马尔堡、埃博拉病毒双重荧光定量PCR检测方法的建立

目的 建立一种快速、敏感、特异的双重实时荧光定量PCR方法,可同时检测马尔堡病毒和埃博拉病毒.方法 通过序列比对挑选出两种病毒基因组中高度保守的序列,分别设计引物及Taqman探针,两条探针分别标记FAM和Texas Red荧光报告基因,建立双重实时荧光定量PCR反应体系.结果 双重荧光定量PCR方法检测两种病毒阳性标准品的灵敏度分别为30.5拷贝/μl和28.6拷贝/μl,通过检测日本脑炎病毒、黄热病毒、登革热病毒无交叉反应,有较好的灵敏度和特异性.结论 建立了马尔堡、埃博拉病毒双重荧光定量PCR检测方法,实现了两种病毒同时实时定量检测,在传染病防控领域有较好的应用前景.     

2012年发现的新冠状病毒分子检测方法的建立与探针优化

目的 建立2012年确定的新冠状病毒的分子检测方法,并筛选优化引物与探针.方法 依据最先发表的208 bp长的新冠状病毒1b片段核酸序列,比对分析后设计合成常规RT-PCR引物1对及荧光定量RT-PCR引物1对与TaqMan探针2条(TZ1,TZ2),同时合成锁核酸修饰的TaqMan探针2条(LNA-TZ1,LNA-TZ2).建立检测新冠状病毒感染的常规RT-PCR方法与4种荧光定量RT-PCR,分析比较其灵敏度与特异性,同时参照欧洲发表的2种荧光定量RT-PCR引物与探针对(upE,ORF1b)建立相应方法,以合成的阳性模板比较6对荧光定量RT-PCR引物与探针的检出灵敏度.结果 所合成的常规RT-PCR与荧光定量RT-PCR引物与探针皆有较好特异性,不能扩增其他人冠状病毒模板及常见呼吸道病毒模板,检出下限达50 ～ 500拷贝/反应.锁核酸修饰TaqMan探针可改善荧光定量PCR检测方法的反应性能,经锁核酸修饰的TaqMan探针与常规TaqMan探针相比,检出率提高10倍左右,其中LNA-TZ1与upE探针对具最佳反应性能.结论 本研究所建立的常规RT-PCR与荧光定量RT-PCR可用于新冠状病毒的分子检测,并推荐使用LNA-TZ1与upE探针对.本研究为新冠状病毒分子检测方法应用与改进奠定了基础.     

阿瓦朗病毒和休斯病毒实时荧光RT-PCR检测方法研究

目的建立可以检测阿瓦朗病毒(Avalon virus,AVAV)和休斯病毒(Hughes virus,HUGV)两种内罗病毒的实时荧光定量RT-PCR检测方法,并进行初步的评价。方法收集、整理、比对、分析在公共数据库发布的两种病毒基因组核苷酸序列,确定检测靶标,设计特异性引物、探针,优化检测程序,建立实时荧光定量RT-PCR检测方法。利用体外转录技术制备的模拟样本、其他病毒感染标本、病毒株和正常人血标本比较评价所建方法的检测限、特异性、重复性特征。结果所建实时荧光定量RT-PCR检测方法可有效扩增检测AVAV和HUGA靶标RNA,检测限分别约为20拷贝/μl和70拷贝/μl,检测科萨努尔森林病毒、乙型流感病毒BV和BY型、甲型流感病毒H3N2、黄热病毒、乙型脑炎病毒、克里米亚-刚果出血热病毒、发热伴血小板减少综合征、内罗毕羊病毒和塔西那病毒样本无非特异性扩增,两种内罗病毒相互间无交叉反应,重复性比较分析显示变异系数小于2%。结论本研究建立的检测AVAV和HUGV的实时荧光定量RT-PCR方法,可用于临床样本检测和媒介生物、宿主动物标本筛查,便于病原的快速识别和疾病诊断。     

TaqMan探针实时PCR检测人MTHFR基因C677T多态性方法的建立

目的建立TaqMan探针实时PCR检测人MTHFR基因C677T多态性的方法。方法设计一对MTHFR基因C677T多态位点的引物及TaqMan探针,采用TaqMan探针实时PCR扩增SNP分型方法检测唇腭裂患者及其父母共100人的MTHFR基因C677T多态性,与常规PCR-RFLP方法进行一致率比较,并对其特异性、敏感性和重复性以及成本-效益等进行评价,同时对部分实时PCR产物样本进行测序验证。结果运用TaqMan探针实时荧光PCR技术对MTHFR基因C677T多态性检测结果准确,特异性好,与常规PCR-RFLP方法结果具有高度一致性,Kappa=0.922>0.75(P=0.000);检测灵敏度可达2×103拷贝;重复性好、高通量、无污染、安全性好;随机样品TaqMan探针分型结果与测序结果完全一致。结论成功建立了TaqMan探针实时PCR检测人MTHFR基因C677T多态性的方法;此方法是常规临床诊断及大规模群体研究的良好平台。     

肝螺杆菌TaqMan MGB探针实时荧光定量PCR快速检测方法的建立及应用研究

目的 建立特异、敏感、快速检测肝螺杆菌的TaqMan MGB探针实时荧光定量PCR方法.方法 针对肝螺杆菌flaB 基因的保守区设计特异性引物和探针,建立肝螺杆菌TaqMan MGB探针实时荧光定最PCR方检测方法,验证方法的特异性、敏感性和稳定性.对2008-2011年期间采集的1081份临床样本中的肝螺杆菌进行检测,同时进行分离培养和常规PCR检测.结果 建立的TaqMan MGB探针实时荧光定量PCR方法对肝螺杆菌的检测具有高度的特异性,对幽门螺杆菌、空肠弯曲菌、泰泽氏菌、侵肺巴斯德氏菌、大肠埃希菌、铜绿假单胞菌均无交叉反应,检测的灵敏度达8.3拷贝.标准曲线显示各浓度范围内具有良好的线性关系,相关系数为0.999,斜率为-3.227,TaqManMGB探针实时荧光定量PCR效率为100％.对1081份临床样本进行检测,TaqMan MGB探针实时荧光定量PCR和常规PCR均能检出86份肝螺杆菌阳性样本,而细菌分离培养则仅检出4份阳性.结果显示,建立的TaqMan MGB探针实时荧光定量PCR方法比细菌分离培养方法更敏感,能够直接从临床样本中检出肝螺杆菌DNA,检测时间仅为2h.结论 研究建立的TaqMan MGB探针实时荧光定量PCR方法具有可靠、特异、敏感的特点,适用于肝螺杆菌的快速检测.     

实时荧光定量RT-PCR监测恒河猴体内人/猴免疫缺陷病毒载量在传代过程中的变化

目的为分析中国SHIV/猕猴AIDS模型的病毒载量变化趋势,建立一种实时、灵敏、特异的针对人/猴免疫缺陷病毒的定量检测方法。方法体外转录制备RNA标准品,利用TaqManEZRT-PCR试剂盒的反应体系和针对SHIVgag保守区91个碱基的TaqMan探针和引物,建立一步法实时荧光定量RT-PCR。提取126份来自SHIV-CN97001感染恒河猴血浆病毒RNA并定量检测。结果利用梯度稀释的RNA标准品对反应体系进行优化,标准曲线下限达到2×102拷贝/ml,相关性(r>0.99)及重复性(CV=4.14%)均能达到测定要求。病毒载量的检测结果表明SHIV-CN97001在猴体内传代过程中病毒载量有先升后降的趋势,病毒载量通常在接种病毒或感染猴的全血后第14天达到高峰。血浆载量可达到105~106拷贝/ml。结论成功地建立了一步法定量SHIVRNA的实时荧光定量RT-PCR,为SHIV/恒河猴AIDS模型的建立与应用提供了灵敏的病毒载量检测方法。SHIV-CN97001的体内繁殖能力在猴体内传代过程中有所增强。     

不同黏膜上皮细胞中巨噬细胞炎性蛋白-3α转录水平的荧光定量RT-PCR比较分析

目的 定量比较分析不同黏膜上皮细胞中人巨噬细胞炎性蛋白-3α(MIP-3α)的转录水平.方法 体外转录制备MIP-3α RNA标准品,人工合成MIP-3α mRNA序列特异的引物及TaqMan探针.利用TaqMan EZ RT-PCR试剂盒的反应体系和ABI实时荧光定量PCR仪进行实时荧光定量RT-PCR.通过对RT-PCR产物测序、使用标准品和质控品进行多次独立测试等评估实时荧光定量RT-PCR方法的特异性、灵敏度和可重复性.随后对不同黏膜上皮细胞系Caco-2、T-84、HeLa和淋巴细胞系PM1中MIP-3α mRNA水平进行了定量检测.结果 建立了可用于MIP-3α mRNA水平定量检测的实时荧光定量RT-PCR方法,该方法特异性好(扩增片段测序结果与参考序列完全一致)、灵敏度高(25 μl反应体系中有5个拷贝就可以检出)、检测样品浓度范围广(103～1010拷贝/ml).对Caco-2、T-84、HeLa和PM1细胞中MIP-3α mRNA水平的定量分析表明,肠黏膜上皮细胞Caco-2和T-84的MIP-3α mRNA水平比HeLa和PM1细胞高.结论 黏膜上皮细胞能表达丰富的MIP-3α,不同黏膜上皮细胞MIP-3α的表达水平可能不同.     

AllGlo探针4重荧光定量PCR技术同时检测4种疱疹病毒

目的 探讨多重荧光定量PCR技术的优化条件,建立基于AllGlo探针技术荧光定量法检测4种疱疹病毒新方法.方法 分别采用单重和多重定性PCR扩增临床常见4种疱疹病毒[单纯疱疹病毒1型(HSV-1)、HSV-2、Epstein-Barr病毒(EBV)、巨细胞病毒(CMV)]并测序鉴定,然后分别采用AllGlo探针和TaqMan探针的单重和多重定量PCR技术对4种疱疹病毒进行单种和多种病毒同时定性定量检测.结果 TaqMan探针和AllGlo探针单种疱疹病毒检测阳性率和特异性均为100％,AllGlo探针单重定量PCR检测比4重探针单种定量PCR的检测Ct值高1～3,AllGlo探针4重定量PCR可以同时检测4种疱疹病毒,相同样品AllGlo探针4重定量PCR检测与单探针单重定量PCR分别检测的结果符合率100％.结论 AllGlo探针荧光定量PCR技术的通量、灵敏度和特异性均高于TaqMan探针,应用前景广阔.     

登革热病毒多重荧光PCR检测及基因分型方法的研究

目的建立一种登革热病毒双靶基因多重荧光PCR检测方法,用于登革热病毒的实验室诊断和基因分型。方法选取登革热病毒Ⅰ-Ⅳ型病毒保守区设计型特异性引物探针和通用型引物探针。评估多重荧光PCR检测方法的特异性、重复性和检测限;并对20份阳性样本进行检测。结果20个登革热阳性核酸标本在通用型检测全部为阳性,特异性型别检测发现登革热病毒Ⅰ型10例、登革热病毒Ⅱ型3例、登革热病毒Ⅲ型3例、登革热病毒Ⅳ型4例;20名正常无症状人群标本提取的核酸和HIV、HCV和HEV通用型和特异性型别检测全部为阴性。梯度检测的变异系数均小于5％。对登革热Ⅰ-Ⅳ型病毒检测最低检测限达10^3 eopies／ml。结论本研究建立的登革热病毒双靶基因多重荧光PCR检测及分型方法具有特异性好、重复性好、快速易操作等优点,可用于登革热病毒的快速检测和基因分型鉴定。     

甲型流感病毒荧光RT-PCR检测方法的设计和检定

目的　设计甲型流感病毒TaqMan荧光RT-PCR检测方法并对其进行检定。方法　用DNAStar和PrimerPremier5. 0软件设计甲型流感病毒荧光PCR检测所用引物和探针 ;在GenBank中进行Blast以及电子对比证明其具有高度的特异性和保守性 ;和标准RT-PCR进行比较 ,检测此方法的灵敏度。结果　所设计的引物和探针高度特异和保守 ,灵敏度比标准RT-PCR方法高 3～ 27倍 ,并且反转录和PCR可合并为一步。结论　设计了甲型流感病毒TaqMan检测方法 ;该方法具有特异、灵敏和简便的特点     

Quantification of hepatitis C virus by TaqMan PCR: comparison with HCV Amplicor Monitor assay.

The quantitation of serum levels of hepatitis C virus RNA in chronic hepatitis C has been regarded as one of the most important indicators for the outcome of interferon therapy. A new method was used for quantitating the copy number of hepatitis C virus RNA using TaqMan polymerase chain reaction and for comparing the ability and usefulness of this assay with Amplicor Monitor assay in 138 patients. The detection range of hepatitis C virus RNA by TaqMan polymerase chain reaction was from 2 x 10(3) to 2 x 10(8) copies/ml. Hepatitis C virus RNA was detectable in 128 cases (92.8%) and undetectable in 10 cases (7.2%) by this method. The RNA levels measured by Amplicor Monitor assay correlated significantly with those measured by TaqMan polymerase chain reaction assay and the sensitivity of the two assays was almost equal. Thus, TaqMan polymerase chain reaction assay appears sufficiently sensitive for the evaluation of hepatitis C virus RNA and would be useful for the diagnosis and management of hepatitis C virus infection.     

一种可用于溶瘤病毒M1体内组织定量的TaqMan RT-qPCR检测法的建立与评价

目的:监测溶瘤病毒M1在组织中的传播情况,以更好地控制给药剂量,确保安全性。方法:建立一种基于TaqMan的实时定量PCR检测法,用于对组织中溶瘤病毒M1的检测和定量,并检测静脉注射病毒后多种实验动物体内的病毒载量和分布。结果:我们以一对特异的引物(Q3)和标准RNA开始SYBR Green RT-qPCR研究。通过优化实验方法发现当退火温度高于62℃时可降低基质效应,但却影响了扩增效率。因此我们建立了一步法TaqMan RT-qPCR实验,重新设计了一对Q3短引物(Q3S)。运用一步TaqMan RT-qPCR检测法和Q3S引物,在混有SD大鼠或食蟹猴基质RNA的背景下,均能特异性检测到低拷贝数的标准RNA。经验证,该方法适用于检测M1病毒在小鼠、SD大鼠和食蟹猴体内的组织分布。结论:利用Q3S引物构建的TaqMan一步法RT-qPCR能够定量检测不同动物不同组织样品中的M1病毒,具有特异性和敏感性,可进一步应用于临床样品的检测。     

扎伊尔型埃博拉病毒的实时荧光RT-PCR检测方法研究

目的 建立扎伊尔型埃博拉病毒的核酸检测方法,以期用于埃博拉出血热临床标本的检测.方法 针对扎伊尔型埃博拉病毒核蛋白和糖蛋白基因设计引物和探针,建立单重和双重实时荧光RT-PCR检测方法,利用体外转录病毒RNA和埃博拉病毒系列参考品RNA评价其敏感性,利用马尔堡病毒、健康人、登革热患者和发热伴血小板减少综合征患者血清评价其特异性.结果 所建立的实时荧光RT-PCR检测方法扩增效率在95％～105％,可特异性地检测扎伊尔型埃博拉病毒核蛋白和糖蛋白基因,与马尔堡病毒、登革热和发热伴血小板减少综合征病毒均无交叉反应,体外转录的病毒RNA可检出10～100拷贝/μl.双重检测方法通过细胞培养的扎伊尔型埃博拉病毒RNA验证,可检出100 pfu/ml病毒.结论 本研究建立的检测扎伊尔型埃博拉病毒的实时荧光RT-PCR方法具有良好的特异性和敏感性,可用于埃博拉出血热临床标本的检测.     

Comparison of TaqMan real-time PCR and p24 Elisa for quantification of in vitro HIV-1 replication

In this study, TaqMan PCR was used to assess viral replication of HIV-1 infected cells in vitro. This PCR technique was compared with p24 ELISA as a standard method to monitor HIV-1 replication in cell culture. Hut78 T-lymphoblastoid cells were infected with different titres of HIV-1(IIIb) (MOI 0.05-0.0005). The course of HIV-1 replication was monitored by determination of p24 concentrations by ELISA in cell culture supernatants and by quantitation of HIV-1 gag RNA by TaqMan RT-PCR. Additionally, the number of HIV-1 proviral copies was assessed by TaqMan PCR. Monitoring of HIV-1 replication by p24 ELISA and TaqMan RT-PCR revealed comparable kinetics of infection. Both methods provided similar data on the exponential increase and on plateauing of HIV-1 replication. Furthermore, both methods were equally sensitive. However, a 7 log linearity of TaqMan HIV-1 gag PCR was demonstrated without dilution of the specimen, in contrast to p24 ELISA, where because of its narrow range of detectable p24 concentrations, sample dilution was necessary. Although determination of the number of proviral copies by TaqMan PCR does not measure HIV-1 replication, the kinetics of proviral copy number following in vitro inoculation of cells with HIV-1 was nearly the same as the kinetics of HIV-1 RNA copy numbers. In conclusion, TaqMan real-time RT-PCR was demonstrated as a reliable and sensitive tool to quantify and monitor HIV-1 replication in cell culture. It is suggested, therefore, that this technique be an alternative method to monitor HIV-1 replication in vitro.     

Comparative evaluation of measles virus specific TaqMan PCR and conventional PCR using synthetic and natural RNA templates

Comparative evaluation of TaqMan RT-polymerase chain reaction (PCR) methodology developed during this study with the conventional RT-PCR-nested PCR methodology developed earlier, using measles virus RNA templates derived from synthetic and natural sources against a number of primer sets belonging to various regions of the genome, revealed the existence of similar assay thresholds for both methods. An exception to this finding was, however, noted using primer sets of the N and M genes regions with RNA templates extracted from the wild type measles virus strain where the nested PCR method proved to be 10- to 100-fold more sensitive than the end points established with the N gene specific TaqMan RT-PCR method with synthetic RNA templates. These differences were not evident when the same primer sets were evaluated with RNA templates extracted from a brain sample of SSPE patient. These findings indicate that the genetic make up of measles virus strain in any given clinical specimen, in relation to the amplifying primers/probe sequences, can have impact on the overall sensitivity and specificity of the methodology applied. Both methods are equally suitable for the molecular detection of measles virus sequences in clinical specimens, although the TaqMan RT-PCR method may be preferred due to its advantages of contamination control, automation, and real-time product quantitation.     

Sensitivity and reproducibility of HCV quantitation in chimpanzee sera using TaqMan real-time PCR assay

The availability of molecular protocols for the detection and quantitation of very low numbers of hepatitis C virus (HCV) particles in biological samples is an issue of interest in both clinical and analytical fields of HCV research. A sensitive and reproducible assay is described for HCV RNA quantitation using the TaqMan PCR fluorogenic real-time detection system to establish the levels of HCV RNA in chimpanzee plasma. Our TaqMan PCR protocol and synthetic full length HCV RNA template show that the threshold of sensitivity for our TaqMan PCR is two copies per reaction. As few as 10 genome copies per reaction could be quantitated maintaining a linear range. The accuracy of the TaqMan PCR test was comparable to commercial bDNA and Amplicor tests. The RNA standards of the laboratory were tested in parallel with a World Health Organization (WHO) International Standard for HCV RNA obtaining ratios of 2.7+/-0.7 RNA copies per HCV international unit (IU). Our method using RNA extracted from chimpanzee samples had an estimated sensitivity of 200 RNA copies/ml of plasma (approximately eight copies/reaction or 74 WHO IU/ml). Serial plasma samples from HCV-infected chimpanzees were analyzed using this methodology to evaluate its applicability, and RNA profiles were observed consistent with the evolution of the pathology in each animal. The present study therefore illustrates the high reproducibility, sensitivity and reliability of our TaqMan methodology, providing a useful method for HCV research to consistently detect and quantify viral RNA throughout a range of concentrations.     

Evaluation of the COBAS TaqMan HCV test with automated sample processing using the MagNA pure LC instrument

The COBAS TaqMan HCV Test (TaqMan HCV; Roche Molecular Systems Inc., Branchburg, N.J.) for hepatitis C virus (HCV) performed on the COBAS TaqMan 48 Analyzer (Roche Molecular Systems) currently relies on a manual sample processing method. Implementation of an automated sample processing method would facilitate the clinical use of this test. In this study, we evaluated the performance characteristics of TaqMan HCV following automated sample processing by the MagNA Pure LC instrument (MP; Roche Applied Science, Indianapolis, Ind.). The analytical sensitivity of TaqMan HCV following sample processing by MP was 8.1 IU/ml (95% confidence interval, 6.1 to 15.2). The assay showed good linearity (R(2) = 0.99) across a wide range of HCV RNA levels (25 to 5 x 10(6) IU/ml), with coefficients of variation ranging from 10% to 46%. Among 83 clinical specimens, the sensitivity and specificity of TaqMan HCV were 100% and 95%, respectively, when compared to the COBAS AMPLICOR hepatitis C virus test, version 2.0 (COBAS AMPLICOR; Roche Molecular Systems), with TaqMan HCV detecting two more HCV RNA-positive specimens than COBAS AMPLICOR. Both specimens were confirmed to be HCV RNA positive by the VERSANT HCV RNA qualitative test (Bayer HealthCare LLC, Tarrytown, N.Y.). There was also strong correlation (R(2) = 0.95) and good agreement between the results from TaqMan HCV and the VERSANT HCV RNA 3.0 assay (bDNA) (Bayer HealthCare LLC) among a group of 93 clinical specimens. The MP is a versatile, labor-saving sample processing platform suitable for reliable performance of TaqMan HCV.