RT-PCR法检测贝类中的甲肝病毒的研究

在世界范围内,甲肝病毒是与食用贝类有关的主要传染性疾病之一。由于贝类中含有PCR抑制剂以及病毒富集过程中病毒的回收率低,阻碍了天然污染的贝类中HAV的PCR检测。研究中建立了一种经苷氨酸缓冲液洗涤,2次PEG沉降富集病毒,然后进行RNA提取和RT-PCR对贝类中的甲肝病毒进行检测的方法。经比较,采用小体系肠道样品检测比采用全贝检测的富集效果更佳,并比较了PEG8000和PEG6000对病毒富集的效果,回收率分别为13.5%和7.6%,此方法可有效地降低PCR抑制剂的影响,最低检测限可达10个TCID50/1.5 g。     

传染性支气管炎病毒实时荧光PCR检测方法的建立

建立特异、敏感、快速的传染性支气管炎病毒(IBV)实时荧光定量PCR检测方法。针对IBV基因序列保守区域设计一对特异性引物和一条特异性的探针,建立实时荧光定量PCR检测方法,并进行特异性、敏感性、重复性检测。结果表明所建立的IBV实时荧光定量PCR检测方法,引物和探针特异性良好,组间组内重复性良好,检出IBV DNA最小拷贝数为5拷贝。因此,本研究建立IBV特异、敏感、快速的实时荧光PCR检测方法,为IBV的诊断奠定基础。     

实时荧光RT-PCR检测贝类中的札幌病毒

建立检测贝类中GⅠ、GⅡ、GⅣ和GⅤ型札幌病毒的实时荧光RT-PCR新方法。首先使用PEG 8000对贝类中的札幌病毒进行富集,然后采用Tri-reagent提取材料中的总RNA,针对札幌病毒RNA 3'端含Poly A尾的特点,使用带有Poly(dT)25的磁珠对病毒RNA进行纯化,用所获的高纯度RNA进行四种型别札幌病毒的实时荧光RT-PCR检测。该方法高效、灵敏,检测下限为101数量级拷贝,能够用于日常检验。     

Norovirus, hepatitis A virus and enterovirus presence in shellfish from high quality harvesting areas in Portugal

This is the first report on the screening of shellfish from Portugal for the presence of human enteropathogenic viruses. Approximately 2000 shellfish (Curbicula fluminea, Ruditapes decussatus, Tellina crassa, Spisula solida, Dosinia exoleta, Ensis spp., Mytilus spp., Ostrea edulis and Cerastoderma edule), organized in 49 batches, were collected between March 2008 and February 2009. They were tested for norovirus (NoV), hepatitis A virus (HAV) and enterovirus (EV) by RT-PCR followed by nucleotide sequencing. Bacterial contamination was also evaluated by Escherichia coli counts. Viral contamination was detected throughout the year in all shellfish species and in all collection areas, independently of their harvesting areas classification. Overall, 67% of all analyzed batches were contaminated by at least one of the studied viruses while the simultaneous presence of two and three viruses was detected in 22% and 6% batches, respectively. Of the three viruses, NoV was detected in 37% of the batches, followed by EV in 35%, and HAV in 33%. Nucleotide sequencing of the NoV and HAV RT-PCR products demonstrated that all strains belonged to NoV genotype GII.4 and HAV subgenotype 1B. The presence of NoV and HAV in shellfish from “A class” harvesting areas of Portugal can represent a potential health risk.     

三种分子生物学方法检测牡蛎中诺如病毒的比较研究

采用RT-PCR、RT- 半巢式PCR(seminested PCR)和RT- 环介导等温扩增(LAMP)三种分子生物学方法分别检测了牡蛎中经粪便污染的诺如病毒。三种方法所采用的特异性引物均针对诺如病毒高度保守的N/S 结构域。结果显示：一步法RT-PCR较两步法结果理想但仍不能有效去除食品中的PCR反应抑制物。RT- 半巢式PCR和RT-LAMP的特异性和敏感性都远远优于RT-PCR,但是RT- 半巢式PCR 操作繁琐费时。RT-LAMP 扩增程序简单、反应时间短,且不需要精密的温度循环装置,在产物中加入SYBR Green Ⅰ染料后可用肉眼直接判断反应结果。因此,RT-LAMP 有望发展成为快速检测牡蛎中诺如病毒的有效手段。     

秦皇岛市售生菜、草莓中诺如病毒监测分析

目的 调查秦皇岛市诺如病毒污染的高风险食品生菜和草莓中诺如病毒的污染状况。方法 2015年9月至2016年6月采集市售的生菜201份、草莓136份, 利用实时荧光RT-PCR法检测诺如病毒, 在样品中添加Mengo病毒进行过程控制, 并根据Mengo病毒标准曲线计算 诺如病毒回收率。结果 Mengo病毒回收率均达到了ISO/TS 15216-2-2013对 病毒回收率＞1%的要求。同时对201份生菜样品进行检测, 检出诺如病毒阳性标本9份, 总检出率为4.5%, 其中GⅠ型检出率为0.5%(1/201); GⅡ型检出率为3.5%(7/201); GⅠ型和GⅡ型同时检出率为0.5%(1/201)。136份草莓样品中检出阳性标本4份, 总检出率为2.9%, 其中GⅠ型检出率为0.7%(1/136); GⅡ型检出率为2.2%(3/136)。结论 秦皇岛市售蔬菜、浆果中部分存在诺如病毒污染, 诺如病毒检出率在不同采样环节以及时间分布比较差异无统计学意义, 需要进一步加强日常监测。     

RT-PCR方法检测贝类中诺沃克样病毒的研究

目的:本研究从病毒富集和核酸提取两方面进行探索,旨在建立一个反转录(RT)PCR技术检测贝类中诺沃克样病毒(NLVs)的方法.方法:利用脊髓灰质炎病毒作为参照毒株,优化了甘氨酸缓冲液-聚乙二醇(PEG)病毒浓缩方法;同时比较了异硫氰酸胍法、SDS-蛋白酶K法、Trizol-异丙醇法、试剂盒法四种RNA提取方法;对市售贝类样品进行了检测,并利用基因测序对阳性样品进行验证.结果:本研究采用的pH9.5甘氨酸缓冲液 -16%聚乙二醇病毒浓缩法,病毒的回收率为16.8%,利用Trizol-异丙醇法提取RNA,检出限为8.1×10 2 RT-PCR50/5g贝肉,实际检测贝类样品25件,其中3件样品为阳性,基因测序结果亦证实为阳性.结论:本研究建立了一个灵敏度较高、较为有效的RT-PCR技术检测贝类中诺沃克样病毒的方法.     

Rapid and Sensitive Detection of Staphylococcus aureus in Food Using Selective Enrichment and Real-Time PCR Targeting a New Gene Marker

Staphylococcus aureus is a bacterial pathogen considered a principal etiological agent of food poisoning. The aim of this study was to develop and evaluate a rapid and sensitive method for the detection of S. aureus in food by using selective enrichment and a new species-specific real-time polymerase chain reaction (PCR). Specific primers and a TaqMan probe targeted to specific S. aureus gene encoding for acriflavine resistance protein were designed. The real-time PCR was highly specific for S. aureus with 100% inclusivity and 100% exclusivity determined using 83 S. aureus strains and 64 non-S.-aureus strains. PCR detection limit of 6.8 × 10¹ and 3.4 × 10¹ CFU ml⁻¹ were obtained with 100% and 70% detection probability, respectively. The single selective enrichment based on the study of different enrichment conditions was selected and a lysis by boiling was used to obtain bacterial DNA. Out of 112 food samples analyzed, 61 were positive by the PCR-based method and 53 by the standard method. Out of ten food matrices artificially contaminated at a level of 10° CFU g⁻¹, ten and six were positive by the respective methods. Moreover, 10° CFU 10 g⁻¹ was detected in all ten artificially contaminated samples after a large-scale enrichment using PCR-based detection, in contrast to seven false negative by standard detection. The developed method facilitated the detection of S. aureus on the next day after the sample reception. This method can be used for S. aureus detection as a faster, highly specific, and more sensitive alternative to microbiological method with the potential for providing of improved food-processing hygiene control.     

实时荧光PCR法快速鉴别狐狸貉子肉源性成分研究

根据狐狸线粒体基因组中的保守序列和貉子线粒体D-loop基因保守序列设计狐狸、貉子特异性引物和Taq Man探针,建立一种基于实时荧光聚合酶链式反应的肉及肉制品狐狸、貉子源性成分测定方法,通过特异性、灵敏性、线性检测对该方法体系进行检验和评价。本研究建立的狐狸、貉子源性成分实时荧光聚合酶链式测定方法体系具有良好的特异性及灵敏性,最低可检测0.5 pg/μL纯狐狸DNA和5 pg/μL纯貉子DNA。本研究建立的狐狸貉子源性成分荧光PCR检测方法,可以用来检测实际样品中是否含有狐狸貉子源性。     

Rapid and Sensitive Determination of L-carnitine and Acetyl-L-carnitine in Liquid Milk Samples with Capillary Zone Electrophoresis Using Indirect UV Detection

The purpose of this study was to establish a new capillary zone electrophoresis method with indirect UV detection for determining the concentration of L-carnitine and acetyl-L-carnitine in liquid milk samples. Orthogonal experimental design was applied to arrange and optimize the experimental conditions. Under selected conditions (using melamine as the additive), both L-carnitine and acetyl-L-carnitine could be detected in 6 min, with satisfied reproducibilities (RSD% of migration times <0.8%), linearities (r ² > 0.995), recoveries (>90%), and relative low LODs (3.0 and 5.0 μmol/L for L-carnitine and acetyl-L-carnitine, respectively). The proposed method was successfully applied to the assay of nine brands of bovine milk samples and shows potential for analyzing L-carnitine and acetyl-L-carnitine in other biosamples.     

Analysis of Porcine Gelatin DNA in a Commercial Capsule Shell Using Real-Time Polymerase Chain Reaction for Halal Authentication

The presence of pig derivatives, such as porcine gelatin, in any products is prohibited to be consumed by Muslim community. This study is intended to develop a specific primer from mytochondrial D-loop capable of amplifying DNA from porcine gelatin in commercial capsule shells. Two pairs of primers designed from mitochondrial D-loop region were tested in order to confirm the primer specificity in gelatin sources (pork, beef, and catfish) and fresh tissue (pig, cows, goat, chickens, and rat). Primers were then used to perform sensitivity test of six dilution series (1000, 200, 100, 10, 5, and 1 pg/µL) of porcine gelatin and porcine capsule shell. The amplification was also performed on capsule shell from porcine-bovine mixture gelatin at 0, 10, 20, 30, 40, 50, and 100% concentration. The repeatability test was performed by measuring amplification capsule shells from porcine–bovine gelatin mixture. Real time polymerase chain reaction method using primers designed was further applied to analyze capsule shells purchased from markets. From two primers have been designed specifically, only primer D-Loop 108 (forward: 5’-CGT ATG CAA AAA ACC ACG CCA-3’; reverse: 5’-CTT ACT ATA GGG AGC TGC ATG-3’) had the capability to identify the presence of porcine DNA in fresh tissue and gelatin sources at optimum annealing temperature of 58.4ºC. Sensitivity of the developed method expressed as limit of detection of DNA in gelatin and capsule shells is 5 pg.     

烟草PVY Real-Time PCR定量检测体系的建立及应用

马铃薯Y病毒是近年来危害烟草生产的重要病毒之一,严重影响烟草的产量与品质。本研究利用DNAMAN软件对GenBank数据库中已登录的马铃薯Y病毒(Potato virus Y,PVY)全基因组序列进行序列比对,设计引物,以烟草肌动蛋白基因为内参,建立了烟草PVY的实时定量检测体系。获得的real-time PCR扩增基线平整,指数扩增明显,斜率大;稳定性和重现性好,变异系数小;循环阈值与PCR起始模板量对数之间存在良好的线性关系。与DAS-ELISA相比,该方法具有高效、灵敏、特异性强等优点,为从分子生物学水平上检测烟草中PVY提供了新的技术手段。     

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。     

Detection and characterization of pathogenic vibrios in shellfish by a Ligation Detection Reaction-Universal Array approach

Vibrios are a group of major foodborne pathogens widely distributed in marine environment. Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus are the pathogenic species of Vibrio that pose the greatest threat to human health. However, other vibrios, e.g. Vibrio alginolyticus, Vibrio mimicus and Grimontia hollisae, apparently less relevant in the group of foodborne pathogens, have been sporadically found in outbreaks. For seafood safety and economic purposes, a rapid and powerful method for the specific identification of harmful Vibrio strains is needed. We developed a PCR-Ligase Detection Reaction-Universal Array (PCR-LDR-UA) assay for the simultaneous identification of pathogenic vibrios and detection of virulence coding genes. The entire procedure was validated on a total of 31 reference strains and isolates from clinical and environmental samples, as well as on bivalve tissue homogenates infected with different strains of target Vibrio species. Twenty-three shellfish samples directed to human consumption were successfully screened, thus demonstrating that the developed microarray-based platform could be a reliable and sensitive detection tool for the identification of harmful Vibrio strains in seafood.     

Practical Molecular Detection Method of Beef and Pork in Meat and Meat Products by Intercalating Dye Based Duplex Real-Time Polimerase Chain Reaction

In this study, a rapid, specific, and low-cost duplex-detection technique of pork and beef was developed by a real-time polimerase chain reaction assay based on fluorescence. Deoxyribonucleic acid was extracted from variable mixtures of pork and beef in sausage and industrial products to develop the duplex assay using the GIDAGEN^® Multi-Fast DNA Isolation Kit. Identification of genomes was accomplished in the same tube by their distinctive melting peak, which was 87.5°C for pork and 80.5°C for beef, respectively. The detection limit of the method was 0.01 ng/µL deoxyribonucleic acidor 0.001% target pork and beef in sausage. The results showed that the intercalating dye based duplex real-time polimerase chain reaction is a potentially sensitive, reliable, and practical assay for the detection of meat species adulterated with beef and pork.