以色列急性麻痹病毒Taq Man荧光定量RT-PCR检测方法的建立及初步应用

Development and application of the real-time RT-PCR assay for the detection of Israeli acute paralysis virus

为建立以色列急性麻痹病毒(IAPV)荧光定量RT-PCR检测方法,本研究根据GenBank中IAPV RNA依赖的RNA聚合酶(RdRp)基因保守区域序列设计了一对特异性引物和一条特异性探针,以体外转录制备的重组质粒标准品为模板,经过反应条件优化,建立了IAPV荧光定量RT-PCR检测方法,并对其进行了特异性、敏感性、重复性试验;利用所建立的方法对70份福建地区临床疑似IAPV感染的蜜蜂样品进行检测,并对阳性样品进行测序验证。结果显示,荧光定量RT-PCR方法的最佳引物和探针浓度分别为0.25μmol/L和0.20μmol/L,最佳退火温度为61℃。该方法仅对IAPV出现阳性扩增信号,对蜜蜂克什米尔病毒、蜜蜂慢性麻痹病毒、蜜蜂残翅病毒、蜜蜂急性麻痹病毒、蜜蜂黑蜂王台病毒、蜜蜂囊状幼虫病毒等6种病毒均未出现扩增,特异性较强;该方法对重组质粒标准品的最低检测限为9.7拷贝/μL,比普通RT-PCR敏感性高约100倍;该方法批内及批间变异系数均小于1.5%,重复性好。利用该方法和普通RT-PCR方法分别对70份临床样品进行检测,结果显示,两种方法的阳性检出率分别为31.4%(22/70)和27.1(19/70),二者的符合率为95.9%。本研究首次建立的IAPV Taq Man荧光定量RTPCR检测方法快速、敏感,检测结果准确可靠,为IAPV的流行病学调查及其分子生物学的快速检测奠定了基础。

A real-time RT-PCR assay for detection of Israeli acute paralysis virus(IAPV)was developed with two primers and a probe which were designed by targeting the RNA dependent RNA polymerase(RdRp)gene sequences of IAPV in GenBank,and the positive template was produced by in vitro transcription of the RNA standards.The specificity,sensitivity and repeatability of the real-time RT-PCR assay were tested.70 clinical samples suspected of IAPV infection in Fujian area were detected by using the established method,and the positive samples were further verified by sequencing.The results showed that the optimal primer concentration and the probe concentration were 0.25μmol/L and 0.20μmol/L,respectively,and the optimum annealing temperature was 61℃.The assay has a good linear relationship at a template range from 9.7×100 copies/μL to 9.7×108 copies/μL,with a coefficient correlation of 0.997;and the lower limit of detection was 9.7 copies/μL;the specificity of this assay showed no cross reaction to KBV,CBPV,DWV,ABPV,BQCV and SBV;the coefficient of variation for intra-assay and inter-assay were both less than 1.5%.22 of 70 clinical samples were identified as positive by using this assay,which was consistent with the sequencing results,while 19 samples were detected as positive by conventional RT-PCR assay,and the coincidence rate of the two methods is95.9%.The developed real-time RT-PCR assay was a promising method for monitoring and surveillance of IAPD.