The Application of Real-Time PCR to Food and Agricultural Systems. A Review

Abstract

Real-time PCR (RT-PCR) allows each cycle of DNA amplification to be observed on a computer screen throughout the sequence of thermal cycling, hence the designation “real-time.” RT-PCR assays have been developed for a variety of target sequences of food borne microbial pathogens, plant pathogens, and genetically modified foods. A major and highly sensitive aspect of RT-PCR is the use of fluorescent reporter dyes that undergo enhanced fluorescence when bound to DNA. Among the fluorescent reporter systems employed are SYBR green, and the molecular probes designated TaqMan, FRET, molecular beacons, and variations of these systems. A major advantage of RT-PCR involves the amplification of short DNA target sequences of 60–70-bp, which allows greatly reduced extension times, which when coupled with the advanced technology of RT thermal cyclers with reduced ramp times, greatly reduces cycle times so that amplified targets can be recognized within 30 min of amplification in some cases. Another major advantage is that agarose-gel electrophoresis is not required to visualize amplified target DNA which greatly reduces assay time.     

PCR-based identification of pathogenic Candida species using primer mixes specific to Candida DNA topoisomerase II genes

For rapid identification of Candida to the species level, degenerated primers and specific primers based on the genomic sequences of DNA topoisomerase II of C. albicans, C. dubliniensis, C. tropicalis (genotypes I and II), C. parapsilosis (genotypes I and II), C. krusei, C. kefyr, C. guilliermondii, C. glabrata, C. lusitaniae and Y. lipolytica were designed and their specificities tested in PCR-based identifications. Each of the specific primers selectively and exclusively amplified its own DNA fragment, not only from the corresponding genomic DNA of the Candida sp. but also from DNA mixtures containing other DNAs from several fungal species. For a simpler PCR-based identification, the specific primers were divided into three groups (PsI, PsII and PsIII), each of which contained four specific primer pairs. PCR with the primer mixes yielded four different sizes of PCR product, corresponding to each Candida sp. in the sample DNA. To obtain higher sensitivity of PCR amplification, sample DNAs were preamplified by the degenerated primer pair (CDF28/CDR148), followed by the main amplification using the primer mixes. By including this nested PCR step, 40 fg yeast genomic DNA was detected in the sample. Furthermore, we applied this nested PCR to a clinical diagnosis, using splenic tissues from experimentally infected mice and several clinical materials from patients. In all cases, the nested PCR amplifications detected proper DNA fragments of Candida spp., which were also identified by the standard identification tests. These results suggest that nested PCR, using primer mixes of the Candida DNA topoisomerase II genes, is simple and feasible for the rapid detection/identification of Candida to species level in clinical materials.     

多重荧光聚合酶链反应对转基因能力验证样品的检测和验证

目的利用多重实时荧光聚合酶链反应(polymerase chain reaction,PCR)对转基因能力验证样品进行检测和验证。方法通过筛选出合适的多重引物和探针、核酸提取及多重扩增和分析方法,分别对9个转基因标准品和11个来自于中国合格评定国家认可委员会(China National Accreditation Service for Conformity Assessment,CNAS)和食品分析水平测试计划(food analysis performance assessment scheme,FAPAS)的能力验证样品进行多重荧光PCR检测,通过SPSS统计软件比较二重、三重实时荧光PCR的循环阈值(cycle threshold,Ct)与单重实时荧光PCR结果的差异性。结果二重实时荧光PCR可同时检测Ca MV35s和NOS 2个基因,三重实时荧光PCR可同时检测Ca MV35s,NOS和NPTII 3个外源基因,各基因之间无信号的交叉干扰。经统计分析,二重和三重实时荧光PCR相对于单重实时荧光PCR的结果无显著性差异。结论建立的二重和三重实时荧光PCR技术可用于能力验证和实验室间比对实验,简单快速,节约试剂成本,且结果准确度可满足日常检测需求。     

The Application of Real-Time PCR to Food and Agricultural Systems. A Review

Real-time PCR (RT-PCR) allows each cycle of DNA amplification to be observed on a computer screen throughout the sequence of thermal cycling, hence the designation “real-time.” RT-PCR assays have been developed for a variety of target sequences of food borne microbial pathogens, plant pathogens, and genetically modified foods. A major and highly sensitive aspect of RT-PCR is the use of fluorescent reporter dyes that undergo enhanced fluorescence when bound to DNA. Among the fluorescent reporter systems employed are SYBR green, and the molecular probes designated TaqMan, FRET, molecular beacons, and variations of these systems. A major advantage of RT-PCR involves the amplification of short DNA target sequences of 60-70-bp, which allows greatly reduced extension times, which when coupled with the advanced technology of RT thermal cyclers with reduced ramp times, greatly reduces cycle times so that amplified targets can be recognized within 30 min of amplification in some cases. Another major advantage is that agarose-gel electrophoresis is not required to visualize amplified target DNA which greatly reduces assay time.     

实时荧光P C R 法检测食品中芝麻过敏原成分

本实验探讨采用实时荧光定量PCR 方法检测食品中的芝麻成分,结果表明：采用芝麻的引物和探针进行扩增时;22 种样品经实时PCR 扩增,只有白芝麻和黑芝麻产生荧光信号,其余样品均不产生荧光信号。灵敏度实验结果表明能检测到10-5 稀释度的4.4 ×10-12g DNA的量,定量关系式为：y=－ 3.472018x+18.851009,R2=0.993088。这两对引物和探针具有极高的灵敏度和扩增效率,符合痕量检测要求。     

4组鱼类DNA条形码引物的筛选与优化

目的通过对DNA条形码氧化酶亚基I基因(cytochrome oxidase subunit I,COI)的4组常用引物进行比较筛选,选择适于舟山鱼类鉴定的最佳引物。方法以舟山主产大黄鱼、小黄鱼、带鱼和乌贼样品DNA为模板,对4组常用DNA条形码引物进行PCR体系优化。通过比较PCR产物量、特异性、灵敏度和测序成功率,综合分析筛选最佳引物组。结果 4组引物均能扩增大黄鱼、小黄鱼和带鱼的DNA,对于乌贼DNA的扩增具有明显差异。COI优化引物具有易于扩增、测序简单等优势,更适于作为海产品鉴定的DNA条形码引物。结论本研究为舟山海产品的市场监管和实验室大量样品检测提供了技术参考。     

一种转基因马铃薯PCR鉴别方法的建立

为建立一种鉴别转基因马铃薯的PCR方法,设计转基因马铃薯常用调控元件的特异性引物,以4种转基因马铃薯标准品系EH92-527-1、AV43-6-G7、AM04-1020、PH05-026-0048为模板进行单基因PCR和多重PCR扩增工艺方法研究。经琼脂糖凝胶电泳结果分析后,从中筛选出一组引物特异性较强,且无交叉污染或非特异性扩增现象的引物组合对。对9种已知转基因马铃薯样品进行多重PCR验证,均得到条带清晰且区分明显的目的条带。成功建立了能够快速、准确鉴别转基因马铃薯中多种调控元件的多重PCR与单基因PCR相结合的检测方法。     

Identification of meats from red deer (Cervus elaphus), fallow deer (Dama dama), and roe deer (Capreolus capreolus) using polymerase chain reaction targeting specific sequences from the mitochondrial 12S rRNA gene

Polymerase chain reaction (PCR) based on oligonucleotide primers targeting the mitochondrial 12S rRNA gene was applied to the specific identification of meats from red deer (Cervus elaphus), fallow deer (Dama dama), and roe deer (Capreolus capreolus). The use of a common reverse primer, together with forward specific primers for red deer, fallow deer, and roe deer, allowed the selective amplification of the desired cervid sequences. The specificity of each primer pair was verified by PCR analysis of DNA from various game and domestic meats. The assay can be useful for the accurate identification of meats from cervid species, avoiding mislabeling or fraudulent species substitution in meat products.     

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。     

应用实时荧光PCR技术量化检测羊肉中猪源性和鸡源性成份

目的建立羊肉中猪源性成份和鸡源性成份的定量检测方法。方法以新鲜羊、猪和鸡瘦肉为样本提取DNA分子作为检测模板,针对基因组中单拷贝基因设计特异性的引物和探针,应用荧光实时定量PCR技术对模板DNA进行扩增。通过绘制扩增标准曲线和确定羊、猪和鸡的质量与DNA比值常数,对4种不同掺混比例的混合肉样进行定量分析。结果检测质量百分比的绝对误差可以控制在7%以内,量化结果基本准确。结论对于组织成份单一的样品,可以通过在基因组单拷贝基因上设计特异性的引物,利用PCR技术实现在质量水平上对食品中动物源性成份的量化分析,该技术方法的建立可以为肉类掺假的监管工作提供有力的技术支撑。     

Selection of Universal Primers for PCR Quantification of Total Bacteria Associated With Fish Fillets

An optimal universal primer pair DG74/RW01 for quantitative PCR detection of the mixed bacterial flora from fish fillets was selected from five pairs of primers. The minimum detection limits with GelStar™ and ethidium bromide (EB) used as agarose stains for DNA bands with these primers were found to be 5 and 1 × 10² CFU/PCR, respectively. The liner range of DNA amplification was from 50 to 1 × 10⁵ with GelStar™ and from 5 × 10² to 1 × 10⁵ with EB.     

Factors Affecting Quantitative PCR Assay of Plesiomonas shigelloides

We have found that there are various factors that can affect the quantitative PCR assays of Plesiomonas shigelloides. Different Taq polymerase preparations, varying sets of primers, different DNA stains, and different cell lysing agents were found to significantly influence the linear relationship between the fluorescent intensities of DNA bands and the log of CFU per PCR. The primer dimers formed in the PCR can be eliminated by using different Taq polymerase preparations and different sets of primers to run the PCR.     

Selection of Universal Primers for PCR Quantification of Total Bacteria Associated With Fish Fillets

An optimal universal primer pair DG74/RW01 for quantitative PCR detection of the mixed bacterial flora from fish fillets was selected from five pairs of primers. The minimum detection limits with GelStar? and ethidium bromide (EB) used as agarose stains for DNA bands with these primers were found to be 5 and 1 × 10² CFU/PCR, respectively. The liner range of DNA amplification was from 50 to 1 × 10⁵ with GelStar? and from 5 × 10² to 1 × 10⁵ with EB.     

免疫磁珠捕获-通用引物PCR快速检测食品中致病菌的研究

为了提高食品中致病菌的检出率和灵敏度,利用免疫磁珠捕获结合经典PCR技术建立免疫捕捉通用引物PCR(IMC-UPPCR)检测食品中的致病菌.结果显示,采用细菌16SrRNA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,IC-UPPCR检测致病菌的最低检测限可达10 cfu,检测致病菌的特异性为100%,无假阳性和假阴性出现.采用本方法与国标方法分别对50种不同样品进行3种致病菌检测,结果显示两种方法的符合率达100%,特异性相当.与国标方法相比,本方法灵敏度更高,并具有简单、快速、特异性好和敏感性高等特点,可以满足大批样品致病菌筛选检测的要求,适用于食品卫生监管、商品检验检疫等领域.     

Application of a polymerase chain reaction to detect adulteration of ovine cheeses with caprine milk

A polymerase chain reaction (PCR) procedure has been applied for the detection of caprine milk in ovine cheeses by using primers targeting the mitochondrial 12S ribosomal RNA gene. The use of a primer pair specific for goat in PCR analysis of cheese samples, enabled the amplification of a goat 122 bp fragment with a sensitivity threshold of approximately 1%, whereas no amplification signal was achieved with sheep's, cow's and water buffalo's milk DNA. This study demonstrates the usefulness of the proposed PCR assay for the qualitative detection of goats’ milk in ewes’ cheeses, and may therefore provide a simple and accurate approach applicable to the authentication of cheese or other dairy products in routine monitoring programs.     

Factors Affecting Quantitative PCR Assay of Plesiomonas shigelloides

We have found that there are various factors that can affect the quantitative PCR assays of Plesiomonas shigelloides. Different Taq polymerase preparations, varying sets of primers, different DNA stains, and different cell lysing agents were found to significantly influence the linear relationship between the fluorescent intensities of DNA bands and the log of CFU per PCR. The primer dimers formed in the PCR can be eliminated by using different Taq polymerase preparations and different sets of primers to run the PCR.     

副溶血弧菌的SYBR Green Ⅰ实时定量PCR检测方法建立

基于副溶血弧菌gyrB基因保守序列设计1对特异性引物,建立SYBR GreenⅠ实时定量聚合酶链式反应(polymerase chain reaction,PCR)检测副溶血弧菌的方法。SYBR GreenⅠ实时定量PCR的Tm为90℃,扩增产物的熔解曲线只出现1个单特异峰,无引物二聚体,表明该引物具有较好的特异性;所制作的实时定量PCR扩增标准曲线在2.06×108～2.06×103拷贝数之间有较好的线性关系,相关系数为0.992,能对副溶血弧进行准确的定量分析。该方法检测时间从核酸抽提到结果分析仅需4～5h,且较传统方法敏感、操作简单,可用于针对副溶血弧菌的进出口检验检疫、食品安全检测及该菌引起的水产动物疾病的诊断与分子流行病学调查。     

转基因玉米品系及转化体成分四重实时荧光PCR快速鉴定

Bt11转基因玉米品系具有抗草铵膦除草剂,鳞翅目昆虫抗性的耐除草剂抗虫玉米,MIR162和Mon89034是鳞翅目昆虫抗性的单抗虫玉米,均是国内外出入境监管主要关注的转基因玉米品系。本研究通过靶标基因筛选,转基因阳性样品采集,核酸样本制备,多重引物和荧光探针组合筛选,反应体系优化以及方法学验证等过程开发建立了四重荧光定量PCR检测技术。结果表明该技术的使用可实现一个反应管中同时检测MIR162、Bt11、Mon89034三个玉米品系的特异性基因序列和一个编码玉米淀粉合成酶异构体zSTSII-2 (zSSIIb) 玉米内源基因。通过阳性对照,阴性对照和空白对照特异性S曲线与对应的阈值大小分析可判定样品中是否含有这三个转基因玉米品系及其转化体成分。经方法学特异性检测,结果与转基因检测金标准的单实时荧光PCR结果一致;经平行样和灵敏度测试,最低检测限为18个拷贝;经标准曲线扩增分析,四个基因的扩增效率均在90%~110%范围内,扩增效果良好。该方法从DNA提取到报告结果不足3小时,可缩短检测时限,节约试剂耗材成本,操作简单易行,满足高通量特征,可为市场流通产品品系和转基因成分的实时监管和快速鉴定提供技术支撑。     

甜菜SSR—PCR反应体系优化及基于DNA混合池的SSR引物快速筛选

应用L16（4^4）正交设计对影响甜菜SSR—PCR的主要参数进行优化,建立适于甜菜的SSR反应体系和扩增程序。20ul反应体系中含有模板70ngDNA,150umol／LdNTP,0．7umol／LSSR引物,0．5TaqDNA聚合酶／U,10×PCRBuffer（含Mg^2＋）2．0ul。以选择的87对SSR引物为对象,对10个甜菜基因组DNA样品等量混合,进行PCR扩增筛选引物,结果表明,与常规筛选方法相比,DNA混合池方法大幅度缩短了实验周期,显著减少了实验资源的消耗,可用于大量甜菜SSR引物的快速高效筛选。     

A simple one-step PCR method for the identification between European and American razor clams species

A specific multiplex polymerase chain reaction (PCR) was applied to differentiate samples of razor clams Ensisarcuatus, Ensissiliqua, Ensisdirectus, and Ensismacha. Universal primers were used for the amplification of internal transcribed spacer 1 (ITS-1) in each species. The alignment of the obtained sequences was the basis for the specific design of species-specific reverse primers (ITSArSil-R, ITSDir-R, and ITSMa-R) located in the ITS-1 region. A multiplex PCR using each specific primer together with a common forward primer allowed identification of razor clam species by means of the different sizes of the species-specific amplicons separated in an agarose gel electrophoresis. This work provides a simple, reliable and rapid protocol for the accurate identification of Ensis species. The present methodology can be very useful for traceability of the species and to reinforce labelling regulations.