No significant difference was found between the two methods. The relative accuracy, relative sensitivity and relative specificity were found to be 96.0, 97.3, and 98.8%, respectively. PCR inhibition was observed for rape seed samples. For the acidified feed samples, more Salmonella-positive samples were found with the PCR method compared to the NMKL method. This study focuses on the growing demand for validated diagnostic PCR methods for routine analysis of animal feed and food samples to assure safety in the food production chain.     

基于G-四链体的PCR-RCA双重扩增技术检测沙门氏菌

将聚合酶链式反应（polymerase chain reaction,PCR）与滚环扩增技术（rolling circle amplification,RCA）联用,并把G-四链体互补序列嵌入到RCA扩增所需的哑铃环状模板上,通过PCR和RCA的双重扩增及特异性结合G-四链体的硫黄素T荧光信号增强的作用,达到基于G-四链体的PCR-RCA技术检测沙门氏菌（Salmonella）的目的。在优化的检测体系下,确定了沙门氏菌基因组DNA浓度对数与486 nm波长处的荧光信号强度具有良好的线性关系,回归方程为y=2.101x＋2.872 3（R2=0.992 5）,线性范围为17 fg/μL～1.7 ng/μL。根据实验的特异性分析,表明此方法适用于沙门氏菌属的检测,对人工污染沙门氏菌牛奶样品进行检测,检出限为4.28 CFU/mL。该方法具有特异性强、灵敏度高、检出限低等优点,为实现食源性致病菌的快速检测提供新的方法。