两种标签引物RT-PCR结合Sanger测序检测登革病毒的研究
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摘要
建立两种标签引物RT-PCR结合Sanger测序的方法,检测登革病毒,包括新变异的登革病毒。一种方法可以快速诊断急性期单一血清型感染的登革热,另一种方法可以诊断双重血清型感染的登革热。
3’末端锚定-标签引物RT-PCR(NAT-PCR)结合Sanger测序法是利用登革病毒(“正链”)3’最末端的保守序列作为锚定靶点进行反转录,合成带有3’最末端序列的cDNA(“负链”)(RT步骤),以登革病毒特异性的简并引物合成cDNA的第二条链(SS步骤)。由于cDNA合成引物与简并引物的5’末端均带有人工设计的“标签”序列,所以新合成的第二条链(“正链”)的两端分别带有彼此互补的“标签”序列,再以“标签”序列作为引物进行PCR扩增(AMP步骤)。扩增产物带有的相同3’最末端序列(cDNA合成引物)作为测序引物进行Sanger测序。并利用SYBR Green I real-time PCR进行监测和筛选NAT-PCR产物及其反应参数,优化出最佳的NAT-PCR扩增效率以及最快速的Sanger测序程序。该方法对A型流感病毒、拉沙病毒、西尼罗病毒、乙型脑炎病毒、肾综合征出血热病毒和黄热病病毒检测不到可读序列,具有较好的特异性;对四个血清型登革病毒RNA的检出限为11-31个拷贝/反应(两步NAT-PCR)或110-310个拷贝/反应(一步NAT-PCR),敏感性较高。对25份临床血清样本RNA的检测结果与临床诊断一致,可获得400-520bp的可读序列,其中包括3份登革I型、5份登革II型、3份登革III型病毒阳性样本,其他样本均为阴性结果。一步NAT-PCR比两步NAT-PCR获得检测结果好:阳性检出率较高、可读序列更长。
随机PCR方法也是利用标签引物进行RT-PCR,扩增的步骤与NAT-PCR方法相同,但以随机引物进行反转录和cDNA第二条链的合成。获得的扩增产物进行TA克隆,以单克隆的M13 PCR产物为模板进行Sanger测序。通过优化反应条件,并利用SYBR Green I real-time PCR和Bioanalyzer DNA质量分析仪进行监测和筛选,优化出无偏嗜扩增的随机PCR反应模式,可对双重感染以及因RNA降解而缺失3’末端序列的所有登革病毒进行检测。该方法不仅可以检测登革病毒,还可以检测样本中其他的单链不分节段RNA病毒以及该类型的未知病毒。其测序鉴定登革病毒的敏感性为100个拷贝的RNA/μl血清。检测DENV-1和DENV-2的混合感染样本,Sanger测序鉴定的阳性克隆率分别为21/92和32/96。对25份临床血清样本RNA的阳性检测结果与临床诊断一致,也与NAT-PCR结合Sanger测序的检测结果一致;但在阴性样本中,有1例被鉴定含有丙型肝炎病毒。在所有的阳性样本中,没有发现双重血清型登革病毒感染的样本。
NAT-PCR结合Sanger测序方法,通过锚定3’末端高度保守的序列,以单管反应同时鉴定四个血清型的登革病毒及其新变异毒株。该方法可在5个小时内完成从样本RNA的提取到序列分析的全过程,检测特异性强、敏感性高,具有广泛的临床应用价值。由于在登革热流行非常严重的地方会出现双重感染的情况,为此本研究建立了随机PCR结合TA克隆和Sanger测序的方法可诊断双重血清型登革病毒的混合感染,并具有检测新的病毒变异株以及未知病毒的能力,有助于发现由单股正链不分节段的RNA病毒引起的人或动物传染病的混合感染。
Dengue virus which causes one of the most prevalent arbovirus diseases can be transmitted to humans by Aedes aegypti and Aedes albopictus mosquitoes in the tropical and subtropical regions of the world. There are four serotypes of Dengue virus (DENV-1, DENV-2, DENV-3, DENV-4), all of which cause dengue fever, dengue hemorrhagic fever and dengue shock syndrome. Clinical diagnosis of Dengue virus infections is difficult due to the non-specific symptoms. Therefore, laboratory diagnosis is essential. Serology detections depend on the existence of antibody and are not suitable for rapid diagnosis. Molecular methods, especially for PCR, have been widely used in early diagnosis of dengue fever. However, new sequence variation can inhibit these specific methods. This study developed a rapid assay for detection of Dengue virus including new strains using 3’-endfragment anchored and tag primed amplification (NAT-PCR) in combination with Sanger sequencing. It also described another tag primed amplification of randomPCR followed cloning M13 PCR products and Sanger sequenging to detect the dual infection of dengure.
For the assay integrating a NAT-PCR with a new Fast Sanger sequencing, the conserved 3’genome end was targeted for specific cDNA synthesis. Degenerated primers were used for second strand synthesis. On all primers a common artificial tag was added to the 5’end, facilitating tag primed amplification. Fast Sanger sequencing was started from the anchored 3’genome end. The resulting sequences were analyzed by BLAST。The assay is specific against Influenza A, Lassa and yellow fever virus with no amplification or sequences got from NAT-PCR respectively Sanger sequencing. The specific SYBR Green I QPCR assays for Dengue cultured strains were applied for quantifying the virual RNA and throughout the whole procedure of NAT-PCR optimization. This assay demonstrated a high sensitivity with a detection limit of 11-31 copies viral RNA/reaction for a two-step protocol and 100-500copies for a one-step protocol. All four serotypes of Dengue can be tested in one reaction in similar to multiplex Real-Time PCR with high efficiency. In this study, sixty samples were run in parallel each time, but for higher throughput a 96-well format can be used for most steps. This assay was evaluated with 25 clinical serum samples. The detection results were consistent with clinical diagnosis reports. The one-step RT-PCR gave better results for the samples tested compared with two-step, since more positive rate and longer sequences were obtained. A random PCR was developed to get uniform and nonbiased amplification for non-segment single-stranded RNA. In combination with TA cloning and Sanger sequencing for M13 PCR products, the detection limit was 100 copies viral RNA/reaction. Twenty-one positive colonies for DENV-1 and thirty-two for DENV-2 were identified from 96 colonies in dual infection of Dengue with 100copies for each type. This assay was not specific for Dengue, but used to detect all the non-segment sigle-stranded RNA in the serum samples. It was also evaluated with the same 25 clinical serum samples. The positive results were the same as clinical diagnosis reports, but one negative sample was found to be Hepatitis C virus infection.
Due to the virus strain differences in virulence, in particular for the emergence of new variant strains may determine the rate and severity of dengue infection, the variation of dengue virus was paid much attention. Dengue virus including new strains can be detected with 5 hours using NAT-PCR in combination with Sanger sequencing. For the endemic regions, dual infections of Dengue two serotypes can be resolved and distinguished by the assay of random PCR in combination with TA cloning and Sanger sequencing.This assy also hase a capability for detection of other non-segment single-stranded RNA in serum samples.
3’末端锚定-标签引物RT-PCR(NAT-PCR)结合Sanger测序法是利用登革病毒(“正链”)3’最末端的保守序列作为锚定靶点进行反转录,合成带有3’最末端序列的cDNA(“负链”)(RT步骤),以登革病毒特异性的简并引物合成cDNA的第二条链(SS步骤)。由于cDNA合成引物与简并引物的5’末端均带有人工设计的“标签”序列,所以新合成的第二条链(“正链”)的两端分别带有彼此互补的“标签”序列,再以“标签”序列作为引物进行PCR扩增(AMP步骤)。扩增产物带有的相同3’最末端序列(cDNA合成引物)作为测序引物进行Sanger测序。并利用SYBR Green I real-time PCR进行监测和筛选NAT-PCR产物及其反应参数,优化出最佳的NAT-PCR扩增效率以及最快速的Sanger测序程序。该方法对A型流感病毒、拉沙病毒、西尼罗病毒、乙型脑炎病毒、肾综合征出血热病毒和黄热病病毒检测不到可读序列,具有较好的特异性;对四个血清型登革病毒RNA的检出限为11-31个拷贝/反应(两步NAT-PCR)或110-310个拷贝/反应(一步NAT-PCR),敏感性较高。对25份临床血清样本RNA的检测结果与临床诊断一致,可获得400-520bp的可读序列,其中包括3份登革I型、5份登革II型、3份登革III型病毒阳性样本,其他样本均为阴性结果。一步NAT-PCR比两步NAT-PCR获得检测结果好:阳性检出率较高、可读序列更长。
随机PCR方法也是利用标签引物进行RT-PCR,扩增的步骤与NAT-PCR方法相同,但以随机引物进行反转录和cDNA第二条链的合成。获得的扩增产物进行TA克隆,以单克隆的M13 PCR产物为模板进行Sanger测序。通过优化反应条件,并利用SYBR Green I real-time PCR和Bioanalyzer DNA质量分析仪进行监测和筛选,优化出无偏嗜扩增的随机PCR反应模式,可对双重感染以及因RNA降解而缺失3’末端序列的所有登革病毒进行检测。该方法不仅可以检测登革病毒,还可以检测样本中其他的单链不分节段RNA病毒以及该类型的未知病毒。其测序鉴定登革病毒的敏感性为100个拷贝的RNA/μl血清。检测DENV-1和DENV-2的混合感染样本,Sanger测序鉴定的阳性克隆率分别为21/92和32/96。对25份临床血清样本RNA的阳性检测结果与临床诊断一致,也与NAT-PCR结合Sanger测序的检测结果一致;但在阴性样本中,有1例被鉴定含有丙型肝炎病毒。在所有的阳性样本中,没有发现双重血清型登革病毒感染的样本。
NAT-PCR结合Sanger测序方法,通过锚定3’末端高度保守的序列,以单管反应同时鉴定四个血清型的登革病毒及其新变异毒株。该方法可在5个小时内完成从样本RNA的提取到序列分析的全过程,检测特异性强、敏感性高,具有广泛的临床应用价值。由于在登革热流行非常严重的地方会出现双重感染的情况,为此本研究建立了随机PCR结合TA克隆和Sanger测序的方法可诊断双重血清型登革病毒的混合感染,并具有检测新的病毒变异株以及未知病毒的能力,有助于发现由单股正链不分节段的RNA病毒引起的人或动物传染病的混合感染。
Dengue virus which causes one of the most prevalent arbovirus diseases can be transmitted to humans by Aedes aegypti and Aedes albopictus mosquitoes in the tropical and subtropical regions of the world. There are four serotypes of Dengue virus (DENV-1, DENV-2, DENV-3, DENV-4), all of which cause dengue fever, dengue hemorrhagic fever and dengue shock syndrome. Clinical diagnosis of Dengue virus infections is difficult due to the non-specific symptoms. Therefore, laboratory diagnosis is essential. Serology detections depend on the existence of antibody and are not suitable for rapid diagnosis. Molecular methods, especially for PCR, have been widely used in early diagnosis of dengue fever. However, new sequence variation can inhibit these specific methods. This study developed a rapid assay for detection of Dengue virus including new strains using 3’-endfragment anchored and tag primed amplification (NAT-PCR) in combination with Sanger sequencing. It also described another tag primed amplification of randomPCR followed cloning M13 PCR products and Sanger sequenging to detect the dual infection of dengure.
For the assay integrating a NAT-PCR with a new Fast Sanger sequencing, the conserved 3’genome end was targeted for specific cDNA synthesis. Degenerated primers were used for second strand synthesis. On all primers a common artificial tag was added to the 5’end, facilitating tag primed amplification. Fast Sanger sequencing was started from the anchored 3’genome end. The resulting sequences were analyzed by BLAST。The assay is specific against Influenza A, Lassa and yellow fever virus with no amplification or sequences got from NAT-PCR respectively Sanger sequencing. The specific SYBR Green I QPCR assays for Dengue cultured strains were applied for quantifying the virual RNA and throughout the whole procedure of NAT-PCR optimization. This assay demonstrated a high sensitivity with a detection limit of 11-31 copies viral RNA/reaction for a two-step protocol and 100-500copies for a one-step protocol. All four serotypes of Dengue can be tested in one reaction in similar to multiplex Real-Time PCR with high efficiency. In this study, sixty samples were run in parallel each time, but for higher throughput a 96-well format can be used for most steps. This assay was evaluated with 25 clinical serum samples. The detection results were consistent with clinical diagnosis reports. The one-step RT-PCR gave better results for the samples tested compared with two-step, since more positive rate and longer sequences were obtained. A random PCR was developed to get uniform and nonbiased amplification for non-segment single-stranded RNA. In combination with TA cloning and Sanger sequencing for M13 PCR products, the detection limit was 100 copies viral RNA/reaction. Twenty-one positive colonies for DENV-1 and thirty-two for DENV-2 were identified from 96 colonies in dual infection of Dengue with 100copies for each type. This assay was not specific for Dengue, but used to detect all the non-segment sigle-stranded RNA in the serum samples. It was also evaluated with the same 25 clinical serum samples. The positive results were the same as clinical diagnosis reports, but one negative sample was found to be Hepatitis C virus infection.
Due to the virus strain differences in virulence, in particular for the emergence of new variant strains may determine the rate and severity of dengue infection, the variation of dengue virus was paid much attention. Dengue virus including new strains can be detected with 5 hours using NAT-PCR in combination with Sanger sequencing. For the endemic regions, dual infections of Dengue two serotypes can be resolved and distinguished by the assay of random PCR in combination with TA cloning and Sanger sequencing.This assy also hase a capability for detection of other non-segment single-stranded RNA in serum samples.
引文
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