Molecular diagnosis and direct quantification of cereal cyst nematode(Heterodera filipjevi) from field soil using TaqMan real-time PCR

Heterodera filipjevi continues to be a major threat to wheat production worldwide.Rapid detection and quantification of cyst nematodes are essential for more effective control against this nematode disease.In the present study,a TaqManminor groove binder(TaqMan-MGB)probe-based fluorescence quantitative real-time PCR(qPCR)was successfully developed and used for quantifying H.filipjevi from DNA extracts of soil.The primers and probe designed from the obtained RAPD-SCAR marker fragments of H.filipjevi showed high specificity to H.filipjevi using DNA from isolatesconfirmed species of 23 Heterodera spp.,1 Globodera spp.and 3 Pratylenchus spp.The qPCR assay is highly sensitive and provides improved H.filipjevi detection sensitivity of as low as 4^(-3) single second-stage juvenile(J2)DNAs,10^(-3) female DNAs,and 0.01μgμL^(-1) genomic DNAs.A standard curve relating to the threshold cycle and log values of nematode numbers was generated and validated from artificially infested soils and was used to quantify H.filipjevi in naturally infested field soils.There was a high correlation between the H.filipjevi numbers estimated from 32 naturally infested field soils by both conventional methods and the numbers quantified using the qPCR assay.qPCR potentially provides a useful platform for the efficient detection and quantification of H.filipjevi directly from field soils and to quantify this species directly from DNA extracts of field soils.

运用Real-time quantification PCR方法建立副溶血性弧菌在即食虾中的生长预测模型

运用Real-time quantification PCR(real-time qPCR)方法建立副溶血性弧菌在即食虾中生长预测模型。首先构建质粒标准品,梯度稀释后建立标准曲线,然后用Real-time qPCR方法检测虾中副溶血性弧菌的数量,最后建立37℃下即食虾中副溶血性弧菌生长预测模型,并与传统涂布计数方法进行比较。结果表明,Real-time qPCR方法和传统计数方法均可建立Gmopertz模型、Logistic模型和Richards模型,模型拟合的相关系数R2均在0.9以上。基于Real-timeqPCR方法省时省力、特异性好等优点,用Real-time qPCR方法建立微生物预测模型是未来预测微生物学领域的一种发展趋势。

Effects of Different Temperature and Time Durations of Virus Inactivation on Results of Real-time Fluorescence PCR Testing of COVID-19 Viruses

Summary:The novel coronavirus SARS-CoV-2 caused an outbreak of pneumonia in Wuhan,Hubei province of China in January 2020.This study aims to investigate the effects of different temperature and time durations of virus inactivation on the results of PCR testing for SARS-CoV-2.Twelve patients at the Renmin Hospital of Wuhan University suspected of being infected with SARS-CoV-2 were selected on February 13,2020 and throat swabs were taken.The swabs were stored at room tempcrature(20-25℃),then divided into aliquots and subjected to different temperature for different periods in order to inactivate the viruses(56℃for 30,45,60 min;65,70,80℃for 10,15,20 min).Control aliquots were stored at room temperature for 60 min.Then all aliquots were tested in a real-time fluorescence PCR using primers against SARS-CoV-2.Regardless of inactivation temperature and time,7 of 12 cases(58.3%)tested were positive for SARS-CoV-2 by PCR,and cycle threshold values were similar.These results suggest that virus inactivation parameters exert minimal infuence on PCR test results.Inactivation at 65℃for 10 min may be sufficient to ensure safe,reliable testing.

Detection of Ratoon Stunting Disease in Virus-free Seedcane via Real-time Fluorescence Quantitative PCR

This study was to develop the real-time fluorescence quantitative PCR technique for detecting the ratoon stunting disease （RSD） in virus-free seedcane seedlings. Healthy tissue culture seedlings were obtained from six plants of sugarcane ROC22, which had been confirmed RSD-positive by detecting the sugarcane juice, by employing the sugarcane seedlings production protocol. Real-time fluorescence quantitative PCR was used to detect RSD pathogens in tissue culture sam- pies. The results showed that target fragment of RSD pathogens was not found in all 10 samples in real-time fluorescence quantitative PCR, with the Ct values of 37 - 39. The healthy tissue culture sugarcane seedlings do not carry RSD pathogens, indicating that adopting healthy seedcane seedlings production technique could thoroughly get rid of RSD pathogens.

Development and Preliminary Application of SYBR Green I Real-Time Fluorescence Quantitative PCR Method for Detecting Porcine Parvovirus Virus

According to VP2 gene sequence of the porcine parvovirus virus strain NADL-2 （NC001718） available in GenBank （NC_001718）, a pair of specific primer was designed, and the target fragment of 431 bp was obtained by PCR amplification. The products were ligated with pMD18- T vector and then transformed into bacteria DH5α for recombinant plasmid extraction. After PCR identification and sequencing, recombinant plasmid was used as a standard template to establish the standard curve of SYBR Green I fluorescence quantitative PCR. Sensitivity test, specificity test and repeatability test were also determined. The results indicated that there was a good linear relationship between threshold cycle of the standard curve and template concentration, R2 =0.997 6. Tm ranged from 82.3 to 82.9 ℃, while the sensitivity was 72.1 copies/μl with good specificity and repeatability. The developed SYBR Green I real-time quantitative PCR method to detect PPV VP2 gene laid the basis for further studies on patho- oenesis, early clinical diaonosis of this virus and quantitative analysis of PPV infection.

Real-time fluorescent quantitative PCR非特异性扩增的研究进展

Real-time fluorescent quantitative PCR(RT-qPCR)因其高效快速、操作简便、高度敏感等优点获得广泛应用,但因其强大的放大功能而容易出现非特异性扩增的问题,尤其是荧光染料法中更加明显。文章对RT-qPCR的原理、非特异性扩增的发生因素、解决方案及该技术的应用前景进行了综述。

Detection of Lactobacillus acidophilus in Fermented Material by Real-time Fluorescent Quantitative PCR

The species distinctive PCR primer of Lactobacillus acidophilus （ L. acidophilus） was designed according to 16S rRNA gene sequences of conunon Lac- tobacillus species in fermented material. Bacterial genome DNA of separated L. acidophilus in fermented sample was taken as template, and L. acidophilus in fer- mented material was conducted the quantitative determination by real-time quantitative PCR （RT-PCR）. Analysis on RT-PCR results shown that contents of L. aci- dophilus in the test sample reached 1.5 billion CFU / g. Test results shown that contents of L. acidophilus in fermented material could be detected accurately by the established RT-PCR method in the test. indicating that the established RT-PCR method could be aookued to the detection of L. acidophilus in fermented material.

Application of Real-time Fluorescent Quantitative PCR in Studies on Plants

Real-Lime fluorescent quantitative PCR is a method for quantitative analysis of gene expression developed in recent years, which has been widely used in various fields such as basic scientific research, clinical diagnosis, disease study, drug research and development since its appearance. It starts relatively late in study on plants, but has already been used for analysis of gene expression in plants and gene identification of exogenous genes. The principles or advantages and dis- advantages of real-time fluorescent quantitative PCR, or its potential problems and condition optimizations in tests were introduced in this study, and then the appli- cation and prospect of real-time fluorescent quantitative PCR in study on plants were also been discussed.

Development of Real-Time Fluorescent PCR for Rapid Detection of Haempohlius parasuis

[ Objective] To develop a real-time fluorescent PCR assay for rapid detection of Haempohlius parasuis （HPS）. [ Method] According to the conservative sequences of 16 S rRNA genes of HPS published in GenBank, a pair of specific primers was designed. The real-time fluorescent PCR was developed by optimizing primer concentration and annealing temperature. And its specificity and reproducibility were evaluated. Ten HPS- suspected samples were detected by the developed method. [ Result] The lowest detection limit of the developed real-time fluorescent PCR was 50 copies/μl. This method had good reproducibility, and its coefficient of variation was lower than 2%. Only HPS rather than Streptococcus suis type 2, Staphylococcus aureus, E. coli DH5 alpha, and swine Salmonella typhi could be detected by the developed real-time fluorescent PCR. The HPS-pesitive samples detected by this method were also positive when they were detected by isolation of bacteria or conventional PCR. [ Conclusion] The developed real-time fluorescent PCR is rapid, sensitive, specific and highly reproducible; thus, it can be used for rapid detection of HPS.

Synchronously Detecting Allergenic Ingredients of Peanut and Sesame in Food by Real-time Fluorescent PCR

Peanut,sesame and other raw materials of food are allergens for special populations.In this study,specific primers and TaqMan probes labeled by different fluorescences were designed targeting Ara h 2 gene of peanut and Ses i 1 gene of sesame.After the optimization of reaction conditions,a real-time fluorescent PCR method was established for simultaneous detection of allergenic ingredients of peanut and sesame in food.Genomic DNA samples of peanut,sesame,rice,wheat,barley,soybean,celery,maize,potato,tomato,walnut,groundnut in shell,cashew nut,sunflower seed,almond,apple,pear and strawberry,pork,beef,mutton and fish were used as templates for PCR amplification with deionized water as negative control template.Results indicated that the established real-time fluorescent PCR method could specifically identify allergenic ingredients of peanut and sesame simultaneously.Sensitivity test showed that the minimum detection limit of this method was 0.01%.Therefore,the established real-time fluorescent PCR method is a specific,sensitive and effective assay for simultaneously detecting allergenic ingredients of peanut and sesame in food.

Establishment of a Real-time Fluorescent Quantitative PCR Assay for Detection of Genetically Modified Maize Line MON88017

In order to improve the standardized technical systems of quantitative analyses for genetically modified organisms （GMOs） and products, ensure bio-safety and reduce ecological risk in China, a real-time fluorescent quantitative PCR assay was established for detection of genetically modified maize line MON88017. The established method was evaluated based on the specificity, sensitivity, accuracy and measurement uncertainty. The results showed that the established method had strong specificity in detection of genetically modified maize line MON88017. 1.50% MON88017 sample was detected with 29 replica- tions. The average measured value （ 1. 541% ） was close to the actual value （ 1.50% ） and the relative deviation was 2.70%. The variation coefficient of the measured value was 0.110 g ; the recovery was 100.00% and the measurement uncertainty was 0. 096. The limit of detection for genetically modified maize line MON88017 with the established method was 5 copies at the 97.5% confidence level. Thus, the real-time fluorescent quantitative PCR assay established in this study exhibited high specificity, accuracy and sensitivity, which could provide technical support for the safety supervision of genetically modified organ- isms and products in China.

猪瘟病毒PCR-ELISA检测方法的初步应用

猪瘟(classicalswinefever,CSF)是危害猪健康的重要传染病之一,具有急性、热性和高度接触性等特性,给世界养猪业造成了巨大的经济损失。PCR-ELISA检测方法是在PCR和ELISA基础上创新的一种新技术,其灵敏性和准确性都高于单纯的PCR检测和ELISA检测。根据GenBank中猪瘟石门株序列,设计特异性引物,分别在5'端标记生物素和地高辛,经过PCR扩增后,加入到包被有链霉亲和素的ELISA反应板上,然后通过DIG-HRP抗体进行显色反应,建立PCR-ELISA检测方法。结果表明:PCR-ELISA最低检测到1.81×10^(4)拷贝/μL,普通PCR最低检测到1.81×10^(6)拷贝/μL,因此PCR-ELISA灵敏性比普通PCR高约100倍;对27份未知样品进行检测,普通PCR检测出阳性样品15份,阳性率为55.5%,PCR-ELISA检出20份阳性样品,阴性7份,阳性率为74%,阳性检出率增加18.5%;用建立的Real-TimePCR进行验证,检测结果与PCR-ELISA一致,两者的符合度为100%;然后用PK15细胞对普通PCR未检测出的阳性样品进行病毒分离培养,通过间接免疫荧光(IFA)及普通PCR检测,结果显示该5份样品均为阳性。说明本研究所建立的PCR-ELISA方法具有灵敏、特异、准确、快速、安全等优势,可应用于CSFV的快速诊断,为CSFV的流行病学监测提供有力保障。

4种致牛腹泻病毒多重荧光定量PCR检测方法的建立及应用

【目的】建立牛病毒性腹泻病毒(BVDV)、牛冠状病毒(BCoV)、牛轮状病毒(BRV)、牛恶性卡他热病毒(MCFV)4种致牛腹泻病毒多重荧光定量PCR检测方法,为BVDV、BCoV、BRV和MCFV的鉴别诊断提供技术支持。【方法】根据BVDV的5′UTR基因、BCoV的N基因、BRV的VP6基因和MCFV的ORF9基因的保守区域设计引物,克隆目的基因构建标准阳性重组质粒,建立可同时检测4种致牛腹泻病毒的多重荧光定量PCR检测方法,并对该方法的特异性、敏感性和重复性进行评价。用建立的多重荧光定量PCR检测方法对76份临床样品进行检测,并与常规PCR检测方法的结果进行比较。【结果】建立了BVDV、BCoV、BRV和MCFV多重荧光定量PCR检测方法,该方法建立的标准曲线线性关系良好,仅可特异性扩增出4种目标基因片段;对BVDV、MCFV、BRV和BCoV的最低检出限分别为9.4×10^(1),1.4×10^(3),9.1×10^(1)和1.2×10^(2)拷贝/μL,与普通PCR相比,灵敏性高出10~1000倍;批内、批间差异均小于5%。76份临床样品检测结果显示,多重荧光定量PCR检测结果与普通PCR检测结果的符合率分别为98.7%(BCoV)、97.4%(BRV)、100.0%(BVDV)和100.0%(MCFV)。【结论】建立了BVDV、BCoV、BRV和MCFV多重荧光定量PCR检测方法,该方法特异性强、灵敏度高、重复性好,可用于牛腹泻病毒病原的实验室检测。

极速热循环荧光定量PCR系统的设计及性能评估

目的:设计一种用于病原体核酸现场检测的极速热循环荧光定量PCR系统,并进行性能评估。方法:该系统由扁平反应杯、极速热循环模块、固化光路荧光检测模块以及智能手机平台数据处理模块组成。极速热循环模块由升温单元和降温单元组成,其中升温单元以陶瓷片和Ag/Pb合金制成,降温单元由高速磁悬浮冷却风扇和双曲线形喉道组成;固化光路荧光检测模块采用注塑工艺制作,由光源激发单元和光探测器单元组成;智能手机平台数据处理模块包括蓝牙串口转接单元和手机应用程序2个部分,其中蓝牙串口转接单元采用TI公司的C2540F256芯片,手机应用程序由Android Studio开发。采用该系统检测甲型/乙型流感病毒和新型冠状病毒,验证该系统的检测性能。结果:该系统可实现对甲型/乙型流感病毒核酸和新型冠状病毒核酸的快速检测,且检测结果与常规实时定量PCR检测结果的一致性较好。结论:该系统体积小、质量轻,可实现病原体核酸快速现场检测。

一种新颖简便的荧光实时RT-PCR相对定量方法的建立

为建立一种新颖、简便的荧光实时RT-PCR相对定量方法,根据实时定量标准曲线,推导出相对定量基因表达的公式.公式显示相对表达指数只与CT值和标准曲线的斜率相关.构建标准曲线的标准品需要通过克隆和体外转录获得,实验过程繁琐.当人为成比例增减标准品各个稀释度的具体拷贝数时,标准曲线的斜率并不改变,说明标准曲线斜率与标准品的具体拷贝数无关.因此,新的相对定量方法可以用任何一个待测样品的总RNA(或cDNA),经系列稀释后作为标准品,来构建相对定量标准曲线,获得斜率.与绝对定量法比较,新方法获得了基本相同的斜率和非常一致的定量结果(差异小于4%),而传统的2-#$CT法却表现出较大的定量误差.这些结果表明,新的相对定量方法是一种简便、准确和高效的定量基因表达的方法.

用细菌16S rRNA荧光定量PCR法检测肠道菌群的变化

目的应用细菌的16S rRNA序列设计双歧杆菌、大肠埃希菌及乳酸杆菌的引物并对肠道的3种细菌进行定量测定。方法收集轮状病毒肠炎患儿及正常对照组的粪便标本提取DNA。取准确定量的3种细菌经系列稀释后抽提细菌的DNA做荧光定量PCR,制作出标准曲线,待测样品同时进行PCR反应并和标准曲线进行比较,获得各样品中3种细菌的量。结果患儿肠道中双歧杆菌和乳酸杆菌的数量较正常儿童明显减低,而大肠埃希菌的数量差异无显著性。与其他文献报道的用细菌培养的方法所得结果一致。结论荧光定量PCR是一种特异性高、敏感性强的定量方法。可正确定量肠道中的细菌数量。

双标准曲线相对定量PCR试验原理与方法

实时荧光定量PCR(FQ-PCR)是一种准确有效的核酸定量分析技术,具有易操作、高通量、高敏感性、高特异性、高度自动化和低污染等优点,并随新定量PCR仪及新操作方法的发展而得到广泛应用,但是,定量PCR的高敏感性特点使得实验操作严格而繁琐。阐述了一种改进的相对定量方法——双标准曲线法的试验原理和特点,描述了定量PCR体系的优化方式,探讨了试验误差分析方法及试验操作技巧,并就试验数据的处理方法进行讨论。试验证明,双标准曲线法是一种经济、简单而准确的定量方法。

实时荧光定量PCR技术及其在水产上的应用

实时荧光定量PCR技术是在PCR技术基础上结合荧光染料发展起来的一种核酸定量技术,能快速、灵敏,并有效地对核酸进行定量检测,已被广泛应用于生命科学的各领域。笔者就实时荧光定量PCR的检测原理、荧光染料,管家基因及其在水产上的应用作一阐述。

实时PCR技术在植物研究上的应用

实时PCR是在常规PCR基础上运用荧光共振能量转移现象,加入荧光标记探针,巧妙地把核酸扩增、杂交、光谱分析和实时检测技术结合在一起的一项新技术,具有快速、灵敏、特异性强、定量准确等特点,广泛应用于医学、检验检疫、军事、农业、基础研究等领域。着重就实时PCR技术的特性及在植物上的应用进行了讨论,并与目前常用的相关技术进行了比较。

羊口疮病毒SYBR Green Ⅰ实时荧光定量PCR的建立及应用

为羊口疮（Orf）的分子流行病学调查、早期快速诊断及细胞培养物的检测等提供参考，根据GenBank 发表的羊口疮病毒（OrfV）B2L 基因序列设计合成1对引物，以 pMD18-T-B2L 重组质粒为阳性标准品，建立了检测 OrfV 核酸的 SYBR Green Ⅰ实时荧光定量 PCR 方法，并进行了临床应用。结果表明：建立的 SYBR Green Ⅰ实时荧光定量 PCR 的线性关系良好，标准曲线的相关系数达到-1；最低检测限为1．0x10^3 copies／μL，比常规 PCR 方法高100倍，与山羊痘和口蹄疫疫苗毒均不发生交叉反应；组内变异系数为1．061％-2．873％，组间变异系数为0．397％-3．829％。用该方法检测5例 OrfV 感染临床病例和8份不同代次的 OrfV 细胞培养物，阳性率均为100％。SYBR Green Ⅰ实时荧光定量 PCR 方法灵敏度高、特异性强、重复性好，可以用于 OrfV 的病原检测及定量分析。