Advances in the Identification of Genetically Modified Rice with Real-time PCR and Multiplex PCR

In recent years, food security and safety have attracted increasing attention due to the worldwide research and development of genetically modified （GM） rice, and the controversy over the commercialization of GM rice. And the identification of GM rice is of great significance. Therefore, in the present study, the po- tential problems in the identification of GM rice with PCR were analyzed both at a technical level and from a theoretical perspective. In addition, PCR detection on the transgenic elements： promoter, terminator, internal reference gene and target gene was discussed, respectively. The possible solutions were proposed based on the principles of plant virology and genetic engineering.

Development of a duplex real-time PCR method for the detection of influenza C and D viruses

Influenza viruses are major respiratory pathogens known to infect human and a variety of animals and are widely prevalent worldwide.Genome structure of influenza D virus(IDV)is identical to that of influenza C virus(ICV),and phylogenetic analyses suggest that IDV and ICV share a common ancestry and high homology.To date,the prevalence of ICV and IDV in China is unclear,but these viruses represent a potential threat to public health due to cross-species transmission and zoonotic potential.To efficiently monitor ICV and IDV,it is necessary to establish a dual detection method to understand their prevalence and conduct in-depth research.A duplex real-time PCR method for the simultaneous detection of ICV and IDV was developed.TaqMan fluorescent probes and specific primers targeting NP gene of ICV and PB1 gene of IDV were designed.This method exhibited good specificity and sensitivity,and the detection limit reached 1 × 10^(1) copies/pL of plasmid standards of each pathogen.Thirty-one clinical swine samples and 10 clinical cattle samples were analyzed using this method.One positive sample of IDV was detected,and the accuracy of clinical test results was verified by conventional PCR and DNA sequencing.The duplex real-time PCR detection method represents a sensitive and specific tool to detect IG/and IDV,It provides technical support for virus research and clinical diagnosis of ICV and IDV.This information will benefit animal and human health.

Rapid quantification of hepatitis B virus DNA by real-time PCR using efficient TaqMan probe and extraction of virus DNA

AIM： To rapidly quantify hepatitis B virus （HBV） DNA by real-time PCR using efficient TaqMan probe and extraction methods of virus DNA. METHODS： Three standards were prepared by cloning PCR products which targeted S, C and X region of HBV genome into pGEM-T vector respectively. A pair of primers and matched TaqMan probe were selected by comparing the copy number and the Ct values of HBV serum samples derived from the three different standard curves using certain serum DNA. Then the efficiency of six HBV DNA extraction methods including guanidinium isothiocyanate, proteinase K, NaI, NaOH lysis, alkaline lysis and simple boiling was analyzed in sample A, B and C by real-time PCR. Meanwhile, 8 clinical HBV serum samples were quantified. RESULTS： The copy number of the same HBV serum sample originated from the standard curve of S, C and X regions was 5.7 × 10^4/mL, 6.3 × 10^2/mL and 1.6 × 10^3/ mL respectively. The relative Ct value was 26.6, 31.8 and 29.5 respectively. Therefore, primers and matched probe from S region were chosen for further optimization of six extraction methods. The copy number of HBV serum samples A, B and C was 3.49 × 10^9/mL, 2.08 × 10^6/mL and 4.40 × 10^7/mL respectively, the relative Ct value was 19.9, 30 and 26.2 in the method of NaOH lysis, which was the efficientest among six methods. Simple boiling showed a slightly lower efficiency than NaOH lysis. Guanidinium isothiocyanate, proteinase K and NaI displayed that the copy number of HBV serum sample A, B and C was around 10^S/mL, meanwhile the Ct value was about 30. Alkaline failed to quantify the copy number of three HBV serum samples. Standard deviation （SD） and coefficient variation （CV） were very low in all 8 clinical HBV serum samples, showing that quantification of HBV DNA in triplicate was reliable and accurate. CONCLUSION： Real-time PCR based on optimized primers and TaqMan probe from S region in combination with NaOH lysis is a simple, rapid and accurate method for quantification of HBV serum DNA.

Evaluation of Clinical Application of Chemiluminescence and Real-time,Fluorescence-based Quantitative PCR in Diagnosis of Epstein-Barr Virus lnfection

Objective:To compare the effects of clinical application of chemiluminescence and real-time,fluorescence-based quantitative PCR in the detection Epstein-Barr virus(EBV).Methods:The data of chemiluminescence and real-time fluorescent quantitative PCR.fromipaEsfwo were suspected of being infectea w1tn rito1 roro January 2016 to January 2019 in our hospital were analyzed.The specific stage of EBV infection was analyzed,and the differences in results of the two detection methods were compared.Results:Chemiluminescence method was used to detect EBV infection during the active phase.The sensitivity of the chemiluminescence method was 76.7%(56/73)and the real-time quantitative PCRmethod was 90.4%(66/73).There was a statistical difference between the two detection methods(P<0.05).Conclusion:There was no statistical difference in positive predictive values between the chemiluminescence method and the real-time,fluorescence-based quantitative PCR method in the detection of EBV infection,but the sensitivity of chemiluminescence method is slightly lower than the real-time quantitative PCRmethod.It is noteworthy that chemiluminescence method is convenient and fast while the real-time,fluorescence-based quantitative PCR method is more accurate,which can provide a more accurate reference for clinical treatment.

One-Step Multiplex PCR for Simultaneous Detection and Identification of Eight Medically Important <i>Candida</i>Species

Recently, the incidence of Candida infections has substantially increased. Conventional identification methods for Candida species are technically difficult to conduct and cannot accurately distinguish each species. The purpose of the present study was to design primers to identify and detect simultaneously eight medically important Candida species using one-step multiplex PCR. PCR primers were designed based on partial sequences of intergenic spacer (IGS) and internal transcribed spacer (ITS) genes of eight medically important Candida species. These primers were able to distinguish each Candida species and did not display cross-reactivity with representative Candida species other than the eight Candida species. Moreover, our developed one-step multiplex PCR method is accurate, specific, cost-effective, time-saving, and worked without requiring DNA extraction.

Advance of Multiplex PCR in Rapid Detecting Transgenic Soybean Oil

Transgenic food safety is a high-profile public health issue in worldwide, especially transgenic soybean (Glycine max L.) oil. To rapidly and effectively detect transgenic components of soybean oil, in the present study, we isolated DNA from transgenic soybean oil by modified method, and employed the multiplex PCR method to identify targeted genes, including CaMV35S promoter, Nos terminator, NPTII, CP4-EPSPS and endogenous gene Lectin. The research aims to build a method which is accurate, rapid and reliable for detection of genetically modified soybeans oil. The targeted gene including DNA was successfully established by the improved method, and then amplified by PCR. Five genes are simultaneously specifically detected. Commercial soybean, genetically modified soy bean and oil were detected with the Multiplex PCR. The improved method of DNA extraction was rapid and accurate to extract high quality total DNA which was amplified by PCR. The method could eliminate the PCR inhibitor. A way of detecting the genetically modified soybean and Oil was set up in this study.

水禽4种常见致病菌多重PCR方法的建立及应用

为建立一种能同时检测大肠杆菌、鸭疫里默氏杆菌、多杀性巴氏杆菌、金黄色葡萄球菌的多重PCR方法,试验根据大肠杆菌phoA基因、鸭疫里默氏杆菌ompA基因、多杀性巴氏杆菌kmt 1基因、金黄色葡萄球菌FemB基因的保守区域设计4对特异性引物,通过优化退火温度和引物浓度建立多重PCR方法,检验该方法的特异性、敏感性性、重复性,应用该方法检测145份病死水禽临床样品,并与传统细菌分离鉴定方法进行比较。结果表明:优化后的最佳退火温度为52℃,引物浓度为10μmol/L。建立的多重PCR方法能特异性地扩增出靶基因,沙门氏菌、链球菌、产气荚膜梭菌等临床常见菌均未扩增出条带,对大肠杆菌、鸭疫里默氏杆菌、多杀性巴氏杆菌、金黄色葡萄球菌4种细菌的检出下限是0.4 pg/mL,单一PCR扩增ompA、kmt 1、phoA和FemB基因的检出下限分别为4×10^(-3),0.4,4×10^(-3),4×10^(-6)pg/mL。多重PCR方法对临床样品的检测结果与传统细菌分离鉴定方法的符合率均在90%以上。说明试验建立的多重PCR方法具有结果稳定、重复性高的特点,适合水禽病原微生物流行病学调查。

Chelex-100提取生物检材DNA实时PCR定量研究

目的研究Chelex-100法提取的生物检材DNA用量与复合STR分型成功率的关系。方法113份各种生物检材采用Chelex-100法提取DNA，应用Quantifiler人类DNA定量试剂盒在ABI 7500荧光定量PCR仪上进行实时PCR定量，同时用Identifiler复合扩增系统在ABI 3100遗传分析仪上对这些DNA样品进行STR分型。结果各种生物检材提取的DNA浓度分别为：37份滤纸、纱布血痕0．042～5．28ng／μl，16份口腔拭子1．15—4．21ng／μl，18份烟头0．016～1．46ng／μl，10份肋软骨0．531—14．40ng／μl，8份肌肉5．75—24．80ng／μl，7份指甲0．788—11．50ng／μl，17份精斑0．79～99．50ng／μl。在建立的8μl扩增体系中，根据上述结果，调整用于复合STR扩增的DNA模板量在0．5—3ng之间，大部分样品可获得完全的STR分型。结论Chelex-100法提取的检材DNA模板用量在0．5—3ng之间可得到有效STR扩增，浓度为0．5ng／μl以上的DNA样品，用小体积模板（1μl）比大体积（3μl）模板扩增效果好。

转基因抗草甘膦油菜的实用PCR检测方法

利用定性PCR技术 ,建立了检测孟山都公司培育的转基因抗草甘膦油菜中的epsps、gox和fmv35s的方法 ,同时设立了阳性内对照napin。该方法的检测灵敏度为 0 .0 5 %。利用复合PCR可以在一个PCR反应中同时检测出epsps、gox、fmv35s和napin基因。这种方法可以有效地用于转基因抗草甘膦油菜中的外源基因的检测。

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hly A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/m L。

实时荧光定量PCR定量方法研究进展

实时荧光定量PCR以其特异性强、灵敏度高、重复性好、定量准确、速度快、全封闭反应等优点而成为了分子生物学研究中的重要工具,综述了实时荧光定量PCR技术及其定量方法的研究进展,并展望了其应用前景。

多重PCR与恒温扩增芯片法在呼吸道病原体中的检测价值

目的评估多重荧光定量PCR法在呼吸道病原体(包括铜绿假单胞菌、肺炎链球菌、金黄色葡萄球菌、肺炎克雷伯菌、结核分枝杆菌、流感嗜血杆菌)检测中的价值。方法以恒温芯片法为参照检测方法,通过检测呼吸道病原体标准菌株以及临床样本,比较多重荧光定量PCR法和恒温芯片法的检出率。结果与恒温芯片法相比多重荧光定量PCR法具有很高的特异性与一致性且呼吸道病原体的检出率差异无统计学意义(χ^(2)=0.410,P=0.522)。同时对单一病原体的检出率显示,两种检测方法的检出率差异无统计学意义(P>0.05),一致性分析(Kappa值)显示两种检测方法具有高度一致性(Kappa>0.75)。结论多重荧光定量PCR法与恒温芯片法相比具有高度一致性,在呼吸道病原体检测中具有良好的应用价值。

同时检测PRRSV美洲型经典株、变异株和TJM-F92疫苗株的多重TaqMan荧光定量RT-PCR方法的建立及应用

为建立猪繁殖与呼吸综合征病毒(PRRSV)的快速鉴别检测方法,本研究针对PRRSV美洲型经典株、变异株及弱毒活疫苗TJM-F92株的基因序列差异,设计了2对特异性引物和3条TaqMan探针,经过反应条件的优化,建立了能够同时检测并区分PRRSV美洲型经典株、变异株及TJM-F92疫苗株的多重TaqMan荧光定量RT-PCR方法。结果显示,该方法具有特异性强、敏感性高、重复性好的特点,与猪其他常见病毒无交叉反应;经典株、变异株、TJM-F92疫苗株重组质粒标准品的检出下限均为2.68拷贝/μL,比常规PCR敏感100倍;组内及组间重复试验的变异系数均小于1.5%。此外,该方法对324份疑似PPRSV感染的临床样品进行检测,结果显示PRRSV阳性114份,其中美洲型变异株105份、经典株6份、TJM-F92疫苗株3份,变异株和经典株混合感染4份。本研究建立的多重TaqMan荧光定量RT-PCR方法可以为PRRSV美洲型野毒株、疫苗株的快速鉴别检测及流行病学调查提供有效的技术手段。

靶序列富集多重PCR-液相芯片联合检测4种常见呼吸道病毒的初步应用

目的呼吸道病毒是引起婴幼儿呼吸道感染的最主要病原体,为快速准确诊断其感染,文中建立一种基于液相芯片系统的4种常见呼吸道病毒快速分子检测方法,为有效防控其发生和流行提供有力手段。方法采集南京总医院儿科就诊的有急性呼吸道感染症状的患儿咽拭子标本120例及30例正常对照标本,分别纳入患儿组和对照组。针对A型流感病毒(Flu A)、B型流感病毒(Flu B)、呼吸道合胞病毒A型(RSVA)和B型(RSVB)的保守区序列分别设计并合成了特异性引物、探针和通用引物,构建4种病原体阳性参比品,通过对扩增和杂交条件的优化,建立了靶序列富集多重PCR(Tem-PCR)和Luminex液相芯片(x MAP)技术相结合的检测系统,并对该检测系统进行了特异性、灵敏度评价。收集以急性上呼吸道感染为主要表现的患儿咽拭子标本,用上述建立的方法进行检测分析,分别与单病毒基因荧光定量PCR检测方法及x TAG液相芯片法进行比对检测。结果建立了Flu A、Flu B、RSVA和RSVB4种呼吸道病毒的特异性快速分子检测方法且灵敏度可达10拷贝/μL。用快速分子检测方法和单病毒基因荧光定量PCR法对120例疑似患儿咽拭子标本进行检测,快速法阳性检出率为31.7%(38/120),荧光定量法阳性检出率为29.2%(35/120)。经一致性检验分析提示2种方法一致性较强(k>0.7)。部分标本同时采用x TAG液相芯片试剂盒进行检测,经一致性检验分析提示2种方法一致性较好(k>0.6)。结论临床的初步应用证实了4种常见呼吸道病毒快速分子检测方法具备灵敏、特异、快速等优点,为临床早期、准确判断呼吸道感染的病原体提供实验依据。

同时检测美洲型及欧洲型PRRSV的多重RT-PCR检测方法的建立及应用

为建立猪繁殖与呼吸综合征病毒(PRRSV)的快速鉴别检测方法,本研究针对PRRSV基因序列设计3对特异性引物,即第1对通用引物扩增美洲型和欧洲型病毒株ORF7基因(433 bp/398 bp)、第2对引物扩增美洲型经典病毒株和高致病性变异株(HP-PRRSV)Nsp2基因(338 bp/248 bp)、第3对引物扩增欧洲型病毒株ORF5基因(614 bp)。经过反应条件的优化,建立了能够同时检测并区分美洲型经典病毒株、HP-PRRSV株以及欧洲型病毒株的多重RT-PCR检测方法。该方法可以特异扩增3种病毒的基因,其重组质粒标准品的检出下限均为1.67×103拷贝;而与猪的其它重要病原均无交叉反应。应用该方法检测176份临床疑似病料样品,结果 PRRSV阳性45份,其中美洲型变异病毒株41份、经典病毒株4份,未检测到欧洲型病毒株。表明本研究建立的多重RT-PCR检测方法可以用于PRRSV的快速鉴别诊断和流行病学调查。

侵染甘薯的SPCSV、SPVG、SPFMV多重RT-PCR检测方法的建立及应用

为建立可同时检测甘薯褪绿矮化病毒（SPCSV）、甘薯G病毒（SPVG）和甘薯羽状斑驳病毒（SPFMV）多重RT-PCR检测方法,本文根据SPCSV热激蛋白基因（hsp70）及SPVG、SPFMV外壳蛋白基因（CP）基因核苷酸序列的保守区域设计特异性引物,通过引物筛选,优化多重RT-PCR反应条件,建立了能同时检测SPCSV、SPVG和SPFMV 3种病毒的多重RT-PCR检测方法。该体系能有效扩增出大小为304、433、601 bp 3个特异性片段。测序结果表明3种病毒与参考序列的一致性达94%-99%。应用建立的多重RT-PCR检测方法可稳定、准确、灵敏地同时检测单一或复合侵染3种甘薯病毒,为甘薯脱毒和病毒病诊断奠定基础。

实验动物皮肤病原真菌多重PCR检测方法的建立与初步应用

目的建立石膏样毛癣菌(Tm)、石膏样小孢子菌(Mg)、犬小孢子菌(Mc)和猴类毛癣菌(Am)的多重PCR检测方法,用于实验动物皮肤病原真菌的快速检测。方法根据Genbank公布的四种皮肤病原真菌的18S-28S rRNA序列设计特异性引物,通过引物、d NTP、Taq DNA聚合酶浓度及退火温度和时间的优化,建立四种皮肤病原真菌的多重PCR体系。经特异性和敏感性检验后,对15份人工感染真菌大鼠毛和260份实验动物毛发样本进行检测。结果所建多重PCR方法能够有效区分四种皮肤病原真菌,产生192 bp(Tm)、460 bp(Mg)、290 bp(Mc)和602 bp(As)的目的片段,最低检测限分别为5.9 pg/μL、6.6 pg/μL、9.5 pg/μL和5.1 pg/μL。被检的15份人工感染样本扩增结果准确,260份毛发样本中未检测出四种真菌。结论所建立的多重PCR方法能够同时对实验动物中的四种皮肤病原真菌进行快速的检测,具有较高的应用价值。

猪群中7种高发传染病多重PCR诊断方法的建立

[目的]建立猪瘟、蓝耳病(又称猪繁殖与呼吸综合征)、圆环病毒2型感染、猪链球菌病、副猪嗜血杆菌病、猪多杀性巴氏杆菌感染和猪支原体感染7种猪病的多重PCR诊断方法。[方法]根据GenBank发表的核苷酸序列设计了分别针对猪瘟、蓝耳病、圆环病毒2型感染、猪链球菌病、副猪嗜血杆菌病、猪多杀性巴氏杆菌感染和猪支原体感染的2套多重PCR特异性引物,优化多重PCR反应条件后进行敏感性与特异性评价。[结果]建立的2套多重PCR诊断方法对其他常见猪病病原的基因组无特异性扩增,检测病原基因组最低浓度可达到pg级。[结论]该研究建立的2套多重PCR诊断方法可以用于猪群中上述7种猪病病原的单一感染或混合感染的鉴别诊断。

鸡传染性支气管炎的多重RT-PCR检测及793／B血清型的鉴定

【目的】建立鸡传染性支气管炎病毒(IBV)快速、准确的检测方法。【方法】根据GenBank中已经发表的793/B型和多株IBV的N基因和S1基因,对比分析后以N基因的保守序列设计合成了1对引物,跨度为582bp;以793/B的S1基因设计合成了1对特异型引物,跨度为891 bp;建立了能检测出IBV并能够同时鉴定出793/B血清型的实验室一次性PCR诊断方法。【结果】多重RT-PCR检测表明,793/B血清型可出现582和891 bp 2条核酸片段,而其他血清型只出现582 bp 1条核酸片段,新城疫病毒、传染性喉气管炎、鹅副粘病毒则无任何条带出现。【结论】所建立的TR-PCR方法具有一定的特异性,可用于IBV的快速检测和793/B血清型的临床诊断。

猪繁殖与呼吸综合征病毒一步多重RT-PCR检测方法的建立及应用

本研究旨在建立猪繁殖与呼吸综合征病毒(PRRSV)的一步多重RT-PCR检测方法。通过多序列比对以及反应条件的优化,从4对引物中精选了3对特异性和敏感性较好、扩增片段分别为228、345和490bp的引物,并应用所建立的方法对4种分离株的细胞毒和组织毒、田间样品及临床症状相似的疾病猪瘟和猪伪狂犬病毒做了检测;并对田间及实验室样品进行符合率试验,对阳性样品进行重复性试验。与常规RT-PCR法进行比较,证明该方法比常规RT-PCR法敏感性提高了10倍;而且利用该方法可使检测时间大大缩短;特异性试验证实该方法无交叉反应;阳性和阴性样本的符合率均为100%;3次阳性样品重复性检测结果完全相同;稳定性试验显示该试剂盒至少可保存1年以上。本研究所建立的PRRSV一步多重RT-PCR检测方法的敏感性高、特异性强、操作简便、省时、结果重复性好,不仅适用于临床病例尤其低微含量样品的诊断,而且在筛查隐形带毒动物及兽医检疫方面具有重要的应用价值。