【摘要】 根据栉孔扇贝(Chlamys farreri)急性病毒性坏死病毒(Acute viral necrobiotic virus,AVNV)的基因序列,选择保守区段,应用Beacon Designer 7.0软件设计了一对能特异性扩增90bp片段的引物和TaqMan探针,建立并完善了AVNV荧光定量聚合酶链式反应(Fluorescent quantitative polymerase chain reaction,FQ-PCR)诊断方法。该方法在108～102病毒拷贝范围内有较好的线性关系,AVNV拷贝数(X)和循环数(C)t的相关关系为:lgX=-0.29Ct+13.28(相关系数R2=0.998);检测AVNV的灵敏度为102拷贝;特异性实验表明只对AVNV基因组呈阳性反应。应用FQ-PCR对76份采自夏季发病期前后的栉孔扇贝样品进行检测,结果发现68份样品检测结果为阳性,阳性检出率为89.47%,高于普通PCR的阳性检出率(80.26%)。研究结果表明,该方法快速、灵敏,特异、重复性良好且能实现AVNV的定量检测,对AVNV的快速诊断和分子流行病学的调查以及AVNV疫情监测具有重要意义。更多还原

【Abstract】 Acute viral necrobiotic virus(AVNV) is an enveloped, spherical virus(130 nm to 170 nm in diameter), with spike-like surface protrusions. This pathogen has been causing acute viral necrobiotic disease(AVND) in scallop Chlamys farreri in China during summer time. Acute mortality of infected scallop can be as high as 90% within 5 to 8 days. Successful therapeutics for scallop against this virus is not available thus far, to the best of our knowledge. Therefore, development of preventive strategies, especially rapid and early detecting assay for this viral pathogen, is of special urgency in disease management of scallop farming. Several methods such as ultrastructural examination and ELISA have been developed for ANAV detection. However, these assays are generally time-consuming, laborious and insensitive. In this paper, based on the DNA sequences of AVNV, a pair of specific primers which can specifically amplify a 90bp fragment, and a fluorescent Taq Man probe were designed. Then, a specific, fluorescence based quantitative polymerase chain reaction(FQ-PCR) assay for real-time detection of AVNV was developed and optimized initially. It could obtain excellent linearity when the AVNV concentration is between 108 and 102 copies, lgX=-0.29 Ct+13.28 (correlation coefficient R2=0.998). After the procedure of fluorescent quantitative polymerase chain reaction(FQ-PCR) assay for acute viral necrobiotic virus was optimized, the specificity, sensitivity and reproducibility of the method were estimated. The detection limit of this assay was 102 copies for AVNV. It was also found that the specificity of this assay was high without any cross-reactions with DNA from AVNV uninfected C. farreri and other viruses such as WSSV, IHHNV, TRBIV and KHV found commonly in China. The coefficients of variance(CV) of cycle threshold were 0.61 %-2.03 % and 0.92 %-2.88 % for the intra-assay and inter-assay tests respectively, which indicated good reproducibility. Seventy-six samples of C. farreri collected from Qingdao, Rongcheng, Changdao in Shandong province and Dalian in Liaoning province during July to September in 2007 were tested by using FQ-PCR and conventional PCR assays. The results showed that the detection rate of FQ-PCR was 89.47 %, higher than that of conventional PCR (80.26%). The FQ-PCR assay demonstrated exceptionally higher sensitivity compared to that of traditional PCR by picking up 7 additional positive cases. None of the positive samples by PCR assay were missed by FQ-PCR assay, thereby showing superior sensitivity of FQ-PCR method. This increased sensitivity makes FQ-PCR a better choice than the traditional PCR for the detection of AVNV in cases when lower concentrations of virus are expected in asymptomatic samples. Thus, virus-carrying C. farreri can be found during early infection stages and effectively controlled before the infection becomes epizootic. Therefore, it is confirmed that the FQ-PCR assay is a powerful tool for the detection of AVNV with rapidity, sensitivity, specificity, accuracy and quantification.更多还原