大鼠冠状病毒和仙台病毒的双重PCR检测方法的建立与应用
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摘要
目的建立快速检测实验大鼠冠状病毒和仙台病毒的双重PCR方法。方法根据大鼠冠状病毒N基因、仙台病毒L基因设计特异性引物;经过双重PCR优化,特异性和敏感性的检测,建立双重PCR体系。应用该PCR体系检测人工感染仙台病毒组织DNA样本和实验动物组织样本,并与ELISA方法比对。结果双重PCR扩增出大鼠冠状病毒(168 bp)和仙台病毒(262 bp)目的条带,PCR扩增产物测序结果利用核酸BLAST功能进行同源序列对比,仙台病毒和大鼠冠状病毒同源性分别为100%和99%。仙台病毒和大鼠冠状病毒的检测下限为1.56×10~2 copies/μL。特异性检测对小鼠肝炎病毒扩增,产生片段大小近似大鼠冠状病毒产物。应用建立的双重PCR体系检测人工感染仙台病毒组织DNA样本,30份DNA标本均被检出;检测94份实验动物肺组织样本,结果均阴性。结论建立的双重PCR方法操作简单、快速、特异性强、灵敏度高,能够实现对实验动物仙台病毒和大鼠冠状病毒病原体的快速检测。
Objective To establish a rapid duplex polymerase chain reaction(PCR) assay for simultaneous detection of Rat coronavirus and Sendai virus. Methods Specific primers were designed based on the N gene of Rat coronavirus and L gene of Sendai virus. A duplex PCR system was established by optimizing the concentration of primers and annealing temperature, and testing the specificity and sensitivity of this system. This was followed by screening DNA samples of artificial infections and tissues of experimental animals through our PCR system and comparing it with the ELISA method. Results Duplex PCR amplification of Rat coronavirus(168 bp) and Sendai virus(262 bp) was done. The results of sequencing of the PCR amplification products were compared with homologous sequences using BLAST functions. The homology of the Sendai virus and Rat coronavirus was 100% and 99%, respectively. The lower limit of detection for Rat coronavirus and Sendai virus was 1.56×10~2 copies/μL. Specific detection of mouse hepatitis virus amplification resulted in a fragment size that similar to the Rat coronavirus product. The established duplex PCR system was used to detect the DNA samples of artificially infected Sendai virus, and 30 positive DNA samples were detected. At the same time, in order to verify the applicability of the system, 94 experimental animal lung tissue samples were detected and the results were negative. Conclusions A rapid PCR assay for simultaneous detection of Rat coronavirus and Sendai virus is successfully established. This duplex PCR assay is specific, sensitive, simple and rapid, and can be used for rapid detection of Sendai virus and Rat coronavirus simultaneously in laboratory animals.
Objective To establish a rapid duplex polymerase chain reaction(PCR) assay for simultaneous detection of Rat coronavirus and Sendai virus. Methods Specific primers were designed based on the N gene of Rat coronavirus and L gene of Sendai virus. A duplex PCR system was established by optimizing the concentration of primers and annealing temperature, and testing the specificity and sensitivity of this system. This was followed by screening DNA samples of artificial infections and tissues of experimental animals through our PCR system and comparing it with the ELISA method. Results Duplex PCR amplification of Rat coronavirus(168 bp) and Sendai virus(262 bp) was done. The results of sequencing of the PCR amplification products were compared with homologous sequences using BLAST functions. The homology of the Sendai virus and Rat coronavirus was 100% and 99%, respectively. The lower limit of detection for Rat coronavirus and Sendai virus was 1.56×10~2 copies/μL. Specific detection of mouse hepatitis virus amplification resulted in a fragment size that similar to the Rat coronavirus product. The established duplex PCR system was used to detect the DNA samples of artificially infected Sendai virus, and 30 positive DNA samples were detected. At the same time, in order to verify the applicability of the system, 94 experimental animal lung tissue samples were detected and the results were negative. Conclusions A rapid PCR assay for simultaneous detection of Rat coronavirus and Sendai virus is successfully established. This duplex PCR assay is specific, sensitive, simple and rapid, and can be used for rapid detection of Sendai virus and Rat coronavirus simultaneously in laboratory animals.
引文
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[12] 李玉恒,赵明慧,赵紫阳,等.利用梯度PCR和降落PCR扩增CIRP基因的比较[J].黑龙江八一农垦大学学报,2011,23(05):46-49+75.Li YH,Zhao MH,Zhao ZY,et al.Comparison of the amplification of CIRP genes with touch down PCR and gradient PCR [J].J Heilongjiang Bayi Agr Univ,2011,23 (05):46-49+75.
[2] 李晓波,付瑞,王吉,等.仙台病毒RT-PCR检测方法的建立及灰仓鼠中流行情况调查[J].中国比较医学杂志,2012,22(12):18-22.Li XB,Fu R,Wang J,et al.Establishment of a RT-PCR detection method for Sendai virus and investigation of its infection status in Cricetulus migratorius[J].Chin J Comp Med,2012,22(12):18-22.
[3] 侯丽波.SV,Reo-3,MHV,MAd,RCV病毒免疫血清制备及ELISA等检测方法的研究[D].中国协和医科大学,2009.Hou LB.Study of the standard serological methods in detecting virus for SV,Reo-3,MHV,MAd,RCV[D].Peking Union Medical College,2009.
[4] 王文.啮齿动物中冠状病毒的分子流行病学研究及登革病毒在非疫区的再现[D].中国疾病预防控制中心,2017.Wang W.Molecular epidemiology of coronavirus sampled from rodents and re-emergence of dengue in non-endemic regions[D].Chinese Center for Disease Control and Prevention,2017
[5] 刘忠华,黄韧,魏社林,等.应用RT-PCR技术检测啮齿类动物冠状病毒[J].上海实验动物科学,2004(04):218-220.Liu ZH,Huang R,Wei SL,et al.Detection of rodent coronavirus by using reverse transcriptase polymerase chain reaction[J].Lab Anim Comp Med,2004(04):218-220.
[6] 王宗耀,谢建云,邵伟娟,等.仙台病毒RT-PCR检测方法的建立与应用[J].中国比较医学杂志,2008,18(01):49-53.Wang ZY,Xie JY,Shao WJ,et al.Establishment and application of reverse transcriptase-ploymerase chain reaction for Sendai virus[J].Chin J Comp Med,2008,18(01):49-53.
[7] 袁文,刘忠华,张钰,等.小鼠肝炎病毒逆转录环介导等温扩增检测技术的建立[J].中国实验动物学报,2009,17(05):354-359+409-410.Yuan W,Liu ZH,Zhang Y,et al.Development of a reverse transcription loop-mediated isothermal amplification (RT-LAMP) technique for detection of mouse hepatitis virus[J].Acta Lab Anim Sci Sin,2009,17(05):354-359+409-410.
[8] Bootz F,Sieber I,Popovic D,et al.Comparison of the sensitivity of in vivo antibody production tests with in vitro PCR-based methods to detect infectious contamination of biological materials[J].Lab Anim,2003,37(4):341-351.
[9] 王宗耀.仙台病毒F基因的表达与间接ELISA检测方法的建立和应用[D].扬州大学,2007.Wang ZY.Expression of SeV-F in E.coli and development and application of an indirect ELISA for detection of Anti-SeV antibodies[D].Yangzhou University,2007.
[10] Hayase Y,Tobita K,Kii M,et al.Detection of nucleoprotein gene of Sendai virus in the lungs of rats by touchdown nested reverse transcription polymerase chain reaction[J].Exp Anim,1997,46(4):307-310.
[11] 李庆超.多重PCR技术同时高效检测食源性致病菌的研究[D].吉林大学,2017.Li QC.Study of multiplex PCR technology in the simultaneously and efficiently detection of food-borne pathogenic bacteria[D].Jilin University,2017.
[12] 李玉恒,赵明慧,赵紫阳,等.利用梯度PCR和降落PCR扩增CIRP基因的比较[J].黑龙江八一农垦大学学报,2011,23(05):46-49+75.Li YH,Zhao MH,Zhao ZY,et al.Comparison of the amplification of CIRP genes with touch down PCR and gradient PCR [J].J Heilongjiang Bayi Agr Univ,2011,23 (05):46-49+75.