基因Ⅰ型和基因Ⅲ型日本脑炎病毒RT-LAMP鉴别检测方法的建立

Establishment of an RT-LAMP assay for identification of Japanese encephalitis virus genotypeⅠand genotype Ⅲ

为建立一种快速、灵敏、特异的鉴别基因Ⅰ型和基因Ⅲ型日本脑炎病毒(JEV)的反转录-环介导等温扩增方法,根据Gen Bank中登录的基因Ⅰ型和基因Ⅲ型JEV毒株基因序列,分别设计了1套基因Ⅰ型和基因Ⅲ型特异性RT-LAMP引物,并以此引物分别建立了基因Ⅰ型和基因Ⅲ型特异性RT-LAMP方法。结果显示,基因Ⅰ型和基因Ⅲ型特异性RT-LAMP方法均能在1 h内完成对相应基因型JEV RNA的扩增,并与猪瘟病毒、猪繁殖与呼吸综合征病毒、猪圆环病毒、猪细小病毒和伪狂犬病病毒无交叉反应。灵敏性试验结果显示,基因Ⅰ型和基因Ⅲ型JEV特异性RT-LAMP方法均能扩增出24 copies的JEV RNA。本研究建立的基因Ⅰ型和基因Ⅲ型JEV特异性RT-LAMP检测方法具有灵敏性高、特异性好、简便、快速的特点,能够快速对JEV进行检测及基因Ⅰ型和基因Ⅲ型的鉴别,具有广泛的应用价值。

To develop a rapid,sensitive and specific method for the detection and differentiation of Japanese encephalitis virus（JEV） genotype Ⅰ（GⅠ） and genotype Ⅲ（GⅢ）,a series of specific reverse-transcriptase loop-mediated isothermal amplification（RT-LAMP）primers were designed based on the sequences of JEV GⅠ and GⅢ strains available in Gen Bank and these primers were used in RT-LAMP for the identification and differentiation of JEV GⅠ and GⅢ. As a result,the GⅠ-JEV RNA and GⅢ-JEV RNA could be amplified within 1 hour by GⅠ-specific and GⅢ-specific RT-LAMP, respectively. In addition,there was no cross reaction with swine fever virus,porcine reproductive and respiratory syndrome virus,porcine circovirus,porcine parvovirus and pseudorabies virus,respectively. The sensitivity of the GⅠ-specific and GⅢ-specific RT-LAMP reached 24 copies for both GⅠ and GⅢ of JEV RNA respectively. The results showed that this genotype-specific RT-LAMP assay was rapid,sensitive, convenient and specific for the detecting and differentiating JEV GⅠ and GⅢ strains.