一种筛选无青蟹呼肠孤病毒(MCRV)种蟹的荧光定量RT-PCR检测方法及其应用

A new SYBR green qRT-PCR diagnostic method for screening MCRV-free breeding mud crabs

中文关键词: MCRV  检测方法  青蟹种蟹  定量RT-PCR

英文关键词: MCRV  Diagnostic method  Breeding mud crab (Scylla paramamonsain)  qRT-PCR

中文摘要:

      青蟹呼肠孤病毒(mud crab reovirus, MCRV)是养殖拟穴青蟹(Scylla paramamosain)致死率最高的病原之一，青蟹种蟹及其幼体携带该病原对青蟹养殖产业的高质量发展造成了重大威胁。为从源头切断该病原的感染和传播，本研究研发了一种筛选无MCRV种蟹的荧光定量检测方法。为最大限度地提高检测灵敏性，首先，本研究分析MCRV在感染青蟹主要组织中的含量，发现血淋巴中病毒载量最高；随后，分析该病毒的13个预测基因在血淋巴中的表达水平，发现病毒VP11基因相对表达量最高；最后，基于VP11基因序列保守区设计特异引物，建立了一种SYBR Green quantitative reverse-transcription PCR (qRT-PCR)检测方法，该方法可精确检测的病毒下限为50 copies/反应。该检测方法具有组织和靶标基因选择优势，灵敏性显著高于早期报道的其他检测方法。使用该方法对含有5种常见甲壳动物病原的样品进行检测，均无特异性扩增。抽取微量血淋巴(大约50 µL/只)提取RNA，随后使用该检测方法对22只种蟹和20只市售青蟹进行MCRV检测，阳性率分别54.44%和85.00%。此外，利用该方法进一步分析了MCRV在较低温度下(21 ℃)在青蟹体内的增殖情况，发现病毒感染早期MCRV呈指数方式增殖，随后进入平台期，实验周期内无青蟹死亡。总之，本研究建立了一种灵敏性高、实用性强的MCRV检测方法，既可用于微创条件下无MCRV种蟹筛选，也能满足病原感染相关研究的需要。

英文摘要:

      Mud crab reovirus (MCRV) is one of the most fatal pathogens of the mud crab Scylla paramamonsain. The outbreak and epidemic of MCRV has seriously affected the healthy development of the mud crab aquaculture industry. To limit MCRV transmission from breeding crabs to larva, we attempt to establish a more sensitive and practical diagnostic method for screening MCRV-free crabs. The primers of the present diagnostic methods for MCRV are based on the VP1 gene (MCRV RNA polymerase gene), and the low expression level of this gene limits the sensitivity of the diagnostic method. Therefore, it is necessary to select the target gene with the highest expression level for the detection primer to improve the sensitivity of the diagnostic method. In addition, the current diagnostic methods require gill samples for virus detection, which requires killing the mud crabs before sampling. This sampling method is obviously not suitable for screening MCRV-free breeding crabs. Therefore, it is necessary to develop a less invasive sampling method for breeding crabs. In this study, a SYBR Green fluorescent quantitative diagnostic method was developed to screen MCRV-free breeding crabs. To improve the sensitivity of the detection, we initially analyzed the relative load of MCRV in the main tissues of infected mud crabs. The viral load in the hemolymph was the highest of all the tissues. The expression levels of 13 putative genes of MCRV were detected in the hemolymph. The relative expression level of the VP11 gene was the highest. Finally, specific primers were designed based on the conserved region of the VP11 gene sequence to establish a SYBR Green qRT-PCR (quantitative reverse-transcription PCR) detection method to accurately detect 50 copies/µL of viral nucleic acid in a sample. Considering the advantages of tissue and target gene selection, the sensitivity of this method should be significantly higher than that of preexisting detection methods. This diagnostic method is very specific for MCRV and no specific amplification was observed using nucleic acid samples containing 5 different kinds of common crustacean pathogens (MCDV, WSSV, DIV1, EHP, and Vibrio parahemolyticus). Compared to other methods of extracting RNA by killing and grinding the gill tissues of crabs, we can select MCRV-free crabs by sampling very small amounts of hemolymph (as low as 20 µL). All of the healthy crabs screened by this method were able to hold eggs that hatched normally. To test the effectiveness of this method, 22 breeding crabs and 20 commercial crabs were screened for MCRV. The positive rates were 54.55% and 85.00%, respectively. In addition, we analyzed the proliferation of MCRV in the mud crabs, and found that MCRV proliferates exponentially in the early stage, then enters a plateau phase, and no crabs died during the infection period of seven days. In conclusion, this study established a highly-sensitive and practical detection method for MCRV in breeding crabs, which can meet the requirements for MCRV-free breeding crab screening with low damage to the breeders. We also investigated the pathogenic infection mechanisms.