TaqMan实时荧光PCR对沙门氏菌能力验证样品的快速检测与鉴定

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TaqMan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12 h,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。     

食品检测能力验证中沙门氏菌的分离鉴定

目的 参加编号为CFAPA-375的食品中沙门氏菌能力验证,提高检验技能,验证实验室沙门氏菌的检测能力。方法 采用国标法对沙门氏菌能力验证样品进行检测,同时采用实时荧光PCR法作为辅助方法。对分离出的可疑菌落,采用全自动微生物鉴定系统和实时荧光PCR法进行全面鉴定。结果 样品N1和N3中检出沙门氏菌,样品N2中未检出沙门氏菌,实时荧光PCR法与国标法检测结果一致。结论 本次实验室的能力验证结果为“满意”,验证了实验室沙门氏菌的检测能力。实时荧光PCR法快速、简便、特异性强,可与国标法配合使用,以确保实验结果的准确快速。     

单核增生李斯特氏菌检测能力验证结果分析

目的验证本实验室对食品中单增李斯特氏菌的检出能力。方法按照能力验证作业指导书、GB4789.30-2016《食品安全国家标准食品微生物学检验单核细胞增生李斯特氏菌》和SN/T 1870-2016《出口食品中食源性致病菌检测方法实时荧光PCR法》进行检验。首先进行2次前增菌,再按照国标法进行选择分离、纯化、生化鉴定进行检验;同时利用增菌液进行实时荧光PCR方法检验。结果国标法和实时荧光PCR法检验结果均为CODE 40样品检出单增李斯特氏菌,CODE 61样品未检出单增李斯特氏菌。结论组织者对本实验室此次能力验证试验结果评价满意,说明本实验室同时具有传统国标法和实时荧光PCR法检测单增李斯特氏菌的能力。     

显色培养基上沙门氏菌及干扰菌的分离鉴定

研究比较沙门氏菌显色培养基(CAS)上干扰菌与沙门氏菌的区别,提高沙门氏菌的分离鉴定效率。使用沙门氏菌和7株干扰菌(铜绿假单胞菌、恶臭假单胞菌、施氏假单胞菌、荧光假单胞菌、斯氏普罗威登斯菌、摩氏摩根菌摩根亚种、粪产碱菌亚种)分别接种CAS、BS、XLD和HE培养基,观察菌落形态特征,分析生化试验结果,考察TTB和SC选择性增菌液对7株干扰菌的选择效果。7株干扰菌在CAS培养基上都是紫色菌落,仔细辨认大部分都可与沙门氏菌区分,无法从菌落形态区分的恶臭假单胞菌和荧光假单胞菌通过在BS上的生长情况或生化试验可以排除,TTB和SC选择性增菌液对7株干扰菌的选择效果各不相同,提示检测过程中TTB和SC两种增菌液须同时使用,相互补充,提高检出率。     

能力验证巧克力样品中肠道沙门氏菌双相亚利桑那亚种的分离鉴定

目的验证国标法和实时荧光定量PCR法2种方法对能力验证中的沙门氏菌双相亚利桑那亚种分离与鉴定的效果。方法按照作业指导书要求及GB 4789.4-2016《食品安全国家标准食品微生物学检验沙门氏菌检验》中的方法分离菌株,以实时荧光定量PCR仪对分离菌株进行快速筛查,再将生化鉴定结果符合沙门氏菌特征的菌株进行血清学鉴定。结果样品CODE 0575在沙门氏菌显色平板上分离得到蓝绿色圆形菌落,在BS平板上分离得到灰绿色圆形菌落,经实时荧光定量PCR仪快速筛查,结果为阳性;再通过VITEK 2 compact鉴定为肠道沙门菌双相亚利桑那亚种;该菌不产硫化氢,ONPG为阳性,确定血清型为60:r:e,n,x,z15。结论以国标法为基准,借助实时荧光定量PCR仪和VITEK 2全自动鉴定系统进行检测,可确保沙门氏菌双相亚利桑那亚种不被漏检;应特别注意沙门氏菌双相亚利桑那亚种在显色培养基上与常见沙门菌表型不一致;建议扩大对沙门氏菌双相亚利桑那亚种的监测范围。     

奶粉样品中沙门氏菌能力验证结果与分析

目的验证分析实验室对奶粉样品中沙门氏菌检测能力。方法依据GB 4789.4-2016《食品安全国家标准食品微生物学检验沙门氏菌检验》、SN/T 1059.7-2010《进出口食品中沙门氏菌检测方法实时荧光PCR法》进行改进,参照能力验证计划参试指导书来进行测定和结果判断。结果通过荧光定量PCR法进行快速初筛,初步判断样品含有阳性沙门氏菌;传统平板法结合生化鉴定套装和VITEK2进行复筛,将可疑沙门氏菌进行血清分型,检出2种血清型O:4,12 H:i,2和O:9,12 H:m,g:-,分别为鼠伤寒沙门氏菌和肠炎沙门氏菌;同时采用Ribo Printer全自动微生物基因指纹鉴定系统对可疑单菌落进行确证性鉴定,结果与血清分型一致。结论本次实验室的奶粉样品中沙门氏菌检测能力验证结果合格。     

奶粉样品中沙门氏菌检测能力验证结果与分析

目的 验证分析实验室对奶粉样品中沙门氏菌检测能力。方法 依据GB 4789.4-2016《食品安全国家标准食品微生物学检验沙门氏菌检验》、SN/T 1059.7-2010《进出口食品中沙门氏菌检测方法实时荧光PCR法》进行改进,参照能力验证计划参试指导书来进行测定和结果判断。结果通过荧光定量PCR法进行快速初筛,初步判断样品含有阳性沙门氏菌;传统平板法结合生化鉴定套装和VITEK2进行复筛,将可疑沙门氏菌进行血清分型,检出2种血清型O:4,12 H:i,2和O:9,12 H:g,m:-,分别为鼠伤寒沙门氏菌和肠炎沙门氏菌;同时采用Ribo Printer全自动微生物基因指纹鉴定系统对可疑单菌落进行确证性鉴定,结果 与血清分型一致。结论 本次实验室的奶粉样品中沙门氏菌检测能力验证结果合格。     

3种方法检测食品中致泻大肠埃希氏菌检测能力验证结果分析

目的提高食品中致泻大肠埃希氏菌的检测能力,促进实验室检测能力的提高。方法参照GB4789.6-2016《食品微生物学致泻大肠埃希氏菌检验》方法进行检测,采用VITEK 2 Compact全自动细菌鉴定系统进行生化鉴定、血清学鉴定,对可疑菌落进行普通PCR确证试验。同时使用多重实时荧光PCR法以及基质辅助激光解析电离飞行时间质谱(matrix-assisted laser desorption ionizationtime-of-flight mass spectrometry,MALDI-TOF-MS)分析鉴定可疑菌落。结果国标法和多重实时荧光PCR法能准确鉴定出目标菌,MALDI-TOF-MS可以检测大肠埃希氏菌,但无法区分致泻大肠埃希氏菌和肠出血性大肠埃希氏菌。结论 3种方法各有优劣,同时使用,综合判断,能确保试验结果准确快速。     

一例沙门氏菌测量审核的回顾与分析

目的通过测量审核提升本实验室检测沙门氏菌能力与自身竞争力。方法以测量审核作业指导书、出入境检验检疫行业标准SN/T1869-2007食品中多种致病菌快速检测方法 PCR法和国家标准GB4789.4-2016食品微生物学检验沙门氏菌检验为依据,采用PCR方法和传统增菌、分离、生化反应、血清学法鉴定沙门氏菌。结果编号091样品检出沙门氏菌,编号123样品PCR检测出现假阳性,经生化鉴定证实检出非沙门氏菌。结论 PCR出现假阳性,需经生化鉴定予以确认。本次测量审核样本检测结果与制样单位反馈结果一致。     

3种方法鉴定鸡肉粉中沙门氏菌的比对研究

目的采用3种方法对鸡肉粉中沙门氏菌进行检测。方法3份样品的前处理依据组织方提供的《作业指导书》进行,每瓶样品直接加入4.5 mL灭菌去离子水复溶,作为初始样本,后续实验依据GB4789.4-2016《食品安全国家标准食品微生物学检验沙门氏菌检验》操作。鉴定分离出疑似菌后,加入了实时荧光定量PCR法、基质辅助激光解吸电离飞行时间质谱法2种方法作为辅助检测,并用全自动微生物鉴定系统进行生化鉴定,再结合生化鉴定与血清学实验综合评判检测结果。结果使用3种鉴定方法在JS015、JS110、JS140样本中均检出沙门氏菌。结论本研究采用的实时荧光定量PCR法、基质辅助激光解吸电离飞行时间质谱法2种方法均能快速准确鉴定出沙门菌,特别是基质辅助激光解吸电离飞行时间质谱法和血清学分型配合使用,对沙门菌的快速鉴定有重要意义。     

The effect of pre-enrichment protocol on the sensitivity and specificity of PCR for detection of naturally contaminated Salmonella in raw poultry compared to conventional culture

Salmonella spp. are the leading cause of foodborne illness worldwide. Conventional culture techniques for the detection of Salmonella spp. are labor intensive and time consuming. Several rapid detection methods have been developed over the past few years. However, standard methods for sample handling and preparation have not been established and limited data are available on the sensitivity and specificity of these methods for detection of Salmonella in naturally contaminated retail meat. Using culture as the gold standard for Salmonella detection in naturally contaminated raw poultry products, the sensitivity and specificity of a polymerase chain reaction (PCR) detection method was determined under varying enrichment protocols. Chicken meat samples (ground, boneless/skinless breast meat, and bone-in breast meat with skin) from retail grocery stores were pre-enriched in buffered peptone water (BPW) and Salmonella specific primers ST 11 and ST 15 were used to amplify a 429 bp region of random fragment target specific to all Salmonella spp. There was a significant decrease (P-value<0.001) in the sensitivity of the PCR test when BPW pre-enrichment alone (85%) was used compared to the sensitivity achieved after both BPW enrichment and selective enrichment with RV and TT-H (100%). PCR failed to detect any positive samples when no pre-enrichment was conducted. A minimum of 12h pre-enrichment was required for detection of Salmonella by PCR at a limit of 100 colony forming unit (cfu)/1 ml of sample. No detectable amplification product was seen in those naturally contaminated meat samples testing negative by culture methods.     

2种检测方法在巧克力中沙门氏菌检验能力验证中的应用

目的比较国标法(生化试剂鉴定法和全自动细菌鉴定仪法)、实时荧光PCR法在能力验证项目巧克力中沙门氏菌检验中的优缺点。方法分析2种方法的不同检测原理,比较生化试剂鉴定法、BD Phoenix-100全自动细菌鉴定法、实时荧光PCR法在细菌鉴定应用中的优缺点。结果 3个能力验证样品检出1阳性2阴性, 2种检测方法结果一致, 3个样品实验均获得满意的结果。结论国标的常规培养法与仪器检测方法相结合的方式,有助于提高结果的准确性;参加能力验证工作有助于提高实验室的检测能力。     

食品能力验证中沙门氏菌的分离和鉴定

目的为了提升实验室食品中沙门氏菌检测能力,增强实验室竞争能力,本实验室参加了中国食品药品检定研究院组织的沙门氏菌检测能力验证活动。方法依据中华人民共和国国家标准GB 4789.4-2010,采用传统分离方法和血清学鉴定,联合全自动微生物生化鉴定系统(VITEK2-compact)对分离出的疑似菌进行生化鉴定。结果编号为CODE1样品+CODE1奶粉混合样检出纽波特沙门氏菌和科林德尔沙门氏菌,CODE3样品+CODE3奶粉混合样检出维普拉沙门氏菌、鼠伤寒沙门氏菌和埃科沙门氏菌,其余8个样品未检出。结论顺利完成本次能力验证活动。     

食品中单核细胞增生性李斯特菌的快速分离鉴定

单核细胞增生性李斯特菌(Listeria monocytogenes,简称单增李斯特菌)是一种重要的食源性致病菌,能引起人畜共患李斯杆菌病。本文采用两种不同的增菌分离方法从散装牛奶和猪肉中共分离得到8株单增李斯特菌。通过革兰氏染色、生化鉴定、PCR扩增hlyA基因、16SrDNA测序、血清分型等一系列实验对可疑菌株进行分析鉴定。综合实验结果和其他单增李斯特菌分离标准,探讨开发了一种快速分离鉴定单增李斯特菌的方法,该方法可在4～5d内完成单增李斯特菌的分离检测过程。     

Detection of low numbers of healthy and sub-lethally injured Salmonella enterica in chocolate

The capacity to detect low levels of healthy and sub-lethally injured Salmonella enterica cells in chocolate by two alternative rapid detection methods iQ-Check(TM)Salmonella II real-time PCR (Bio-Rad) and VIDAS? Easy SLM (BioMérieux) was assessed and compared with ISO 6579:2005. Chocolate, a low moisture food known to support the survival of Salmonella, was challenged as food matrix. Buffered peptone water (BPW) did not support the recovery of low levels of sub-lethally injured S. enterica independent of the detection method, while BPW supplemented with milk powder enabled detection by the three examined methods. However, inhibition of real-time PCR was observed since for one out of three repetitions of chocolate inoculated with a low number of sub-lethally injured S. enterica cells, no PCR signal was obtained. Therefore, attention should be paid to the enrichment step to avoid false negative results due to the presence of especially sub-lethally injured Salmonella cells in chocolate. An appropriate sample preparation (such as enrichment media and conditions for incubation) remains the key factor for reliable detection including sub-lethally injured cells and should be evaluated, if necessary optimized, for each detection assay.     

Using a portable real-time PCR assay to detect Salmonella in raw milk

The purpose of this study was to determine the efficacy of a portable real-time PCR system in detecting Salmonella spp. in raw milk. The 200 bulk milk samples chosen for this study constituted a subset of the samples for a larger study; this subset contained 24 samples that were culture positive for Salmonella and 176 that were culture negative. Milk was both plated directly on selective agar and plated after enrichment in selective media. Presumptive Salmonella colonies were isolated by direct culturing of five samples, while Salmonella was isolated from the remaining 19 positive samples only after enrichment. Presumptive Salmonella isolates were serotyped, and isolates from 22 samples were confirmed to be Salmonella isolates. PCR assays of culture-positive milk prior to enrichment yielded no evidence of Salmonella. DNA extracts of bacterial pellets from the enriched samples were analyzed for Salmonella by real-time PCR with the Ruggedized Advanced Pathogen Identification Device (RAPID). Fifty-four samples from the enrichment pellets tested positive for Salmonella by real-time PCR. Two samples that tested positive for Salmonella by culture and serotyping tested Salmonella negative by real-time PCR. Serotyping identified isolates from these samples as Salmonella Montevideo. All DNA extracts of Salmonella Montevideo isolates tested positive for Salmonella by real-time PCR. Thirty-three samples tested negative by culture and positive by real-time PCR. These results indicate that the portable real-time PCR system appears to be a useful tool for detecting Salmonella in raw milk. Additionally, the combination of enrichment and real-time PCR techniques used in this study can yield results in 24 h, compared with the 48 to 72 h required for traditional culture.