五种大鼠和小鼠重要病毒荧光定量PCR检测方法的建立

发布时间：2018-06-15 19:49

本文选题：大鼠微小病毒 + 大鼠细小病毒　；参考：《扬州大学》2017年硕士论文

【摘要】：大鼠和小鼠是生物医学科研实验中用途最广和使用量最大的实验动物,实验动物的质量决定了科研结果的准确性和可靠性。目前,我国国家标准规定的SPF级实验大鼠和小鼠必须或必要检测的病毒项目是15种,除此之外,依然有不少病毒危害着实验鼠的健康。本研究选取5种暂未列入我国SPF级实验动物检测标准的大鼠和小鼠病毒,分别为大鼠细小病毒(Ratparvovirus,RPV)、大鼠微小病毒(Ratminute virus,RMV)、鼠脑心肌炎病毒(Encephalomyocarditis virus,EMV)、小鼠轮状病毒(Murine Rotavirus,MRV)和大鼠疑似泰勒氏病毒(Theiler's-likevirusofRats,TLV),以这些病原为研究对象,分别建立快速、准确的诊断方法,为实验动物的健康监护提供技术支撑。1.RMV和RPV双重TaqMan荧光定量PCR检测方法的建立和应用以实验大鼠中常见的两种细小病毒RMV和RPV为研究对象,根据GenBank中已公布的RMV的NTU1株全基因组序列(GenBak登录号:JX627317.1),在3389～3589bp处设计引物和TaqMan探针。以及RPV的NTU1毒株全基因组序列(GenBank登录号:JX827169),在863-967 bp处设计引物和Taqaan探针。以构建的质粒标准品为模板建立荧光定量PCR检测方法,并对该鉴别检测方法的灵敏度、稳定性和特异性进行评价;用建立的荧光定量方法对50份临床样品进行检测,并用ELISA的商品试剂盒进行验证,验证该方法的可靠性。结果显示,建立的RMV和RPV的双重荧光定量PCR方法标准曲线的线性关系良好,R2值可达0.99,最低检测限可以达到30拷贝/μL,使用建立的荧光定量PCR方法和血清学ELISA检测试剂盒,同时对50只大鼠的肝脏样本和血清样品进行对应的检测,检测准确性为100%。因此,建立的RMV和RPV双重TaqMan实时荧光定量PCR方法具有特异、灵敏的特点,适用于实验大鼠临床的监测。2.EMV的TaqMan探针荧光定量RT-PCR检测方法建立根据EMV的PEC9毒株全基因组序列(GenBank登录号:DQ288856.1),设计一对特异性引物和TaqMan探针,建立EMV的TaqMan探针荧光定量RT-PCR方法,通过条件优化,对其特异性,敏感性,稳定性进行分析,结果显示,建立的荧光定量PCR标准曲线的线性关系良好,R2值可达0.99;敏感性最低检测限达到1拷贝/μL,高于普通PCR方法1000倍:其特异性强,常见的小鼠病毒株均无特异性扩增;重复性好,批内和批间变异系数均小于2%。利用该方法对上海市120份小鼠脑组织样品进行检测,未检测到阳性感染鼠,本研究为EMV病毒感染的分子检测、制定国家标准提供良好的方法。3.MRV的TaqMan探针荧光定量RT-PCR检测方法建立根据MRV的EB-C8/G16P毒株VP1基因(Genbank登录号:KJ477127.1),设计引物和Taqman探针,构建扩增曲线,制作标准曲线,建立MRV荧光定量RT-PCR方法,并检测其特异性、敏感性和稳定性,从而初步建立检测方法并应用于动物房小鼠的检测。结果显示:建立的MRV荧光定量RT-PCR方法标准曲线的线性关系良好,R2值可达0.99;最低检测到10拷贝/μL,是常规PCR的100倍;与常见的小鼠病毒株均无特异性扩增;批内和批间变异系数均小于2%,表明该方法重复性好。所建立的MRV荧光定量RT-PCR方法具有特异、灵敏、准确的特点。对上海市120份小鼠肠道样品的检测的结果,为MRV的流行病学调查、及国家/地方标准的制定提供了可靠的借鉴。4.TLV的荧光定量RT-PCR检测方法的建立为了建立一种能在临床上快速、准确地检测TLV的方法,我们运用荧光定量聚合酶链式反应(qRT-PCR)的技术,特异性地针对TLV病毒核酸进行检测。根据GenBank中TLV代表毒株NGS910的全基因组序列(GenBank登录号:AB090161.1),通过基因合成序列作为质粒标准品的模板,同时根据3622～3729 bp的特异性序列,设计引物和TaqMan性探针,优化反应体系及条件,进行qRT-PCR扩增,从而建立起了 TLV的TaqMan探针qRT-PCR方法,并对其灵敏度、稳定性和特异性进行评价。结果显示,建立的TLV的qRT-PCR检测方法,标准曲线线性关系良好,R2值可达到0.99,灵敏度可达30个拷贝/μL,灵敏度是普通PCR的100倍;且该qRT-PCR方法特异性强,重复性好,批内和批间变异系数均小于1%。因此,利用TaqMan探针法的优势所建立的TLV检测方法,具有操作简便、灵敏度高、特异性好等特点。本研究方法为TLV国家或地方标准的制定,提供了可靠的数据。本研究运用了荧光定量PCR技术在病毒检测学上的优势,对上述五种未列入国标的大、小鼠病毒,进行了检测方法的建立。同时结合血清学检测和普通PCR检测,校验本研究方法的可靠性和灵敏性。最终分别建立起了五种病毒的可靠荧光定量PCR检测方法。
[Abstract]:Rats and mice are the most widely used and most used experimental animals in the biomedical research experiment. The quality of the experimental animals determines the accuracy and reliability of the scientific research results. At present, 15 kinds of virus items must be or must be detected in the SPF level experimental rats and mice stipulated by the national standard of our country. In addition, there are still a lot of viruses. In this study, 5 kinds of rat and mouse virus, which were not included in the test standard of SPF experimental animals in China, were selected as rat parvovirus (Ratparvovirus, RPV), rat parvovirus (Ratminute virus, RMV), rat brain myocarditis virus (Encephalomyocarditis virus, EMV), mouse rotavirus (Murine Rotavirus, MR). V) and rats suspected to be Theiler's-likevirusofRats (TLV), taking these pathogens as research objects, establishing rapid and accurate diagnostic methods respectively, providing technical support for the health monitoring of experimental animals, the establishment and application of.1.RMV and RPV double TaqMan fluorescence quantitative PCR detection methods for the two common parvovirus R in experimental rats. MV and RPV were used to design primers and TaqMan probes at 3389 to 3589bp, according to the whole genome sequence of NTU1 strain of RMV (GenBak login number: JX627317.1) published in GenBank, and the whole genome sequence of NTU1 virulent strain of RPV (GenBank login number). The primers and probes were designed at 863-967. The fluorescence quantitative PCR detection method was established for the template, and the sensitivity, stability and specificity of the differential detection method were evaluated. 50 clinical samples were detected by the established fluorescence quantitative method, and the reliability of the method was verified by the commercial kit of ELISA. The results showed that the dual fluorescence of RMV and RPV was established. The linear relationship of the standard curve of the PCR method is good, the R2 value can reach 0.99, the minimum detection limit can reach 30 copies / L. The established fluorescence quantitative PCR method and the serological ELISA detection kit are used, and the liver and serum samples of 50 rats are detected at the same time, the detection accuracy is 100%., and the established RMV and RPV are double Ta. The qMan real-time fluorescence quantitative PCR method has specific and sensitive characteristics. It is suitable for the clinical monitoring of experimental rats by.2.EMV TaqMan probe fluorescence quantitative RT-PCR detection method to establish a pair of specific primers and TaqMan probes based on the whole genome sequence of the PEC9 strain of EMV (GenBank login number: DQ288856.1), and to establish a EMV TaqMan probe fluorescence determination. RT-PCR method was used to analyze its specificity, sensitivity and stability by optimization of conditions. The results showed that the linear relationship between the established fluorescence quantitative PCR standard curve was good, the R2 value could reach 0.99, the minimum detection limit of sensitivity reached 1 copies / L, which was 1000 times higher than the ordinary PCR method: its specificity was strong and the common mouse virus strains had no specific expansion. Increase, good reproducibility, the coefficient of variation in both batch and batch is less than 2%., and 120 samples of mouse brain tissue in Shanghai are detected by this method. The positive infected mice are not detected. This study is the molecular detection of EMV virus infection, and the national standard provides a good method of.3.MRV TaqMan probe fluorescence quantitative RT-PCR detection method based on MRV The EB-C8/G16P strain VP1 gene (Genbank login number: KJ477127.1), designed primers and Taqman probes, constructed the amplification curve, made the standard curve, established the MRV fluorescence quantitative RT-PCR method, and detected its specificity, sensitivity and stability. The detection method was initially established and applied to the detection of mice in the animal room. The results showed that the established MRV fluorine was established. The linear relationship of the standard curve of the light quantitative RT-PCR method is good, the R2 value can reach 0.99, the lowest detection to 10 copies / L, is 100 times of the conventional PCR; it has no specific amplification with the common mouse virus strain, and the coefficient of variation in both batch and batch is less than 2%, indicating that the method is repeatable. The MRV fluorescence quantitative RT-PCR method built is specific, sensitive and accurate. The results of the detection of 120 mice intestinal samples in Shanghai, the epidemiological investigation of MRV, and the establishment of national / local standards for the establishment of a reliable.4.TLV fluorescence quantitative RT-PCR detection method to establish a rapid and accurate method for detecting TLV in the clinic, we use fluorescence quantitative polymerization. The technique of enzyme chain reaction (qRT-PCR) is specific for detection of TLV virus nucleic acid. According to the whole genome sequence of NGS910 in GenBank (GenBank login number: AB090161.1), TLV is used as the template of the plasmid standard, and the primers and TaqMan probes are designed according to the specific sequence of 3622~3729 BP. The reaction system and conditions are optimized and qRT-PCR amplification is carried out. The method of TLV TaqMan probe qRT-PCR is established and its sensitivity, stability and specificity are evaluated. The results show that the established TLV qRT-PCR detection method has a good linear relationship, the R2 value can reach 0.99, the sensitivity can reach 30 copies / mu L, and the sensitivity is common. 100 times of PCR, and the qRT-PCR method has a strong specificity, good reproducibility, the coefficient of variation in both batch and batch is less than 1%., so the TLV detection method established by the advantage of TaqMan probe has the characteristics of simple operation, high sensitivity and good specificity. This research method provides reliable data for the establishment of home or local standards of TLV countries. Using the advantages of the fluorescence quantitative PCR technology in the detection of the virus, we have established the detection methods for the five kinds of mice that are not included in the national standard. At the same time, the reliability and sensitivity of the method are checked by serological detection and common PCR detection. Finally, the reliable fluorescence quantitative PCR of five kinds of viruses has been established. Detection method.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.91

【参考文献】