不同基因型草鱼呼肠孤病毒通用RT-PCR检测方法的建立及应用

Establishment and application of a universal RT-PCR assay for detection of different genotype grass carp reovirus

根据Gen Bank登录的草鱼呼肠孤病毒(grass carp reovirus,GCRV)不同基因型毒株编码VP2蛋白的S2节段保守序列,设计1对简并引物,通过优化反应条件及灵敏度和特异性评价,建立了一种能同时检测三种基因型GCRV的通用RT-PCR检测方法。结果显示:该方法在三种基因型GCRV中均扩增出大小约590 bp的特异性条带;灵敏度试验结果显示,该方法对于I、Ⅱ、Ⅲ型GCRV的检测限分别为380、250和380拷贝/μL。该方法可特异性地检测目前已知的三种基因型GCRV,而在大鲵虹彩病毒(GSIV)、锦鲤疱疹病毒(KHV)、鲤疱疹病毒Ⅱ型(Cy HV-2)、鲤春病毒血症病毒(SVCV)和传染性脾肾坏死病毒(ISKNV)中无扩增产物。利用该方法检测49份草鱼出血病疑似样品,结果显示均为GCRV阳性,其中I型阳性率为8. 2%,Ⅱ型阳性率为85. 7%,Ⅲ型阳性率为2%,I、Ⅱ型混合感染阳性率为4. 1%,其他类型的混合感染未检测到。结果表明,本研究建立的针对I、Ⅱ和Ⅲ型GCRV的RT-PCR检测方法具有通用性好、省时高效的突出特点,同时还兼具较高的灵敏度和特异性。

A universal detection method for three genotypes of grass carp reovirus(GCRV)based on reverse-transcription polymerase chain reaction(RT-PCR)was developed.A pair of degenerate primer was designed to target the multiple conserved sequences alignment of segment 2 encoding VP2 protein of different GCRV genotype strains deposited in GenBank.Using this primer pair,only one specific product,approximately 590 bp in length was obtained when the cDNA of the three genotypes of GCRV was used as template by RT-PCR amplification.The sensitivity test results showed that the detection limit was 380,250 and 380 copies/μL for types I,Ⅱ,andⅢ,respectively.This method exhibited good specificity detection of GCRV.However,no products were amplified for PCR templates of GSIV,KHV,CyHV-2,SVCV and ISKNV nucleic acids.Totally 49 suspected grass carp hemorrhagic disease samples were detected by this method.The results showed that all the tested samples were positive of GCRV.The positive rates of genotypes I,Ⅱ,andⅢwere 8.2%,85.7%,and 2%,respectively.The positive co-infection rate of types I andⅡwas 4.1%.Meanwhile,no co-infection of other types were detected.Collectively,the developed RT-PCR method could detect the three genotypes GCRV simultaneously with high sensitivity and specificity.