两种标签引物RT-PCR结合Sanger测序检测登革病毒的研究
发布时间：2018-08-24 19:20
【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。
[Abstract]:To establish two methods of RT-PCR and Sanger sequencing with labeled primers for detection of dengue virus, including newly mutated dengue virus. One method can rapidly diagnose acute single serotype infection of dengue fever, the other can diagnose double serotype infection of dengue fever.
3'-terminal anchoring-tagging primer RT-PCR (NAT-PCR) combined with Sanger sequencing is a method of reverse transcription using the conserved sequence of the 3'-terminal end of dengue virus ("positive chain") as anchoring target to synthesize a 3'-terminal sequence of cDNA ("negative chain") (RT step), and to synthesize a second DNA strand (SS step) with a dengue virus-specific degenerate primer. Because the 5'end of the synthetic primer and degenerate primer of the cDNA contain the designed "tag" sequence, the two ends of the newly synthesized second strand ("positive strand") have complementary "tag" sequence respectively, and then the "tag" sequence is used as the primer for PCR amplification (AMP step). The same 3'end sequence of the amplified product is carried out. Sequencing of influenza A virus, Lassa virus, West Nile virus and encephalitis B virus was carried out by using SYBR Green I real-time PCR to monitor and screen NAT-PCR products and their reaction parameters. Hemorrhagic fever with renal syndrome virus and yellow fever virus can not detect the readable sequence, with good specificity; the detection limit of four serotypes of dengue virus RNA is 11-31 copies / reactions (two-step NAT-PCR) or 110-310 copies / reactions (one-step NAT-PCR), with high sensitivity. The detection results of RNA in 25 clinical serum samples and clinical diagnosis 1. As a result, 400-520 BP readable sequences were obtained, including 3 dengue type I, 5 dengue type II, 3 dengue type III virus positive samples, and all the other samples were negative.
Random PCR was also used for RT-PCR with labeled primers. The amplification procedure was the same as that of NAT-PCR, but the reverse transcription and the synthesis of the second strand of the cDNA were carried out with random primers. The amplified product was cloned by TA and sequenced by Sanger using the template of the monoclonal M13 PCR product. The e-PCR and Bioanalyzer DNA quality analyzer were used to monitor and screen the dengue virus and optimize the random PCR reaction mode of unbiased amplification. The method can detect all dengue viruses with double infection and deletion of 3'terminal sequence due to RNA degradation. The positive cloning rates of DENV-1 and DENV-2 were 21/92 and 32/96 respectively. The positive detection results of RNA in 25 clinical serum samples were consistent with clinical diagnosis and NAT-PCR. The results were consistent with Sanger sequencing, but one of the negative samples was identified as having hepatitis C virus. No double serotype dengue virus infection was found in all positive samples.
Four serotypes of dengue virus and its new variant strains were simultaneously identified by single-tube reaction by anchoring highly conserved 3'-terminal sequences with NAT-PCR combined with Sanger sequencing method. The method can complete the whole process from RNA extraction to sequence analysis within 5 hours. The method has strong specificity and high sensitivity, and has wide clinical application. Value. Because double infection occurs in places where dengue fever is very serious, a random PCR combined with TA cloning and Sarger sequencing method was established to diagnose mixed infection of double serotype dengue virus. It has the ability to detect new virus variants and unknown viruses, and is helpful to detect positive single strand dengue virus. A mixed infection of human or animal infectious diseases caused by RNA virus.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R373
本文编号：2201783
[Abstract]:To establish two methods of RT-PCR and Sanger sequencing with labeled primers for detection of dengue virus, including newly mutated dengue virus. One method can rapidly diagnose acute single serotype infection of dengue fever, the other can diagnose double serotype infection of dengue fever.
3'-terminal anchoring-tagging primer RT-PCR (NAT-PCR) combined with Sanger sequencing is a method of reverse transcription using the conserved sequence of the 3'-terminal end of dengue virus ("positive chain") as anchoring target to synthesize a 3'-terminal sequence of cDNA ("negative chain") (RT step), and to synthesize a second DNA strand (SS step) with a dengue virus-specific degenerate primer. Because the 5'end of the synthetic primer and degenerate primer of the cDNA contain the designed "tag" sequence, the two ends of the newly synthesized second strand ("positive strand") have complementary "tag" sequence respectively, and then the "tag" sequence is used as the primer for PCR amplification (AMP step). The same 3'end sequence of the amplified product is carried out. Sequencing of influenza A virus, Lassa virus, West Nile virus and encephalitis B virus was carried out by using SYBR Green I real-time PCR to monitor and screen NAT-PCR products and their reaction parameters. Hemorrhagic fever with renal syndrome virus and yellow fever virus can not detect the readable sequence, with good specificity; the detection limit of four serotypes of dengue virus RNA is 11-31 copies / reactions (two-step NAT-PCR) or 110-310 copies / reactions (one-step NAT-PCR), with high sensitivity. The detection results of RNA in 25 clinical serum samples and clinical diagnosis 1. As a result, 400-520 BP readable sequences were obtained, including 3 dengue type I, 5 dengue type II, 3 dengue type III virus positive samples, and all the other samples were negative.
Random PCR was also used for RT-PCR with labeled primers. The amplification procedure was the same as that of NAT-PCR, but the reverse transcription and the synthesis of the second strand of the cDNA were carried out with random primers. The amplified product was cloned by TA and sequenced by Sanger using the template of the monoclonal M13 PCR product. The e-PCR and Bioanalyzer DNA quality analyzer were used to monitor and screen the dengue virus and optimize the random PCR reaction mode of unbiased amplification. The method can detect all dengue viruses with double infection and deletion of 3'terminal sequence due to RNA degradation. The positive cloning rates of DENV-1 and DENV-2 were 21/92 and 32/96 respectively. The positive detection results of RNA in 25 clinical serum samples were consistent with clinical diagnosis and NAT-PCR. The results were consistent with Sanger sequencing, but one of the negative samples was identified as having hepatitis C virus. No double serotype dengue virus infection was found in all positive samples.
Four serotypes of dengue virus and its new variant strains were simultaneously identified by single-tube reaction by anchoring highly conserved 3'-terminal sequences with NAT-PCR combined with Sanger sequencing method. The method can complete the whole process from RNA extraction to sequence analysis within 5 hours. The method has strong specificity and high sensitivity, and has wide clinical application. Value. Because double infection occurs in places where dengue fever is very serious, a random PCR combined with TA cloning and Sarger sequencing method was established to diagnose mixed infection of double serotype dengue virus. It has the ability to detect new virus variants and unknown viruses, and is helpful to detect positive single strand dengue virus. A mixed infection of human or animal infectious diseases caused by RNA virus.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R373
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1 胡哲;两种标签引物RT-PCR结合Sanger测序检测登革病毒的研究[D];吉林大学;2009年
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