目的建立一种基于叠氮溴化丙锭(Propidium monoazide,PMA)的定量反转录聚合酶链式反应(RT-qPCR)的方法,以快速检测甲型肝炎病毒(Hepatitis A virus,HAV)的感染性。方法将热灭活的HAV样品经PMA处理和曝光,通过RT-qPCR检测体系进行检测,优...目的建立一种基于叠氮溴化丙锭(Propidium monoazide,PMA)的定量反转录聚合酶链式反应(RT-qPCR)的方法,以快速检测甲型肝炎病毒(Hepatitis A virus,HAV)的感染性。方法将热灭活的HAV样品经PMA处理和曝光,通过RT-qPCR检测体系进行检测,优化PMA预处理浓度、孵育时间以及光解时间,建立甲肝病毒PMART-qPCR检测方法。以建立的检测方法对感染性病毒与灭活病毒混合样品、不同浓度的灭活病毒以及感染性病毒进行检测,评估其检测病毒感染性的效果。结果PMA-RT-qPCR检测方法的PMA浓度确定为50μmol/L,4℃避光孵育30min,光解30min。对热灭活HAV的RT-qPCR检测结果显示,PMA处理组与未处理组Ct值差异(ΔCt)>3.78;而对活病毒(10~2CCID_(50)/mL~10~7CCID_(50)/mL)的ΔCt值<0.5。该方法可最低分辨9 CCID_(50)的活病毒,在感染性病毒与灭活病毒混合物中检出最低27 CCID_(50)的活病毒。结论本研究建立的PMA-RT-qPCR无细胞培养方法为判断HAV的感染性提供了快速、便捷的检测方法。展开更多
Introduction: Arbovirus diseases such as dengue and chikungunya threaten public health worldwide. Early and rapid diagnosis and surveillance of dengue virus (DENV) and chikungunya virus (CHIKV) infections are essentia...Introduction: Arbovirus diseases such as dengue and chikungunya threaten public health worldwide. Early and rapid diagnosis and surveillance of dengue virus (DENV) and chikungunya virus (CHIKV) infections are essential to the control of these diseases. In this study, we evaluate the diagnostic performance of our new in-house multiplex RT-qPCR method for detecting DENV serotypes and CHIKV in an external laboratory. Methodology: The evaluation study was conducted on 200 clinical samples of suspected patients for arbovirus disease infection, collected in Centre de Recherche Biomoléculaire Pietro Annigoni (CERBA), Ouagadougou, Burkina Faso. Our new multiplex RT-qPCR was compared to the commercial kit, the Zika, Dengue, and Chikungunya (ZDC) Real-Time PCR Assays kit (Bio-Rad, California, USA). Results and Conclusions: Among 200 samples, 21.5% (43/200) were DENV-positive by multiplex RT-qPCR, and 21.5% (43/200) were also DENV-positive by reference real-time RT-PCR. 157 (78.5%) samples tested negative for DENV by both tests (new mRT-qPCR and reference test). The sensitivity and specificity of mRT-qPCR were 100%. The DENV serotypes detected were DENV-1 60.5% (26/43) and DENV-3 39.5% (17/43). CHIKV was not detected in this study. Our new mRT-qPCR is sensitive, cost-effective, simple, and can be used in developing country laboratories.展开更多
Real time RT-qPCR(real-time quantitative reverse transcription-PCR)是一种快速、简便、准确、灵敏、成本低廉的基因检测技术,被认为是目前检测基因在转录水平表达的金标准。研究发现Real time RT-qPCR的检测结果会受到实验设计、...Real time RT-qPCR(real-time quantitative reverse transcription-PCR)是一种快速、简便、准确、灵敏、成本低廉的基因检测技术,被认为是目前检测基因在转录水平表达的金标准。研究发现Real time RT-qPCR的检测结果会受到实验设计、引物、模板的质量、内参的选择及数据分析的方法等多个因素的影响,规范实验设计及操作是得到可靠结论的前提和必要条件。对近年来国内外涉及Real time RT-qPCR整个实验流程及数据发表的一些规范及标准进行综述,以便规范实验设计及操作,加强实验质量控制,促进研究结果的推广,和更好地发挥其应用价值。展开更多
文摘目的 基于ICH Q2(R2)和Q14,建立冻干甲型肝炎减毒活疫苗(简称冻甲疫苗)感染性滴度细胞培养-RT-qPCR快速检测方法,并对方法进行优化及验证。方法 采用分析质量源于设计(Analytical Quality by Design,AQbD)理念,通过分析目标概况(Acceptance Test Procedure,ATP)设定、风险识别与实验设计(Design of Experiment,DoE),建立细胞培养-RT-qPCR联合检测平台。以病毒核酸扩增循环阈值(cycle threshold,Ct)为响应指标,优化细胞密度、病毒培养温度和时间等关键参数。验证方法的专属性、线性、中间精密度,确定定量范围,Bland-Altman分析与传统滴度检测方法的一致性。结果 共识别13个主要风险因素,确定方法的最适反应条件为:细胞密度(1~2)×10^(5)个/mL;病毒稀释梯度间距4倍;96孔细胞培养板有边缘效应;病毒吸附温度和时间为37℃吸附60 min,病毒培养温度和时间为35℃培养72 h;PCR无位置效应;变性温度和时间为90℃变性63 s;逆转录温度和时间为60℃逆转录15 min;计算模型为双对数线性模型。该方法具有良好的专属性(灭活病毒对照Ct值> 28)、线性(R2=0.966)及中间精密度[几何变异系数(geometric coefficient of variation,GCV)≤9.28%],定量范围覆盖5.0~8.0 lgCCID50/mL。该方法与传统方法检测结果差值的95%一致性界限(95%limits of agree-ment,95%LoA)为-0.06~0.41 lgCCID50/mL。结论 建立的细胞培养-RT-qPCR检测方法将检测周期由传统方法的21~28 d缩短至3 d,且具有定量准确、通量高等优势,为冻甲疫苗工艺过程中及成品甲型肝炎病毒(hepatitis A virus,HAV)滴度检测和批签发效率提升提供了关键技术支持。
文摘Introduction: Arbovirus diseases such as dengue and chikungunya threaten public health worldwide. Early and rapid diagnosis and surveillance of dengue virus (DENV) and chikungunya virus (CHIKV) infections are essential to the control of these diseases. In this study, we evaluate the diagnostic performance of our new in-house multiplex RT-qPCR method for detecting DENV serotypes and CHIKV in an external laboratory. Methodology: The evaluation study was conducted on 200 clinical samples of suspected patients for arbovirus disease infection, collected in Centre de Recherche Biomoléculaire Pietro Annigoni (CERBA), Ouagadougou, Burkina Faso. Our new multiplex RT-qPCR was compared to the commercial kit, the Zika, Dengue, and Chikungunya (ZDC) Real-Time PCR Assays kit (Bio-Rad, California, USA). Results and Conclusions: Among 200 samples, 21.5% (43/200) were DENV-positive by multiplex RT-qPCR, and 21.5% (43/200) were also DENV-positive by reference real-time RT-PCR. 157 (78.5%) samples tested negative for DENV by both tests (new mRT-qPCR and reference test). The sensitivity and specificity of mRT-qPCR were 100%. The DENV serotypes detected were DENV-1 60.5% (26/43) and DENV-3 39.5% (17/43). CHIKV was not detected in this study. Our new mRT-qPCR is sensitive, cost-effective, simple, and can be used in developing country laboratories.
文摘Real time RT-qPCR(real-time quantitative reverse transcription-PCR)是一种快速、简便、准确、灵敏、成本低廉的基因检测技术,被认为是目前检测基因在转录水平表达的金标准。研究发现Real time RT-qPCR的检测结果会受到实验设计、引物、模板的质量、内参的选择及数据分析的方法等多个因素的影响,规范实验设计及操作是得到可靠结论的前提和必要条件。对近年来国内外涉及Real time RT-qPCR整个实验流程及数据发表的一些规范及标准进行综述,以便规范实验设计及操作,加强实验质量控制,促进研究结果的推广,和更好地发挥其应用价值。
