嗜肠型小鼠肝炎病毒RT-PCR诊断方法的建立及其应用
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摘要
目的建立小鼠肝炎病毒(MHV)RT-PCR检测方法,为小鼠的日常生产提供一种快速的监测手段。方法提取小鼠结肠组织的总RNA,通过反转录反应合成第一链c DNA,以其为模板,利用MHV的4对特异性引物,进行PCR扩增。对PCR反应条件(包括引物、引物浓度、退火温度、模版浓度等)进行优化。对PCR扩增出的基因片段进行克隆测序,进一步确定扩增结果的特异性。结果其中2对MHV引物具有良好的扩增性和特异性,利用这两对引物进行的PCR检测结果与ELISA检测结果相符。结论 MHV的RT-PCR检测方法具有良好的特异性,并且操作相对简单,可以作为小鼠生产过程中肝炎病毒发生的一种快速的监控(测)方法。
Objective To establish a method for RT-PCR detection of mouse hepatitis virus(MHV)and provide a rapid method for monitoring the daily production process of mice. Methods The total RNA form mouse colon tissue was extracted. The first strand c DNA was synthesized by reverse transcription reaction. Then using the c DNA as the template, a DNA fragment of MHV was amplified by PCR with four pairs of specific primers. The PCR reaction conditions(including primers, primer concentration,annealing temperature, template concentration, etc.) were optimized. Results The two pairs of MHV primers were effective and specific. The PCR results of these two pairs of primers were consistent with the results of ELISA. Conclusion The RT-PCR detection method of MHV was specific and relatively simple. It can be used as a rapid method for MHV detection in the production process of mice.
Objective To establish a method for RT-PCR detection of mouse hepatitis virus(MHV)and provide a rapid method for monitoring the daily production process of mice. Methods The total RNA form mouse colon tissue was extracted. The first strand c DNA was synthesized by reverse transcription reaction. Then using the c DNA as the template, a DNA fragment of MHV was amplified by PCR with four pairs of specific primers. The PCR reaction conditions(including primers, primer concentration,annealing temperature, template concentration, etc.) were optimized. Results The two pairs of MHV primers were effective and specific. The PCR results of these two pairs of primers were consistent with the results of ELISA. Conclusion The RT-PCR detection method of MHV was specific and relatively simple. It can be used as a rapid method for MHV detection in the production process of mice.
引文
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[3]赵婷婷,王会娟,王蕾,等.检测小鼠肝炎病毒的SNa Pshot分型技术及其应用[J].动物学杂志,2016,51(6):1084-1091.
[4]王翠娥,陈立超,周倩,等.实验大鼠和小鼠多种病毒的血清学检测结果分析[J].实验动物科学,2014,31(2):20-24.
[5]赵婷婷,严磊,魏立雯,等.小鼠肝炎病毒反向斑点杂交检测方法的建立及初步应用[J].第三军医大学学报,2014,36(16):1679-1683.
[6]Mathieu PA,Gomez KA,Coutelier JP,et al.Detection of mouse hepatitis virus infection by assay of anti-liver autoantibodies[J].J Virol Methods.2002,106(2):159-166.
[7]孙凤萍.不同品系小鼠感染小鼠肝炎病毒后Ig G、Ig M抗体变化及肝脏病毒检测[D].南京:南京农业大学,2007.
[8]周艳.小鼠肝炎病毒N基因的表达与间接ELISA检测方法的建立和应用[D].扬州:扬州大学,2008.
[9]许宝华,张玉珍,穆松牛,等.两种不同组织嗜性的小鼠肝炎病毒组合抗原检测抗体的间接ELISA方法[J].中国比较医学杂志,2006,16(8):495-498.
[2]赖国旗,何明忠,谭毅,等.小鼠肝炎病毒RT-PCR检测方法的研究[J].西南农业大学学报:自然科学版,2004,26(6):762-763.
[3]赵婷婷,王会娟,王蕾,等.检测小鼠肝炎病毒的SNa Pshot分型技术及其应用[J].动物学杂志,2016,51(6):1084-1091.
[4]王翠娥,陈立超,周倩,等.实验大鼠和小鼠多种病毒的血清学检测结果分析[J].实验动物科学,2014,31(2):20-24.
[5]赵婷婷,严磊,魏立雯,等.小鼠肝炎病毒反向斑点杂交检测方法的建立及初步应用[J].第三军医大学学报,2014,36(16):1679-1683.
[6]Mathieu PA,Gomez KA,Coutelier JP,et al.Detection of mouse hepatitis virus infection by assay of anti-liver autoantibodies[J].J Virol Methods.2002,106(2):159-166.
[7]孙凤萍.不同品系小鼠感染小鼠肝炎病毒后Ig G、Ig M抗体变化及肝脏病毒检测[D].南京:南京农业大学,2007.
[8]周艳.小鼠肝炎病毒N基因的表达与间接ELISA检测方法的建立和应用[D].扬州:扬州大学,2008.
[9]许宝华,张玉珍,穆松牛,等.两种不同组织嗜性的小鼠肝炎病毒组合抗原检测抗体的间接ELISA方法[J].中国比较医学杂志,2006,16(8):495-498.