常规PCR、实时定量PCR检测传染性皮下及造血器官坏死病毒的效果分析

对虾传染性皮下及造血器官坏死病毒(IHHNV)可感染世界各地养殖对虾,给对虾养殖业造成严重经济损失。本实验首次采用实时定量PCR法对广西地区的84份凡纳滨对虾样品进行检测,同时以常规PCR检测作对照。实时定量PCR检测阳性率为79·8%,常规PCR检测阳性率为40·5%,表明广西地区养殖的凡纳滨对虾IHHNV的感染率较高。将二者检测均呈阳性的30份样品扩增产物进行序列分析测序,测序结果通过DNA STAR软件包进行分析,并通过NCBI Blast与GenBank中的序列进行比对。结果证明,测定的是IHHNV序列。30份样品的IHHNV序列很保守,可以分为4种类型,仅有两个碱基的位置发生变异。实时定量PCR检测IHHNV,快速、灵敏、准确,特异性好,可以作为检测对虾感染病毒的有效方法。

Evaluation of conventional PCR and real-time quantitative PCR test for infectious hypodermal and hematopoietic necrosis virus

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) can infect cultured shrimp worldwide and cause serious economic losses to the shrimp culture industry. 84 Litopenaeus vannamei samples from Guangxi Province were tested with real-time quantitative PCR (rt-qPCR) for the first time, and compared to the test result of conventional PCR. The positive ratio of rt-qPCR was 79.8% and that of the conventional PCR was 40.5%, indicating that the IHHNV infection rate of L.vannamei was high in Guangxi Province. Nucleotide sequencing was performed on 30 samples that were IHHNV positive in both tests. The results were analyzed with the DNA STAR software and compared with the GenBank using NCBI Blast program, which indicated that they were IHHNV sequences. The IHHNV sequences of the 30 samples were conservative; they were classified into four types, with mutation found for only two base pairs. rt-qPCR was rapid, sensitive and accurate for IHHNV test, and can effectively diagnose IHHNV infections in shrimps.