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目的建立登革1、2型病毒、黄热病毒及流行性乙型脑炎病毒的多重RT-PCR快速检测方法。方法参照病毒核酸序列设计多重RT-PCR引物,并检索GenBank国际基因序列数据库初步验证其特异性,随后对Mg2+、dNTP及引物浓度,RT-PCR反应条件进行优化,建立稳定、特异的多重RT-PCR快速检测4株病毒方法 ,并以同属于黄病毒科的登革3型病毒、登革4型病毒及甲病毒属基孔肯亚病毒、辛德毕斯病毒为对照,验证其特异性。结果应用多重RT-PCR反应体系,对引物的相关性实验结果表明引物之间不会因相互干扰而出现假阳性结果 ;采用多重RT-PCR引物对登革1、2型病毒、黄热病毒及流行性乙型脑炎病毒进行扩增,分别获得574、251、879、422 bp片段,与设计相符,而对照病毒组均无非特异性扩增条带。结论实验证明,所建立的多重RT-PCR方法能够快速地检测登革1、2型病毒、黄热病毒及流行性乙型脑炎病毒,为其检测提供了一种方便易行的方法 。
Objective To establish a rapid multiplex RT-PCR method for detecting dengue 1,2 virus, yellow fever virus and Japanese encephalitis virus. Methods According to the viral nucleic acid sequence, multiple RT-PCR primers were designed and the GenBank international gene sequence database was searched to verify its specificity. Subsequently, Mg2 +, dNTP, primer concentration and RT-PCR reaction conditions were optimized to establish stable and specific multiple RT- PCR was used to detect four viruses rapidly. The specificity of dengue virus type 3, dengue type 4 virus, alphavirus Chikungunya virus and Sindbis virus in the same genus as Flaviviridae was tested. Results The results of multiplex RT-PCR reaction showed that there was no false-positive result between the two primers due to the mutual interference between the primers. Multiple RT-PCR was used to detect the dengue 1,2 virus, yellow fever virus and The Japanese encephalitis viruses amplified 574, 251, 879 and 422 bp, respectively, in line with the design, but there was no non-specific amplification band in the control virus group. Conclusion The results of experiments show that the established multiplex RT-PCR method can rapidly detect dengue 1,2 virus, yellow fever virus and epidemic encephalitis virus, providing a convenient and convenient method for the detection.