应用Taq Man荧光定量RT-PCR快速检测肠道病毒的研究

目的建立一种快速、灵敏、特异的荧光定量RT-PCR检测肠道病毒的方法。方法根据GenBank中肠道病毒5′UTR序列,应用生物软件在保守区设计与筛选特异引物和TaqMan探针,对荧光定量RT-PCR反应体系与条件进行优化,验证方法的特异性、敏感性和重复性。并通过对急性临床样本的检测,评价该方法的实际应用价值。结果该方法对肠道病毒包括脊髓灰质炎病毒Ⅰ、Ⅱ、Ⅲ型及柯萨奇病毒和埃可病毒等的检测有高度特异性,甲肝病毒、乙脑病毒、登革病毒、腺病毒、单纯疱疹病毒、诺如病毒等均呈阴性。该方法的检测下限达10-1TCID50/100μl。12份急性结膜炎病例结膜拭子标本中7份肠道病毒核酸阳性,普通RT-PCR方法5份阳性。结论TaqMan荧光定量RT-PCR方法快速、敏感、特异,适于肠道病毒的快速检测。     

两种检测尼帕病毒的实时荧光定量PCR方法的比较与效果验证

目的 建立稳定可靠的尼帕病毒特异性检测技术和方法,对该病毒进行监测和检测、预警预报.方法 本研究对已建立的两种尼帕病毒的实时荧光定量PCR检测方法的特异性与敏感性进行了比较和验证.结果 研究结果显示两种方法检测体外转录RNA时灵敏度为10 copies/μL,检测活病毒的灵敏度为10 TCID50/mL.特异性检测结果...     

实时荧光定量PCR在手足口病肠道病毒快速检测中的应用

目的建立快速检测手足口病肠道病毒的实时荧光定量PCR法,并作出应用分析。方法采用卫生部《手足口病预防控制指南》(2008年版)推荐的RT-PCR法,对丽水市2008年4～5月间发生的172例临床诊断手足口病患者的324份标本进行人肠道病毒、柯萨奇病毒A组16型和肠道病毒EV71型特异性核酸的检测,同时采用实时荧光定量PCR法进行平行检测,比较两者的实验结果,分析实时荧光定量PCR方法的特异性和灵敏度。结果实时荧光定量PCR检测324份标本,肠道病毒通用核酸阳性70份,柯萨奇A16核酸阳性22份,肠道EV71病毒核酸阳性15份,与RT-PCR结果一致,符合率100%。结论实时荧光定量PCR法具有特异性强、灵敏度高等优点,是一种理想的手足口病肠道病毒的检测方法。     

实时荧光RT-PCR与普通RT-PCR检测手足口病病原体的比较分析

目的对比分析实时荧光RT-PCR和普通RT-PCR方法在检测手足口病病原体的差别。方法选择临床诊断为手足口病患者粪便256份,提取RNA,用实时荧光RT-PCR和普通RT-PCR检测手足口病肠道病毒71型(EV71)、柯萨奇病毒A组16型(CoxA 16)和总肠道病毒(PE),对实验结果进行统计学分析。结果实时荧光RT-PCR和普通RT-PCR检测手足口病患者粪便标本,CoxA 16结果差异有统计学意义(P<0.01),CoxA 16阳性率分别为62.22%和16.67%;EV71、PE结果差异无统计学意义(P>0.05),实时荧光RT-PCR方法EV71和PE阳性率为42.22%和65.00%,普通RT-PCR方法为37.78%和55.00%。普通RT-PCR检测的36份EV71、CoxA 16和PE全阴性标本,荧光RT-PCR方法检出PE阳性9份、EV71阳性4份、CoxA 16阳性14份。结论实时荧光RT-PCR方法比普通RT-PCR方法更灵敏、快捷,适用于临床手足口病病原体的检测。     

狂犬病病毒和犬瘟热病毒疫苗株混合感染Vero细胞的实验研究

目的研究狂犬病病毒(RV)及犬瘟热病毒(CDV)共同感染Vero细胞及混合感染对产毒量的影响,比较病毒检测方法的敏感性。方法将单独培养的RV、CDV及RV-CDV混合培养物进行连续10倍稀释后同步接种Vero细胞,37℃5%CO2培养5d,分别以直接免疫荧光(DFA)、RT-PCR及荧光定量RT-PCR方法检测病毒的半数细胞感染量,并对各病毒检测方法进行比较。结果RV和CDV可同时感染Vero细胞,并在其中增殖。CDV单独感染Vero细胞的TCID50为10-5.8/0.05ml,RV和CDV混合感染Vero细胞的TCID50为10-5.5/0.05ml。DFA、RT-PCR和荧光定量RT-PCR阳性的RV-CDV感染最大稀释度分别为10-6、10-5和10-6。结论混合培养对RV和CDV的感染滴度及产毒量影响很小。免疫荧光灶检测与RT-PCR及荧光定量RT-PCR方法检测的敏感性相当。犬瘟热-狂犬病毒联合疫苗的病毒滴度检测可不经中和其中的一个病毒直接将联合疫苗接种Vero细胞,然后分别进行免疫荧光检测。     

鼠类携带淋巴细胞脉络丛脑膜炎病毒实时荧光定量RT-PCR检测方法的建立

目的建立检测鼠类携带淋巴细胞脉络丛脑膜炎病毒（lymphocytic choriomeningitis virus,LCMV）的实时荧光定量RT-PCR方法。方法根据LCMV核蛋白编码基因序列设计合成特异性引物对和TaqMan荧光探针,经优化反应体系和条件,建立LCMV实时荧光定量RT-PCR检测方法,然后进行灵敏度、特异性和重复性试验,并对79份宁波口岸捕获的鼠样品进行检测。结果建立实时荧光定量RT-PCR方法对鼠肺总RNA检测的灵敏度为20pg,是常规PCR方法的100倍;试验的重复性良好,CV值为0.85%;试验的特异性为100%。用该方法检测79份鼠样品有3份LCMV阳性,与常规RT-PCR结果一致。结论成功建立了鼠LCMV实时荧光定量RT-PCR检测方法,对于监测和防控鼠传LCM有重要意义。     

Taq Man荧光定量RT-PCR快速检测甲3型流感病毒

目的建立一种特异、灵敏、快速的荧光定量RTPCR方法用于检测甲3型流感病毒核酸。方法根据GenBank登录的流感毒株序列,应用生物学软件进行序列比对,在甲3型流感病毒血凝素(HA)基因的保守区设计引物和TaqMan探针、并进行筛选。对荧光RT-PCR反应条件进行优化,检测该方法的特异性和灵敏度。并对疑似流感含漱液标本进行检测。结果该方法对甲3型流感病毒的检测有高度的特异性,对甲1型、乙型、禽流感病毒H5、SARS病毒及其他呼吸道病毒均无交叉反应,检测的灵敏度达0.01TCID50,可从疑似流感患者含漱液中直接检测流感病毒核酸,从病毒核酸提取至完成检测仅需3h左右。结论本研究建立的TaqMan荧光定量RTPCR是一种快速检测甲3型流感病毒特异、敏感的新方法。     

狂犬病病毒荧光定量RT—PCR检测方法的建立与初步应用

目的建立狂犬病病毒快速检测法。方法根据狂犬病病毒核蛋白基因的保守序列分别设计两对引物及其相应的TaqMan探针。构建pMD-N重组质粒,建立荧光定量RT-PCR绝对定量标准品,并对反应体系进行优化,建立绝对定量的标准曲线。利用10倍稀释法检验方法的灵敏度并与普通RT-PCR法进行比较;作重复性和特异性检验后进行临床标本的检测。结果构建了绝对定量的参照质粒,标准曲线相关系数为0.998。狂犬病病毒荧光定量RT-PCR检测反应的灵敏度为10个TCID_(50);5种非狂犬病病原体检测均为阴性。结论建立的狂犬病病毒的荧光定量RT- PCR检测方法快速、特异性强、灵敏度高、稳定性好,可以应用于临床样品的检测。     

实时荧光定量PCR检测GI型诺瓦克样病毒方法的建立

目的建立临床粪便中GI型诺瓦克样病毒的实时荧光定量PCR检测方法。方法针对诺瓦克样病毒GI型保守序列,用序列比对软件设计特异性引物与探针,建立诺瓦克样病毒实时荧光定量PCR检测方法。并用常规RT-PCR和本文建立的实时荧光定量PCR对137份临床腹泻标本进行检测。结果该方法对诺瓦克样病毒检测准确,重复性好,标准曲线的线性范围为102～107拷贝,相关系数为0.9991。并且对临床标本的检出率显著高于普通RT-PCR。结论本研究建立的实时荧光定量PCR方法可用于检测临床腹泻粪便标本中的GI型诺瓦克样病毒,从而有效预防和控制该病毒的传染。     

2010-2015年广西崇左市手足口病患者病原学检测结果分析

目的分析广西崇左市手足口病病原学检测结果,为该地区今后防制提供科学依据。方法收集整理该市2010-2015年各县(市、区)送检的2 401例疑似手足口病患者的粪便、肛拭子、咽拭子、疱疹拭子标本,应用RT-PCR检测样本中肠道病毒(PE)、肠道病毒71型(EV71)、柯萨奇病毒A组16型(CoxA16)的数据。结果该市6年间共检测疑似手足口病患者标本共2 401份,核酸检测阳性1 586份,阳性率66.06%(1 586/2 401)。在1 586份患者中EV71病毒占25.03%(397/1 586);CoxA16病毒占24.78%(393/1 586);其他肠道病毒占50.19%(796/1 586)。结论该市手足口病患者EV71和CoxA16病毒隔年交替出现高峰,上半年以EV71和CoxA16患者为主,而下半年以其他肠道病毒感染患者为主。     

一种汉滩病毒和汉城病毒双重实时荧光定量 RT-PCR检测方法的建立

目的 建立一种汉滩病毒(Hantaan virus,HTNV)和汉城病毒(Seoul virus,SEOV)双重实时定量荧光RT-PCR检测方法.方法 根据HTNV和SEOV S基因设计引物和探针、优化反应条件,建立2种汉坦病毒的双重实时荧光定量RT-PCR方法.以甲型流感病毒、登革热病毒、新布尼亚病毒、寨卡病毒、新冠...     

The evaluation of three diagnostic tests for the detection of equine influenza nucleoprotein in nasal swabs

Background

Equine influenza (EI) is a highly contagious respiratory disease of horses.

Objectives

The aim of this study was to evaluate two rapid antigen detection kits (Directigen or DFA, and Espline) and a commercial ELISA for the detection of EI nucleoprotein in nasal swabs.

Method

Nasal swab samples from naturally and experimentally infected horses were used to compare the sensitivity and specificity of these assays to virus isolation (VI) and real-time RT-PCR.

Results

If real-time RT-PCR was considered as the gold standard, the sensitivity of the other tests in field samples was 68% (DFA), 35% (ELISA), 29% (Espline), and 9% (VI). These tests had 100% specificity when compared to real-time RT-PCR. A receiver operating characteristic (ROC) curve indicated that decreasing the cutoff of the ELISA would increase sensitivity with some loss of specificity. In samples from experimentally infected horses, the sensitivity of the tests compared with real-time RT-PCR was 69% (VI), 27% (DFA), 6% (Espline), and 2% (ELISA). The specificity was 100% for Espline and ELISA and 95% for VI and DFA.

Conclusions

This study illustrated that DFA is the most sensitive antigen detection test evaluated for the diagnosis of EI and that it can detect virus in some subclinical infected and vaccinated horses. The results suggest that DFA is a useful adjunct to laboratory tests and may be effective as a screening test in a quarantine station or similar facility where horses are monitored daily.     

2009～2010年泰安市手足口病病原体基因型特征分析

目的检测泰安市手足口病的主要病原体EV71和CoxA16,并进行分子生物学分析,了解泰安市手足口病致病病毒的基因型特征。方法采用Real-time PCR法检测肠道病毒EV71和CoxA16;采用Nested PCR和RT-PCR扩增VP1区片段并测序。用DNASTAR、Bioedit、MEGA4等软件进行序列分析。结果与各基因亚型参考株的VP1序列进行对比,泰安市分离株EV71的基因亚型为C4a型,CoxA16可分为B1a、B1b两个基因亚型。结论泰安市分离株EV71属于我国的优势基因型C4a型。CoxA16可分为B1a、B1b两亚型,表明泰安市存在不同的传播链,对公众健康构成威胁,提示应加强对泰安市手足口病的监测及其流行规律的研究。     

One-step real-time PCR assay for detection and quantification of RNA HCV to monitor patients under treatment in Brazil

The Brazilian Public Health Service provides freely αPEG-IFN to treat patients infected with HCV. The primary goal of HCV therapy is the long-term elimination of HCV from the blood to reduce the risk of HCV associated complications and death. Patient viremia affects the treatment duration and response, thus influencing clinical decisions. We developed a high-throughput method to perform the quantification of RNA hepatitis C virus (HCV) virus load in plasma samples to monitor patients under treatment. The method is based on a duplex detection, in a one-step real-time RT-PCR assay and it has been validated according to the rules established by the official Brazilian regulatory agency (ANVISA). This new method was compared to a commercial kit (Cobas/Taqman HCV Test v2.0 - Roche), showing virus load results with significant correlation between them (p?=?0,012) using commercial and clinical panels. In addition, 611 samples from patients treated with peguilated alfa-interferon (αPEG-IFN) from different regions of Brazil were analyzed. Our one-step real-time RT-PCR assay demonstrated good performance in viral load measurement and in treatment course monitoring, with acceptable sensitivity and specificity values     

Direct and indirect evaluation of lesions obstructing the carotid bifurcation. A comparison of Doppler spectrum analysis with angiography

The diagnostic accuracy of real-time continuous wave (c.w.) Doppler spectrum analysis and of indirect periorbital Doppler examination for detecting carotid bifurcation disease was evaluated. The results of non-invasive studies were compared with contrast arteriograms of patients studied for symptomatic cerebrovascular disease (91%) or for asymptomatic bruits (9%). Periorbital examination was insensitive to less than 75% carotid stenoses and sensitivity to severe stenoses or occlusions was respectively 56% and 85% (mean 73%). Mean sensitivity of real-time C.W. Doppler spectrum was 93% and sensitivity to stenoses of less than 45% diameter reduction was equal to 76%, while specificity in identifying normal carotid arteries was 89% for an accuracy of the method of 91%. Positive and negative predicting values were respectively 89% and 93%. Direct Doppler interrogation of the carotid arteries with real-time C.W. Doppler spectrum analysis was able to distinguish operable stenoses from inoperable occlusions in 87% of the cases.     

Multiplex real-time RT-PCR for detecting chikungunya virus and dengue virus

ObjectiveTo develop diagnostic test for detection chikungunya virus (CHIKV and Dengue virus (DENV) infection.MethodsWe have performed a rapid, accurate laboratory confirmative method to simultaneously detect, quantify and differentiate CHIKV and DENV infection by single-step multiplex real-time RT-PCR.ResultsThe assay's sensitivity was 97.65%, specificity was 92.59% and accuracy was 95.82% when compared to conventional RT-PCR. Additionally, there was no cross-reaction between CHIKV, DENV, Japanese encephalitis virus, hepatitis C, hepatitis A or hepatitis E virus.ConclusionsThis rapid and reliable assay provides a means for simultaneous early diagnosis of CHIKV and DENV in a single-step reaction.