为建立十足目虹彩病毒1(decapod iridescent virus 1,DIV1)的SYBR Green Ⅰ荧光定量PCR检测方法,根据DIV1的MCP和ATPase基因序列,设计并筛选出引物,以制备的DIV1阳性质粒标准品为模板构建标准曲线,建立DIV1的SYBR Green Ⅰ qPCR方法,并对该方法进行临床初步应用.结果显示,建立的qPCR方法阈值循环数(cycle threshold value,Ct)与标准品拷贝数的对数线性关系良好,标准曲线相关系数(R2)为0.999;对DIV1阳性的虾蟹核酸样本能够进行特异性扩增,但对传染性脾肾坏死病毒(infectious spleen and kidney necrosis virus,ISKNV)和白斑综合征病毒(white spot syndrome virus,WSSV)阳性核酸样本均无扩增;最低检测限为9.77copies/μL;Ct值的组内和组间变异系数均小于1％.运用该方法对70份疑似感染DIV1的虾蟹类样本进行DIV1检测,该方法阳性率为48.57％,与套式PCR检测方法的阳性率一致;利用建立的方法对DIV1阳性三疣梭子蟹的血淋巴、肝胰腺及心脏等组织进行定量检测分析,结果显示各组织中均存在DIV1,其中血淋巴中DIV1平均拷贝数最高.研究表明,建立的SYBR Green Ⅰ荧光定量PCR方法特异性强、灵敏度高、重复性好,可用于对DIV1的快速、定量检测,对十足目虹彩病毒病的诊断和防控具有重要意义.
ESTABLISHMENT AND PRELIMINARY APPLICATION OF SYBR GREEN Ⅰ FLUORESCENCE QUANTITATIVE PCR METHOD FOR DETECTION OF DECAPODAIRIDOVIRUS 1 IN PORTUNUS TRITUBERCULATUS
To establish a SYBR Green Ⅰ qPCR method for the detection of decapod iridescent virus 1(DIV1),five primers were designed according to the conserved MCP and ATPase sequence of DIV1,and the optimal primer M205 was screened out,the 205 bp MCP target gene sequence of DIV1 was amplified by PCR and cloned into pEASY-T1 vector to construct the standard plasmid of DIV1.A SYBR Green Ⅰ qPCR method for DIV1 detection was developed by constructing a standard curve using a continuously diluted standard plasmid as a template.The results showed that the cycle threshold value(Ct)of the established SYBR Green Ⅰ qPCR assay had a good linear relationship with the copy number of the standard plasmid,and the linear range was wide.The correlation coefficient(R2)of the standard curve was 0.999,and the amplification efficiency was 101％.Amplification specificity analysis indicated that the method could specifically detect DIV1,had no amplification of infectious spleen and kidney necrosis virus(ISKNV),and white spot syndrome virus(WSSV).The coefficient of variation within and between groups was less than 1％,respectively,which indicated that the method was repeatable.The qPCR method could detect a minimum of 9.77 copies/pL with higher sensitivity than the conventional PCR method.The nested PCR method and SYBR Green Ⅰ qPCR method were used to detect DIV1 in 70 suspected DIV1-infected Portunus trituberculatus(35 samples)and Penaeus vannamei(35 samples)samples.The results showed that the positive detection rate of both methods was 48.57％.Application analysis indicated that DIV1 could be detected in the main tissues of DIV1 infected P.trituberculatus with 4.37×10 7 average copies in hemolymph.The average amounts of DIV1 virions in hemolymph were significantly higher than those in other tissues.In conclusion,the established SYBR Green Ⅰ qPCR method has high sensitivity,strong specificity,and good repeatability,and can be used for rapid and quantitative detection of DIV1,which would be helpful for the prevention and control of decapod iridescent virus 1 disease.
Decapod iridescent virus 1(DIV1)fluorescence quantitative PCRSYBR Green Ⅰdetection method
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