纳米免疫磁分离-实时荧光聚合酶链式反应快速检测海产品中副溶血性弧菌

采用副溶血性弧菌单克隆抗体、纳米免疫磁分离技术,结合实时荧光聚合酶链式反应（polymerase chain reaction,PCR）检测方法,建立海产品中副溶血性弧菌纳米免疫磁分离-实时荧光PCR检测方法。副溶血性弧菌纳米免疫磁珠在菌体浓度为103 CFU/mL水平时,对副溶血性弧菌的捕获率达到74%。在纯培养、无需增菌情况下,该方法检测灵敏度达到140 CFU/mL;通过134 株副溶血性弧菌和74 株非目标菌的测试,实时荧光PCR技术具有良好的特异性;在食品基质添加实验中,其检测限为2 CFU/25 g样品,增菌时间缩短到8 h。     

3种快速检测柳州螺蛳粉中热损伤沙门氏菌方法的比较分析

目的比较实时荧光PCR法、全自动免疫分析仪法(vitek immune diagnostic assay system, VIDAS)和环介导等温扩增法(loop-mediatedisothermalamplification, LAMP)检测柳州螺蛳粉中热损伤沙门氏菌。方法以预包装柳州螺蛳粉为样品,利用3种热损伤沙门氏菌以人工添加的方式模拟污染样品,污染水平为4CFU/25 g和12 CFU/25 g,通过一步增菌和二步增菌后分别用实时荧光PCR、VIDAS和LAMP检测,并与国家标准法比较。结果当3种快速检测方法对只经过一步增菌的3种热损伤沙门氏菌进行检测时,7组VIDAS检测结果和2组LAMP检测结果出现假阴性;对40份模拟样品检测结果显示, VIDAS无法检测柳州螺蛳粉中低污染水平的热损伤亚利桑那沙门氏菌。结论实时荧光PCR法相对VIDAS法和环介导等温扩增法更适合基质复杂且沙门氏菌污染水平低样品的快速检测。     

肉及肉制品中沙门氏菌活细胞的PCR检测研究

将荧光染料叠氮溴化丙锭(propidium monoazide,PMA)与普通聚合酶链式反应(polymerase chain reaction,PCR)结合,通过对PMA的曝光时间、浓度进行优化,确定PMA-PCR区别死活细胞的最佳条件,并制作活细胞定量标准曲线,建立肉及肉制品中沙门氏菌活细胞的PMA-PCR检测方法。结果表明：使插入死细胞DNA中的PMA活化并且光解溶液中游离PMA的最佳曝光时间为15min;不抑制沙门氏菌活细胞DNA扩增的最大PMA质量浓度为10μg/mL;完全抑制热致死细胞DNA扩增的最小PMA质量浓度为4μg/mL。经PMA处理,含有不同比例的沙门氏菌热致死细胞和活细胞的混合液中活的沙门氏菌能够通过PCR被选择性的检测,最小检测限为20CFU/PCR。而且,经研究发现在20～2×105CFU/PCR范围内,电泳条带相对荧光强度与活细胞数的对数具有线性关系。采集30份肉及肉制品样品,利用PMA-PCR方法检测出两份生肉样品中存在沙门氏菌,经过6h的富集培养后的活菌浓度分别为2.5×103CFU/mL和3.4×103CFU/mL。     

实时荧光PCR法检测食品中亚利桑那沙门氏菌

沙门氏菌（Salmonella）是食品检测过程中最常见的致病菌之一,亚利桑那沙门氏菌（Salmonella arizonae）又是沙门氏菌中比 较难鉴定的亚种。该实验通过实时荧光聚合酶链式反应（PCR）方法快速准确的检测亚利桑那沙门氏菌和其他沙门氏菌。根据GenBank 公布的亚利桑那沙门氏菌和其他沙门氏菌gud D基因序列, 分别设计引物和Taqman探针。 使用10株不同血清型的沙门氏菌标准菌 株、88株沙门氏菌分离株和29株食品中常见食源性致病菌进行实时荧光PCR特异性实验。结果显示,该实验所设计的引物探针特异 性非常好。 实时荧光PCR灵敏性试验结果表明,检测灵敏度可达到1～10 CFU/mL的添加浓度。 经模拟污染样品验证,所建立的实时 荧光PCR方法与传统方法的检测结果相一致,具有检测周期短、操作简便的优势。     

磁分离结合qPCR快速检测酱卤肉中的沙门氏菌

验证噬菌体磁分离结合实时荧光定量聚合酶链式反应,快速检测方法对食品中沙门氏菌的检测效果。以一株鼠伤寒沙门氏菌的特异性噬菌体T102为分子识别元件,首先将其与羧基化磁珠偶联,制备获得噬菌体磁性颗粒（Phage T102 Magnetic Beads）复合物,利用噬菌体磁性颗粒复合物从食品中特异性分离富集沙门氏菌,然后利用实时荧光定量聚合酶链式反应检测富集后的沙门氏菌。该沙门氏菌快检方法检出限为100 CFU/mL（0.1 CFU/PCR）,线性范围为1×102~1×109 CFU/mL,变异系数2.1%,特异性强,检测时间为6 h。实验选取300批食品安全抽检样品与GB 4789.4-2016标准《食品安全国家标准食品微生物学检验沙门氏菌检验》进行比对,均未检出阳性样品,结果一致。该方法可为噬菌体偶联纳米磁珠在食源性致病菌检测领域的应用提供参考依据。     

荧光定量环介导等温扩增法快速检测食品中沙门氏菌

目目的 建立一种快速简易检测食源性沙门氏菌的实时荧光定量环介导等温扩增（quantitative loop-mediated isothermal amplification, qLAMP）方法。方法 依据沙门菌属invA基因序列设计引物, 并结合短时间增菌构建沙门氏菌快速检测qLAMP法, 使用人工污染样品进行验证。结果 建立的qLAMP法最佳反应时间为40 min, 最佳反应温度是65 ℃, 最佳Mg2+浓度为6 mmol/L, Bst 2.0 WarmStart聚合酶的浓度为0.40 U/μL, 反应特异性良好, 纯培养实验表明方法检出限为100 CFU/mL。将沙门氏菌人工添加至鸡胸肉、果蔬沙拉、山泉水中, 经过7 h BPW有效增菌后所建立的qLAMP法在食品基质中的检出限达到5 CFU/25 g。结论 该方法准确、简单易行, 实验成本低, 可用于食品中沙门氏菌的快速检测。通过WarmStart Colorimetric LAMP试剂盒无需昂贵仪器即可快速检测,为沙门氏菌现场即时检测的场景提供技术支持。     

预包装柳州螺蛳粉沙门氏菌实时荧光PCR快速检测

目的建立实时荧光PCR法快速检测预包装柳州螺蛳粉中沙门氏菌的分析方法。方法根据沙门氏菌invA基因设计引物和探针,优化反应体系中的探针浓度后对人工添加沙门氏菌和干扰菌模拟受污染的预包装柳州螺蛳粉样品进行检测。结果设计的引物和探针只对阳性菌株有荧光反应,具有良好的特异性,最佳反应探针终浓度为0.2μmol/L,检测灵敏度为100CFU/mL,扩增效率103.92%;对模拟污染的预包装柳州螺蛳粉经过一步增菌18 h后最低能检测出4 CFU/25 g沙门氏菌。结论实时荧光PCR检测灵敏度高、特异性强,可应用于预包装柳州螺蛳粉中沙门氏菌的快速高效检测。     

荧光免疫层析法结合免疫磁珠分离技术快速检测单增李斯特菌

为建立单增李斯特菌简单快速、灵敏度高和特异性强的检测方法,本研究以抗单增李斯特菌单克隆抗体偶联磁珠制备免疫磁珠;以羧基荧光微球标记的抗单增李斯特菌多克隆抗体及鼠IgG为标记抗体,抗单增李斯特菌多克隆抗体和羊抗鼠二抗分别作为检测线和质控线制备荧光免疫层析试纸条。将免疫磁珠分离与荧光免疫层析法相结合应用于单增李斯特菌的现场快速检测中。结果表明:荧光免疫层析试纸条对纯培养单增李斯特菌的检测限为4×105CFU/mL,联合检测方法10倍、100倍浓缩时,检测限分别为4×104CFU/mL和1×104CFU/mL。联合检测体系特异性较好,与实验室保存的10株细菌无交叉反应。人工污染样本检测限为1×104CFU/mL,同纯培养物相比检测灵敏度并没有降低。本方法的建立对于食品中单增李斯特菌的现场快速检测具有重要意义。     

荧光假单胞菌、沙门氏菌和单增李斯特菌多重PCR检测方法的建立

针对肉制品中易污染的荧光假单胞菌、沙门氏菌和单增李斯特菌等有害微生物,通过3 种标准菌株及肉制品建立多重聚合酶链式反应方法,实现肉制品中这3 种菌的同时、快速检测。利用荧光假单胞菌的gyrB基因、沙门氏菌的invA基因和单增李斯特菌的hlyA基因设计3 对特异性引物,在确定引物特异性的基础上,对3 种标准菌株及在冷却肉上过夜富集后进行灵敏度检测。结果表明,该多重聚合酶链式反应方法对于同时检测这3 种有害微生物具有高度的特异性;同时检测这3 种菌时,纯菌DNA检测限可达1 pg/μL;将3 种菌一起接种到冷却肉中35 ℃过夜培养后,荧光假单胞菌、沙门氏菌和单增李斯特菌的检测限分别可达到9、5、70 CFU/mL。     

基于侧向流免疫的单增李斯特氏菌检测方法的建立

传统微生物方法以及实时荧光聚合酶链式反应(Polymerase chain reaction, PCR)方法都不能完全满足重大活动食品安全保障工作中现场检测的要求,因此研究将重组酶聚合酶等温扩增(Recombinant polymerase isothermal amplification, RPA)与侧向流免疫试纸(Lateral flow immunoassay,LFS)技术结合用于检测单增李斯特氏菌。RPA-LFS方法在纯菌、人工污染熟肉制品和新鲜水果中的检出限分别为3.0×101、3.0×10¹ CFU/g和3.0×10² CFU/g。结合叠氮溴化丙啶(Propidium monoazide,PMA)处理,建立了检测活菌的PMA-RPA-LFS检测方法,该方法可以抑制104 CFU/mL浓度水平的死菌的扩增。将2种方法和GB 4789.10—2016用于人工污染和实际样品的检测,结果发现PMARPA-LFS可减少死菌带来的假阳性。建立的方法分别适用于未经或经热处理的食品。该研究为不同类型食品提供不同的检测方法选择,同时为重大活动食品安...     

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。     

Evaluation of universal preenrichment broth for growth of heat-injured pathogens.

Universal preenrichment broth (UPB) was developed to enable enrichment of injured foodborne pathogens of different genera simultaneously in lieu of having to undergo separate simultaneous enrichment cultures for subsequent detection or isolation of each pathogen. Enrichment conditions in UPB for growth of injured pathogens to populations that will enable pathogen detection by rapid immuno-based or polymerase chain reaction (PCR)-based assays have not been defined. Hence, studies were done to determine recovery and growth rates of heat-injured Escherichia coli O157:H7, Salmonella enterica ser. Typhimurium, Salmonella enterica ser. Enteritidis. and Listeria monocytogenes in UPB. Bacterial cells were heat injured in tryptic phosphate broth at 57.2 degrees C and inoculated at populations of ca. 0.17 to 63 injured cells per ml with raw ground beef, fresh chicken, lettuce, and environmental sponge samples. Enrichment cultures were sampled at 1, 2, 3, 4, 5, 6, and 24 h at 37 degrees C postinoculation, and pathogens were enumerated on appropriate selective media. Results revealed that recovery and growth of pathogens during the first 6 h of enrichment were not sufficient to ensure adequate numbers of bacteria (> 10(3) CFU/ ml) for detection by most immunoassays or PCR assays. Cells often required 3 to 4 h for recovery before growth was initiated. Salmonella Typhimurium, Salmonella Enteritidis, E. coli O157:H7, or L. monocytogenes cell populations in enrichment cultures with ground beef or lettuce at 6 h were 0.5 to 2.9 log10 CFU/ml. At 24 h of incubation, cell counts of enrichment samples for the three pathogens from all food and environmental sponge samples ranged from 4.0 to 8.3 log10 CFU/ml. Enrichment in UPB at 37 degrees C of foods or environmental sponge samples containing heat-injured cells of Salmonella Typhimurium, Salmonella Enteritidis, E. coli O157:H7, or L. monocytogenes reliably provides at 24 h of incubation-but not at 6 h-sufficient cell populations for detection by rapid immunoassay or PCR assay procedures that can detect at least 4 log10 CFU/ml. These results raise questions regarding the sensitivity of rapid detection methods that employ an abbreviated enrichment protocol of 6 h or less.     

Evaluation of a 5'-nuclease (TaqMan) assay with the thin agar layer oxyrase method for the detection of Yersinia enterocolitica in ground pork samples

A 5'-nuclease (TaqMan) assay was evaluated for its capability to recover and detect stressed Yersinia enterocolitica. Sensitivity studies of a 5'-nuclease assay for detecting Y. enterocolitica 0:8 in a pure culture system and spiked ground pork samples demonstrated that the assay has reliable sensitivity with a detection limit of 3 to 4 log CFU/ml or CFU/g. The PCR 5'-nuclease (TaqMan) assay was evaluated with the Thin Agar Layer Oxyrase method (TALO, overlaying 14 ml of Trypticase soy agar with a 1:30 dilution of "Oxyrase for Agar" onto a prepoured pathogen-specific, selective medium), and it was compared against the selective medium cefsulodin-irgasan-novobiocin (CIN) for recovering and detecting Y. enterocolitica from inoculated nonfrozen and frozen (-15 degrees C, 2 days) ground pork samples. The TALO method showed more sensitivity (detection limit, 2 log CFU/ml), and it has greater recovery capability (0.5 to 1 log CFU/ml) than CIN (P < 0.05). The 5'-nuclease assay provided rapid detection processing (5 versus 24 h after an 18-h enrichment). The sensitivity per PCR was calculated to as low as 0 to 1 log CFU per PCR reaction; however, in the method's current developmental stage, target pathogens should be enriched to 3 to 4 log CFU/ml or CFU/g to show consistent results. In a survey of 100 ground pork samples using TALO, CIN, and PCR methods, no Y. enterocolitica was recovered. A combined cultivation and an automated PCR TaqMan could be used as a presumptive screening test for detecting Y. enterocolitica in food samples.     

婴幼儿奶粉中阪崎肠杆菌双重荧光PCR快速检测方法的建立

旨在建立针对阪崎肠杆菌的双重荧光PCR快速检测方法。以阪崎肠杆菌局部大分子合成(MMS)操纵子和外膜蛋白A(ompA)为靶基因,建立双重荧光PCR反应体系,探讨该体系的特异性、灵敏度和抗干扰能力。结果表明,双重荧光PCR体系对阪崎肠杆菌的灵敏度为4.3×103 CFU/mL,人工污染初始菌量为2 CFU/100 g奶粉样品增菌24 h即可检出;39株实验菌中的15株阪崎肠杆菌出现特异性扩增,24株非阪崎肠杆菌未出现特异性扩增。本研究所建立的双重荧光PCR体系特异好、灵敏度较高及抗干扰能力强,可用于婴幼儿奶粉中阪崎肠杆菌的快速检测。     

Sequencing of an internal transcribed spacer region of 16S-23S rRNA gene and designing of PCR primers for the detection of Salmonella spp. in food

DNA sequences of an internal transcribed spacer (ITS) region for 40 Salmonella serovars were determined and compared with ITS sequences of Salmonella spp., and non-Salmonella spp. already available on the GenBank database. From such comparison, two Salmonella-specific ITS based PCR primers, ITSF and ITSR, were designed. When Salmonella strains with various serotypes were PCR assayed with primers ITSF/ITSR, all generated PCR products with molecular weight bands equal to 312 bp. On the other hand, 48 non-Salmonella isolates, including strains of Enterobacteriaceae and other food pathogens generated negative results. Detection limits of this PCR method was 1-9 CFU per assay. These PCR primers were used for the detection of Salmonella cells in artificially contaminated foods, including chicken meat and whole milk. The detection limit was 1-9 x 10(3) CFU per assay. With an 8-h enrichment step performed prior to the PCR assay, however, the detection limit became 1-9 CFU per gram of the food sample.     

基于荧光微球的免疫层析法结合免疫磁珠分离技术快速定量检测鼠伤寒沙门氏菌

为了建立一种能快速、灵敏、特异检测鼠伤寒沙门氏菌(Salmonella typhimurium,ST)的方法,本研究以荧光微球(FM)为标记物建立了荧光微球免疫层析检测法(FM-ICA),将羧基化的磁性微球与抗体偶联制备了抗-ST免疫磁珠(IMB),并将FM-ICA与IMB联合用于富集和检测三种食品样本中的ST。在优化条件下,FM-ICA检测的线性范围为3.16×10~6~2×10~9 CFU/m L,回收率为80%~120%,变异系数均小于5%。当菌液浓度大于1×10~8 CFU/m L时,结果显示与同属沙门氏菌的交叉反应;而当菌液浓度小于或等于1×108CFU/m L时,常见的12株非目标菌检测结果为阴性,特异性较好。FM-ICA+IMB联合检测三种模拟食品时的回收率为38%~55%,其中,西红柿对回收率影响较小,鸡蛋和猪肉影响较大。然而,联合检测的灵敏度可达到1×10~5 CFU/m L,比单一FM-ICA方法提高了30倍,同时,整个检测过程可在2 h内完成。本检测方法的建立对于快速筛查鼠伤寒沙门氏菌具有重要意义。     

双抗夹心ELISA方法检测食品中单核细胞增生李斯特氏菌

采用0.5% 福尔马林灭活的单核细胞增生李斯特氏菌作为免疫原免疫日本大耳兔,获得抗单核细胞增生李斯特氏菌多克隆抗体,并以此多克隆抗体作为捕获抗体,以抗单核细胞增生李斯特氏菌Internalin A(InlA)单克隆抗体作为检测抗体,建立快速、特异的检测该菌的双抗夹心ELISA 方法。结果表明：该方法对单核细胞增生李斯特氏菌纯培养液的最低检测量为1.7 × 105CFU/mL。在检测食品样品的实验中,食品样本对本方法干扰较小,运用选择性增菌液进行前增菌可提高该方法的准确性。     

Detection of viable Salmonella in lettuce by propidium monoazide real-time PCR

Contamination of lettuce by Salmonella has caused serious public health problems. Polymerase chain reaction (PCR) allows rapid detection of pathogenic bacteria in food, but it is inaccurate as it might amplify DNA from dead target cells as well. This study aimed to investigate the stability of DNA of dead Salmonella cells in lettuce and to develop an approach to detecting viable Salmonella in lettuce. Salmonella-free lettuce was inoculated with heat-killed Salmonella Typhimurium cells and stored at 4 °C. Bacterial DNA extracted from the sample was amplified by real-time PCR targeting the invA gene. Our results indicate that DNA from the dead cells remained stable in lettuce for at least 8 d. To overcome this limitation, propidium monoazide (PMA), a dye that can selectively penetrate dead bacterial cells and cross-link their DNA upon light exposure, was combined with real-time PCR. Lettuce samples inoculated with different levels of dead or viable S. Typhimurium cells were treated or untreated with PMA before DNA extraction. Real-time PCR suggests that PMA treatment effectively prevented PCR amplification from as high as 10(8) CFU/g dead S. Typhimurium cells in lettuce. The PMA real-time PCR assay could detect viable Salmonella at as low as 10(2) CFU/mL in pure culture and 10(3) CFU/g in lettuce. With 12-h enrichment, S. Typhimurium of 10(1) CFU/g in lettuce was detectable. In conclusion, the PMA real-time PCR assay provides an alternative to real-time PCR assay for accurate detection of Salmonella in food.     

免疫磁珠-环介导等温扩增快速检测牛肉中的鼠伤寒沙门氏菌与金黄色葡萄球菌

建立免疫磁珠分离（immunomagnetic separation,IMS）联合环介导等温扩增（loop-mediated isothermal amplification,LAMP）技术检测牛肉中鼠伤寒沙门氏菌与金黄色葡萄球菌的方法。用生物素标记的鼠伤寒沙门氏菌抗体和生物素标记的金黄色葡萄球菌菌体蛋白抗体对链霉亲和素磁珠进行功能化,从牛肉中捕获和分离目标致病菌。结果表明：经优化后,每毫克磁珠与10 μL鼠伤寒沙门氏菌抗体偶联,400 μL鼠伤寒沙门氏菌免疫磁珠在45 min内对104 CFU/mL鼠伤寒沙门氏菌的捕获率为52.16%;每毫克磁珠与6 μL金黄色葡萄球菌抗体偶联,300 μL金黄色葡萄球菌免疫磁珠在30 min内对104 CFU/mL金黄色葡萄球菌的捕获率为56.80%;建立的IMS-LAMP方法特异性高,对牛肉中鼠伤寒沙门氏菌的检测灵敏度为1.2×103 CFU/mL,对金黄色葡萄球菌的检测灵敏度为4.4×104 CFU/mL;富集5 h后,鼠伤寒沙门氏菌的检测限可至1.2 CFU/mL,富集7 h后,金黄色葡萄球菌的检测限可降至4.4 CFU/mL。建立的IMS-LAMP方法用时短,灵敏度高,操作简单,可以有效检测牛肉中的鼠伤寒沙门氏菌和金黄色葡萄球菌。