实用WSSV定量检测方法的建立及其应用于脊尾白虾病毒感染规律的研究

摘要

White spot syndrome virus( WSSV) can infect many kinds of crustaceans and thus has caused huge economic losses to the crustacean farming. In order to detect WSSV precisely and sensitively, quantify WSSV conveniently, and further investigate the WSSV multiplication patterns in crustaceans, a practical realtime PCR for the quantification and diagnosis of WSSV is required to be developed. However, currently, the harvested quantitative real-time PCR for WSSV was either costly for those which were developed based on the Taqman probe technology, or insensitive for a latest method which was set up with the SYBR green dye. In this study,a practical,sensitive,and specific quantitative real-time PCR for WSSV based on SYBR Green was developed, and the tissue distributions and the profiles of WSSV proliferation in ridgetail white prawns {Exopalaemon carinicauda) were examined with this method. The details were as follows. First, a pair of primers(F1, R1 ) was designed to amplify the target fragment, which would be used to construct WSSV standard plasmid,and then another pair of primers( RF2,RR2) located inside the above target fragment was synthesized to produce amplicons in real-time PCR reaction, thereby developing a new diagnostic and quantitative real-time PCR for WSSV with SYBR Green dye. The results demonstrated that this quantitative method for WSSV was specific, sensitive, and precise. Subsequently, we further investigated WSSV distribution and proliferation pattern in ridgetail white prawns. The results of WSSV distribution showed that WSSV infected all detected tissues, among which, the connective tissues under the carapace possessed the highest virus load(l. 1 ×107 copies/μg tissue) ,and the second highest tissue were gills with the virus load of 4. 1 × 106 copies/μg tissue; The data of WSSV proliferation pattern illustrated that the copies of WSSV began to increase dramatically at 48 h after challenge,but before that there was no obvious change for virus copies. With the increase of infection time, the copies of WSSV increased more and reached the highest numbers at 120h post infection. In addition,we found that during this process the mortality of ridgetail white prawns caused by WSSV was approximately 20% . All these results suggest that WSSV is a deadly pathogen to ridgetail white prawns,and the new quantitative real-time PCR for WSSV is practical.%为优化当前白斑综合征病毒( WSSV)定量检测方法,实验结合当前WSSV诊断与定量方法应用的实际,试图通过设计两对普通PCR引物来建立一种实用的WSSV绝对定量方法,并利用此方法进一步探明WSSV在脊尾白虾体内的增殖规律及致病性.首先根据WSSV的囊膜蛋白VP28基因序列设计了两对特异引物(F1,R1)和(RF2,RR2),分别用于标准品质粒的构建和扩增子的扩增,以开展SYBR Green染料法定量检测WSSV的研究；随后利用该定量检测方法分析了WSSV在脊尾白虾体内的组织分布及感染规律.研究结果显示:(1)以定量引物RF2和RR2建立的SYBR Green绝对定量检测方法具有敏感度高、特异性强、定量范围广且准确等特点；(2)WSSV能感染脊尾白虾鳃、肝胰腺、肌肉、头胸甲下结缔组织、胃及肠等组织,其中头胸甲下结缔组织病毒含量最高(1.1×107 copies/μg),其次是鳃组织(4.1×106copies/μg)；正常脊尾白虾感染WSSV,首先经过一个约48 h潜伏期,而后开始大量增殖并造成部分虾死亡,死亡率约为20％.