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。     

4种分离培养基检测志贺氏菌效果比较

志贺氏菌是一类具有高度传染性的肠道致病菌。文中基于GB 4789.5—2012《志贺氏菌检验》标准,比较了4种分离培养基检测志贺氏菌的效果。通过4种分离培养基对福氏志贺氏菌、宋内氏志贺氏菌的回收率、最低检出限及对不同样品检测效果的比较,对不同培养基的检测效果进行评价。结果显示4种分离培养基最低检出限相近,显色培养基01检测特异性优于其他3种;针对实际检测样品,应灵活选择培养条件,以提高检测的准确性及效率。     

志贺氏菌实时荧光单引物等温扩增方法的建立及应用

以志贺氏菌ipa H基因特异序列为靶序列,设计RNA-DNA组合引物和链终止序列,优化反应体系,建立实时荧光单引物等温扩增检测志贺氏菌的方法,反应时间为44 min。通过对4株不同群志贺氏菌和12株其他食源性致病菌进行实时荧光单引物等温扩增检测,结果表明,除4株志贺氏菌外,其他细菌均未扩增出荧光曲线。进一步研究表明,采用普通热裂解法提取DNA,实时荧光检测福氏志贺氏菌DNA的灵敏度为1.16 fg/μL,纯培养菌液的灵敏度为1.3 CFU/m L;对牛奶模拟样品中福氏志贺氏菌的检出限是1.8 CFU/m L。研究结果表明,实时荧光单引物等温扩增检测志贺氏菌灵敏度高、特异性强、耗时短、方法简便。     

奶粉中病原菌质控考核结果分析

目的 对质控考核样品进行检测, 分离鉴定肠杆菌科志贺氏菌属A群痢疾志贺氏菌和金黄色葡萄球菌, 并分析其中特别的生物学现象。方法 按照《食品安全国家标准 食品微生物学检验 (GB 4789.5-2012)》对考核检样进行检测。结果 分离出宋内志贺氏菌而未分离出金黄色葡萄球菌, 并在检验过程中观察到特别的生物学现象。结论 志贺氏菌在志贺氏菌属显色培养基上并非显示一样颜色, 有白色菌落周围培养基显示紫红色,也有黄色菌落周围培养基显示黄色。     

辣椒制品大肠杆菌O157及志贺氏菌污染调查与风险评估

掌握辣椒制品大肠杆菌O157及志贺氏菌的污染状况,为开展辣椒制品安全风险评估,企业及产品分类管理提供必要的依据。参照GB/T4789.36—2008全自动酶联荧光免疫分析仪筛选法对辣椒制品大肠杆菌O157进行快速筛选试验,大肠杆菌0157检验结果为阳性时,参照GB/T4789.36—2008常规培养法进行大肠杆菌O157分离鉴定确认。参照GB/T4789.5—2003对辣椒制品志贺氏菌进行检验。可疑菌株用API20E生化鉴定试剂盒及VITEK2compact30全自动细菌鉴定分析系统进行生化鉴定,综合生化试验和血清学的试验结果报告。共抽890份样品、包括4类辣椒制品、涉及18个省(区、市)的194家生产企业,进行了大肠杆菌O157和志贺氏菌检验,均未检出目标菌,表明大肠杆菌O157和志贺氏菌在辣椒制品中污染风险较小。     

牛奶样品中志贺氏菌胶体金免疫试纸条检测方法的建立

通过用超声波破碎的宋内氏志贺氏菌和福氏志贺氏菌的菌体碎片为免疫原免疫BALB/c小鼠,获得4株能稳定分泌抗志贺氏菌单克隆抗体的杂交瘤细胞株,选取其中一株制备单抗,并用胶体金标记。同时抗志贺氏菌的兔多抗和驴抗鼠抗体(二抗)分别喷涂于硝酸纤维素膜作为检测线(T线)和质控线(C线),研制了志贺氏菌胶体金快速检测试纸条。结果显示试纸条的灵敏度为105mL-1,并只与两株志贺氏菌有阳性反应外,而与其它23株常见的细菌无交叉反应。同时在检测添加有阪崎肠杆菌和长双歧杆菌的牛奶样品中,结果显示试纸条的灵敏度为106mL-1。志贺氏菌免疫胶体金试纸条的建立,为加强食品中志贺氏菌的检测工作,建立一种快速、简便的筛查方法具有重要的意义。     

志贺氏菌噬菌体空间杀菌效力的研究

将福氏志贺氏菌噬菌体SF-2A、痢疾志贺氏菌噬菌体SD-11及宋氏志贺氏菌噬菌体SS-92以等比例混合后进行气雾喷洒分析其在空间的杀菌效力,以期在食品生产和加工中控制食源性志贺氏菌的污染。首先在空间分析不同噬菌体气雾喷洒后的杀菌活性和最佳作用浓度,其次将福氏、痢疾及宋氏志贺氏菌以等量进行混合,以3×105cfu/mL的终浓度进行人工喷洒污染,并以3×108cfu/mL的噬菌体混合物进行气雾喷洒,分别在喷洒后1 h,2 h和3 h检测宿主菌的数量。结果显示,噬菌体SF-2A、SD-11及SS-92均能在空间有效灭活其相应宿主菌,其混合溶液作用3 h后已检测不到志贺氏菌。由此可见,混合噬菌体能够有效控制志贺氏菌的污染,这为食品生产及加工中环境中的病原菌的污染控制提供了新途径。     

牛奶样品中志贺氏菌的快速PCR检测技术研究

建立牛奶中快速检测志贺氏菌的有效方法。根据GenBank公布的志贺氏菌ipaH基因的保守序列设计特异性引物,筛选合适的DNA模板制备方法,采用快速常规PCR和定量实时PCR结合,对培养液中及牛奶阳性样品中的志贺氏菌进行检测,检测敏感度可达到2CFU/ml,检出时间小于20h。新建的PCR方法具有特异性好,灵敏度高等特点,适用于快速、准确检测牛奶中志贺氏菌的需要。     

抗志贺氏菌IgY的提纯及建立间接ELISA检测志贺氏菌

采用水稀释法提纯抗志贺氏菌IgY,检测10mg/mL纯化抗志贺氏菌IgY的效价为1∶320,并以此IgY为基础建立间接ELISA检测志贺氏菌,测定志贺氏菌纯培养液的检出限为105～106cfu/mL。对蜡样芽孢杆菌等10株不同菌株的检测结果表明,该方法对志贺氏菌有明显的检测特异性,对所测定的其它菌株无交叉反应。     

反转录-环介导等温扩增技术快速检测志贺氏菌

建立了反转录-环介导等温扩增技术的志贺氏菌快速检测方法。针对志贺氏菌特异保守基因ipa H设计多套引物,建立优化了的反应体系,并评价其准确性、特异性、灵敏度。以人工污染脱脂乳样品比较RT-LAMP与RT-PCR的灵敏度。结果表明,在65℃等温条件下,RT-LAMP反应可在30 min内完成。在活菌/损伤菌模型中,该方法表现出比国标法更好的准确性。在23株细菌标准菌株中仅对4株志贺氏菌有特异性检出。志贺氏菌RNA模板的检测灵敏度为7 fg/μL。人工污染脱脂乳样品检测灵敏度达到40 CFU/g,比RT-PCR方法高出2个数量级。表明所建立的志贺氏菌RT-LAMP扩增方法可以准确的检测志贺氏菌,同时具有快速、特异、灵敏的优势,可用于志贺氏菌的快速筛查和现场监控。     

动物源弯曲菌分离纯化鉴定方法优化

【目的】弯曲菌是重要的食源性人畜共患病原菌,通常难以诊断,而动物源方向缺少系统的检测分析方法及相应的优化。为了获得更适宜分离动物来源的空肠弯曲菌和结肠弯曲菌,提高分离效率,降低分离成本,特开展方法优化,填补动物源弯曲菌分离方法建设的空白,为耐药菌株的有效追踪溯源和风险评估提供依据。【方法】对已有的空肠弯曲菌和结肠弯曲菌分离纯化鉴定方法Preston肉汤和Bolton肉汤增菌;CCDA选择性培养分离、弯曲菌显色培养分离、Skirrow选择性培养分离;生化鉴定和分子生物学鉴定做了筛选优化,针对目前存在的分离纯化和鉴定方法进行了调查比较,得到了更适合于健康动物来源的粪便和盲肠中的分离方法。【结果】牛血清预增菌、CCDA选择性分离、弯曲菌显色培养基纯化、哥伦比亚血琼脂进行扩增及分子学方法鉴定,同时建议采用厌氧条件进行培养。该流程简单易行,成本较低,适合于绝大多数实验环境条件,是获取弯曲菌的首选流程,便于推广实施。【结论】通过将现研究阶段存在的大多数检测方法进行了综合分析,整理出一套针对于动物来源的粪便及肠道内容物的检测分析方法,对健康动物中弯曲菌的分离效率能够提高30%,同时空肠弯曲菌和结肠弯曲菌的分离效率无明显差异。     

食品微生物测试片的研究进展

食品安全是人们赖以生存和发展的基础。食品微生物是影响食品安全的重要因素之一。传统的培养计数法主要依赖于微生物富集培养、选择性分离、生化鉴定,存在操作步骤繁杂、检测周期长等缺点,难以满足当今高速发展的现代化食品行业的检测要求。快速测试片作为一种新型微生物检测工具,凭借操作简单、节省空间、成本低、便于现场检测等优点,在多个领域得到了广泛应用。本文阐述了不同种类微生物测试片的作用原理和检测范围,并对当前微生物快速检测卡的优缺点进行了比较和评述。我国微生物快速检测技术研究开始较晚,对微生物测试片的研发和制作也处于初级阶段。因此,研发具有我国自主知识产权、具有高灵敏度和高特异性的测定产品仍是今后的发展方向。     

运用实时荧光PCR法辅助鉴定能力验证样品中的沙门氏菌

目的运用实时荧光PCR法辅助鉴定能力验证样品中的沙门氏菌,以提高检验准确度。方法采用传统的增菌、选择性培养、生化鉴定方法进行样品检验,实时荧光PCR法作为辅助手段对BPW增菌液及可疑菌落进行扩增,最后用API20E鉴定系统对可疑菌落进行确认。结果 3个样品经增菌、选择性培养、生化鉴定后均无显著符合沙门氏菌菌落特征及生化特征的可疑菌落;缓冲蛋白胨水增菌液实时荧光PCR法扩增结果表明样品S02为可疑样品,其分离得到的菌落S02-4有典型扩增曲线;经API20E鉴定系统确认,样品S02为阳性样品,阳性菌为猪霍乱沙门氏菌亚利桑那亚种。结论实时荧光PCR法操作简单,准确度高,可作为传统检验方法的有效补充。     

乳粉能力验证中沙门氏菌的分离鉴定和血清分型

目的参加编号为NIFDC-PT-220的能力验证,考核从乳粉中检出沙门氏菌及血清分型能力。方法参照国标方法对中国食品药品检定研究院提供的随机乳粉样品(CODE140、CODE121)进行沙门氏菌的检验,并将荧光酶联免疫法和荧光定量PCR技术作为辅助方法。对分离出的疑似菌进行生化鉴定,并且采用全自动微生物鉴定系统进行全面鉴定。结果编号CODE140血清分型为O:4,5,12, H:i; 1,2被确定为鼠伤寒沙门氏菌。编号CODE121未检出。结论荧光酶联免疫法和荧光定量PCR技术缩短了检测时间,特异性和敏感性好,与国标方法配合可确保实验结果的准确性。     

Comparison of the BAX System with a multiplex PCR method for simultaneous detection and identification of Campylobacter jejuni and Campylobacter coli in environmental samples

The Campylobacter detection is performed by conventional culture methods and the identification of Campylobacter jejuni and Campylobacter coli is principally based on the hippurate hydrolysis test. The two major drawbacks of this biochemical test for species identification include the inconsistency of the results and the presence of atypical strains, which can lead to the misidentification of an isolate. As an alternative, multiplex polymerase chain reaction (mPCR) protocols for the simultaneous detection and identification of different Campylobacter species have been developed. This study examined the performances of an experimental BAX System assay for the C. jejuni and C. coli identification in comparison to a multiplex PCR protocol recently published. The samples tested were represented by 106 environmental swabs collected on Teflon strips and tables, stainless steel saws, hooks and trays, ceramic floors and walls, as well as equipment surfaces, located in a swine (N=50) and a poultry (N=56) slaughterhouse. The highest Campylobacter detection rate was obtained after 48 h of enrichment by using both the PCR procedures. After 24 h, the BAX System provides a more rapid and accurate Campylobacter detection and identification assay than the multiplex PCR. Except for two samples, all the broths where Campylobacter cells were detected after 24 or 48 h of enrichment, with at least one of the PCR protocols, gave Campylobacter colonies using the culture method.     

Direct detection and identification of Arcobacter species by multiplex PCR in chicken and wastewater samples from Spain

Twenty-two chicken livers, 10 chicken carcasses, and 15 wastewater samples were processed and analyzed for Arcobacter by PCR and traditional culture methods. Samples were enriched for 24 and 48 h, incubated at 30 degrees C under aerobic conditions, and streaked on blood selective media. To determine the best isolation conditions, 20 samples also were processed under microaerophilic conditions at 37 degrees C. Simple and multiplex PCR assays were used directly with enrichment broths and isolated strains. Seventeen Arcobacter strains were isolated from chicken samples, and A. butzleri was the only Arcobacter species identified. The direct PCR assay revealed that 29 of the 32 chicken samples were contaminated with Arcobacter. A. butzleri was the most frequently detected species, although Arcobacter cryaerophilus also was present in some of the samples and Arcobacter skirrowii occasionally was detected. All the wastewater samples were positive by PCR assay for Arcobacter after 24 h of enrichment. A. butzleri and A. cryaerophilus were detected with the multiplex PCR assay. Fourteen Arcobacter strains were isolated from 10 of the 15 water samples analyzed; 7 were identified as A. butzleri and the remaining 7 were A. cryaerophilus. Both for chicken and water samples, Arcobacter detection rate for PCR amplification was higher than for culture isolation. These results indicate the high prevalence of Arcobacter in chicken and wastewater and the inadequacy of available cultural methods for its detection. The species-specific multiplex PCR assay is a rapid method for assessing Arcobacter contamination in chicken and wastewater samples and is a viable alternative to biochemical identification of isolated strains.     

克罗诺杆菌检测方法的研究进展

克罗诺杆菌是一种食源性致病菌,能够感染婴幼儿并导致坏死性小肠结肠炎、脑膜炎和菌血症,死亡率最高可达80%。克罗诺杆菌广泛地存在于食品和自然环境当中,并且具有极强的抗干燥能力,因此容易污染乳粉和其原料并在其中长期存在。控制克罗诺杆菌的污染需要增强食品生产质量控制,也需要开发相应的检测技术。本研究主要从生理生化检测、免疫学检测技术、核酸检测技术等对克罗诺杆菌的检测方法进行综述,对上述各种检测方法的原理和优劣势进行了分析和总结,并且对克罗诺杆菌检测方法的未来发展进行展望。需要指出的是,由于克罗诺杆菌在婴幼儿食品中污染量低、婴幼儿食品基质成分复杂且检测要求高的特点,增菌培养是不同检测方法不可缺少的步骤并且是检测的限速步骤,未来还需要对这一环节进行更为深入的研究以提升检测效率。     

The VIT technology for rapid detection of Listeria monocytogenes and other Listeria spp

Detection of Listeria monocytogenes is generally performed in a two-step cultural enrichment process and takes on average 1 week until the biochemical identification of a L. monocytogenes suspicious colony is completed. However, food processing companies depend increasingly on test methods, which attempt to generate results comparable to standard methods but in reduced time-frame and which allow to release produced batches dependent on such results. In the present study, the vermicon identification technology (VIT), a rapid commercial test system using fluorescently labelled gene probes, was compared to a cultural standard method. In total, 298 naturally contaminated samples were analysed. The sensitivity and the specificity of the VIT system were 100% for the detection of L. monocytogenes and 97.1% and 100%, respectively, for the detection of the genus Listeria.     

Comparison of a real-time PCR and an IMS/culture method to detect Escherichia coli O26 and O111 in minced beef in the Republic of Ireland

This study compared an immuno-magnetic separation (IMS)/culture method and a real-time PCR method to detect Verocytotoxigenic Escherichia coli (VTEC) serovar O26 and/or O111 in minced beef. A total of 65 samples were examined, 40 of which were frozen beef samples previously established as containing E. coli O157, and 25 were samples of fresh minced beef, purchased from butcher shops in the Dublin area. After selective enrichment, all samples were (a) subjected to IMS, plated on differential media and identified as E. coli O26 or O111 using biochemical and immuno-logical methods; and (b) subjected to DNA extraction and real-time PCR analysis using primers and probes against E. coli O111 and O26 serovar specific genes, and verotoxin genes. Overall, from the 65 minced beef samples collected, three were positive for E. coli O26 by real-time PCR, with only one of these samples positive for E. coli O26 by the culture method. One sample was positive for E. coli O111 by both real-time PCR and the culture method. The two samples found positive for E. coli O26 by real-time PCR method but not by culture method belonged to the group of frozen beef samples, indicating that the previously developed culture method for the detection of E. coli O26 may not be suitable for the detection of freeze injured cells. In conclusion, this study highlights the role of beef meat in the transmission of non-O157 VTEC. The results of the study emphasize that the analyses for emergent pathogens should be included in food safety surveillance systems and that the development of standard methods for the detection of E. coli O26 and O111 in routine food testing is needed in order to reduce the consumer exposure to contaminated food.     

An evaluation of rapid methods for detecting Escherichia coli O157 on beef carcasses

Numbers of Escherichia coli O157 in food may be low and sensitive techniques are therefore needed for its detection. The objectives of this study were to use carcass meat samples artificially inoculated with various strains of E. coli O157 to compare the sensitivity of enrichment in three different media and to compare immunomagnetic separation followed by culture of magnetic beads to cefixime tellurite sorbitol MacConkey agar with three immunoassays for the detection of E. coli O157 in the enrichment cultures. Duplicate 250, 25 and 2-3 CFU of each of 16 strains of E. coli O157 added to 25-g samples of beef carcass meat were used to compare the sensitivity of (1) enrichment in supplemented tryptone soya broth (sTSB), Reveal 8-h and Reveal 20-h media, and (2) immunomagnetic separation and culture to cefixime tellurite sorbitol MacConkey agar (IMS/CT-SMAC) with Reveal, VIP and STAT immunoassays for detecting the organism. An initial inoculum of 250 CFU/25 g meat was detected in all 32 samples by IMS/CT-SMAC performed on all enrichment media and by Reveal performed on Reveal 8-h and Reveal 20-h media, but in only 30, 19 and 9 samples by Reveal, VIP and STAT, respectively, performed on sTSB medium. An initial inoculum of 25 CFU/25 g meat was detected in 28, 32 and 30 of 32 samples by IMS/CT-SMAC performed on sTSB, Reveal 8-h and Reveal 20-h media, respectively, and in 32 and 30 samples by Reveal performed on Reveal 8-h and Reveal 20-h media, but in only 22, 11 and 2 samples by Reveal, VIP and STAT, respectively, performed on sTSB medium. An initial inoculum of 2-3 CFU/25 g meat was detected in 25, 31 and 28 of 32 samples by IMS/CT-SMAC performed on sTSB, Reveal 8-h and Reveal 20-h media, respectively, and in 25 and 23 samples by Reveal performed on Reveal 8-h and Reveal 20-h media, but in only 14, 1 and 0 samples by Reveal, VIP and STAT, respectively, performed on sTSB medium.