本文選題：大鼠微小病毒 + 大鼠細小病毒�。� 參考：《揚州大學(xué)》2017年碩士論文

【摘要】：大鼠和小鼠是生物醫(yī)學(xué)科研實驗中用途最廣和使用量最大的實驗動物,實驗動物的質(zhì)量決定了科研結(jié)果的準確性和可靠性。目前,我國國家標準規(guī)定的SPF級實驗大鼠和小鼠必須或必要檢測的病毒項目是15種,除此之外,依然有不少病毒危害著實驗鼠的健康。本研究選取5種暫未列入我國SPF級實驗動物檢測標準的大鼠和小鼠病毒,分別為大鼠細小病毒(Ratparvovirus,RPV)、大鼠微小病毒(Ratminute virus,RMV)、鼠腦心肌炎病毒(Encephalomyocarditis virus,EMV)、小鼠輪狀病毒(Murine Rotavirus,MRV)和大鼠疑似泰勒氏病毒(Theiler's-likevirusofRats,TLV),以這些病原為研究對象,分別建立快速、準確的診斷方法,為實驗動物的健康監(jiān)護提供技術(shù)支撐。1.RMV和RPV雙重TaqMan熒光定量PCR檢測方法的建立和應(yīng)用以實驗大鼠中常見的兩種細小病毒RMV和RPV為研究對象,根據(jù)GenBank中已公布的RMV的NTU1株全基因組序列(GenBak登錄號:JX627317.1),在3389～3589bp處設(shè)計引物和TaqMan探針。以及RPV的NTU1毒株全基因組序列(GenBank登錄號:JX827169),在863-967 bp處設(shè)計引物和Taqaan探針。以構(gòu)建的質(zhì)粒標準品為模板建立熒光定量PCR檢測方法,并對該鑒別檢測方法的靈敏度、穩(wěn)定性和特異性進行評價;用建立的熒光定量方法對50份臨床樣品進行檢測,并用ELISA的商品試劑盒進行驗證,驗證該方法的可靠性。結(jié)果顯示,建立的RMV和RPV的雙重?zé)晒舛縋CR方法標準曲線的線性關(guān)系良好,R2值可達0.99,最低檢測限可以達到30拷貝/μL,使用建立的熒光定量PCR方法和血清學(xué)ELISA檢測試劑盒,同時對50只大鼠的肝臟樣本和血清樣品進行對應(yīng)的檢測,檢測準確性為100%。因此,建立的RMV和RPV雙重TaqMan實時熒光定量PCR方法具有特異、靈敏的特點,適用于實驗大鼠臨床的監(jiān)測。2.EMV的TaqMan探針熒光定量RT-PCR檢測方法建立根據(jù)EMV的PEC9毒株全基因組序列(GenBank登錄號:DQ288856.1),設(shè)計一對特異性引物和TaqMan探針,建立EMV的TaqMan探針熒光定量RT-PCR方法,通過條件優(yōu)化,對其特異性,敏感性,穩(wěn)定性進行分析,結(jié)果顯示,建立的熒光定量PCR標準曲線的線性關(guān)系良好,R2值可達0.99;敏感性最低檢測限達到1拷貝/μL,高于普通PCR方法1000倍:其特異性強,常見的小鼠病毒株均無特異性擴增;重復(fù)性好,批內(nèi)和批間變異系數(shù)均小于2%。利用該方法對上海市120份小鼠腦組織樣品進行檢測,未檢測到陽性感染鼠,本研究為EMV病毒感染的分子檢測、制定國家標準提供良好的方法。3.MRV的TaqMan探針熒光定量RT-PCR檢測方法建立根據(jù)MRV的EB-C8/G16P毒株VP1基因(Genbank登錄號:KJ477127.1),設(shè)計引物和Taqman探針,構(gòu)建擴增曲線,制作標準曲線,建立MRV熒光定量RT-PCR方法,并檢測其特異性、敏感性和穩(wěn)定性,從而初步建立檢測方法并應(yīng)用于動物房小鼠的檢測。結(jié)果顯示:建立的MRV熒光定量RT-PCR方法標準曲線的線性關(guān)系良好,R2值可達0.99;最低檢測到10拷貝/μL,是常規(guī)PCR的100倍;與常見的小鼠病毒株均無特異性擴增;批內(nèi)和批間變異系數(shù)均小于2%,表明該方法重復(fù)性好。所建立的MRV熒光定量RT-PCR方法具有特異、靈敏、準確的特點。對上海市120份小鼠腸道樣品的檢測的結(jié)果,為MRV的流行病學(xué)調(diào)查、及國家/地方標準的制定提供了可靠的借鑒。4.TLV的熒光定量RT-PCR檢測方法的建立為了建立一種能在臨床上快速、準確地檢測TLV的方法,我們運用熒光定量聚合酶鏈式反應(yīng)(qRT-PCR)的技術(shù),特異性地針對TLV病毒核酸進行檢測。根據(jù)GenBank中TLV代表毒株NGS910的全基因組序列(GenBank登錄號:AB090161.1),通過基因合成序列作為質(zhì)粒標準品的模板,同時根據(jù)3622～3729 bp的特異性序列,設(shè)計引物和TaqMan性探針,優(yōu)化反應(yīng)體系及條件,進行qRT-PCR擴增,從而建立起了 TLV的TaqMan探針qRT-PCR方法,并對其靈敏度、穩(wěn)定性和特異性進行評價。結(jié)果顯示,建立的TLV的qRT-PCR檢測方法,標準曲線線性關(guān)系良好,R2值可達到0.99,靈敏度可達30個拷貝/μL,靈敏度是普通PCR的100倍;且該qRT-PCR方法特異性強,重復(fù)性好,批內(nèi)和批間變異系數(shù)均小于1%。因此,利用TaqMan探針法的優(yōu)勢所建立的TLV檢測方法,具有操作簡便、靈敏度高、特異性好等特點。本研究方法為TLV國家或地方標準的制定,提供了可靠的數(shù)據(jù)。本研究運用了熒光定量PCR技術(shù)在病毒檢測學(xué)上的優(yōu)勢,對上述五種未列入國標的大、小鼠病毒,進行了檢測方法的建立。同時結(jié)合血清學(xué)檢測和普通PCR檢測,校驗本研究方法的可靠性和靈敏性。最終分別建立起了五種病毒的可靠熒光定量PCR檢測方法。
[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.
【學(xué)位授予單位】：揚州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S858.91

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