| 摘要: |
| 【目的】建立鳜弹状病毒（SCRV）和传染性脾肾坏死病毒（ISKNV）的双重PCR检测方法。【方法】分别针对SCRV N基因和ISKNV MCP基因的保守区域设计特异性引物，通过优化退火温度和引物用量（μL）比，建立SCRV和ISKNV的双重PCR检测方法，并对该方法的敏感性和特异性进行检验。应用建立的双重PCR方法对20份临床样品进行单一和双重PCR检测，比较单一和双重PCR的检测结果。【结果】成功建立了SCRV和ISKNV双重PCR方法，该法特异性较强，对草鱼呼肠孤病毒（GCRV）、锦鲤疱疹病毒(KHV)、神经坏死病毒（NNV）、鲤春病毒血症病毒（SVCV）、新加坡石斑鱼虹彩病毒(SGIV)、罗非鱼湖病毒（TiLV）等鱼类常见病毒无扩增；敏感性较好，对2种病毒DNA的检测下限均为0.01 ng/μL。20份临床样品的双重PCR样品检测结果显示，3份为SCRV阳性，11份为ISKNV阳性，2份为SCRV和ISKNV混合感染，结果与单一PCR检测结果一致。【结论】建立的SCRV和ISKNV双重PCR检测方法特异性较强，敏感性较好，可用于鳜鱼SCRV与ISKNV的快速鉴别诊断和分子流行病学调查。 |
| 关键词: 鳜弹状病毒 传染性脾肾坏死病毒 双重PCR 病毒检测 |
| DOI： |
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| 基金项目:中国-东盟海上合作基金项目；广东省促进经济发展专项资金（海洋经济发展用途）项目(GDME-20181007)；广州市珠江科技新星项目（201710010087） |
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| Establishment of duplex PCR assay for detecting Siniperca chuatsi rhabdovirus and infectious spleen and kidney necrosis virus |
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LIANG Hongru,FAN Zhiyi,CAI Xiuzhu,et al
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| Abstract: |
| 【Objective】This study aimed to establish a duplex reverse transcription-polymerase chain reaction (PCR) assay for detecting the the Siniperca chuatsi rhabdovirus (SCRV) and infectious spleen and kidney necrosis virus (ISKNV) in Mandarin fish at the same time.【Method】Two pairs of specific primers were designed from the conserved sequence of N gene in SCRV and MCP gene in ISKNV.By optimizing the reaction conditions and system,a duplex PCR was established to detect the two genotypes simultaneously.After further testing the sensitivity and specificity,the duplex PCR and sequencing methods were used to detect 20 clinical samples.【Result】The duplex PCR for detecting SCRV and ISKNV was established successfully with high sensitivity for SCRV and ISKNV and no amplification for Grass Carp Reovirus (GCRV),Koi herpesvirus (KHV),Nervous necrosis virus (NNV),Spring Viremia of Carp Virus (SVCV),Singapore grouper iridovirus (SGIV) and Tilapia Lake Virus(TiLV).The DNA detection limit was 0.01 ng/μL for both SCRV and ISKNV.Among the 20 samples,3 were SCRV positive,11 were ISKNV positive and 2 were SCRV and ISKNV positive.The results of the duplex PCR were consistent with those of single PCR.【Conclusion】The established duplex PCR method had high sensitivity for SCRV and ISKNV and could be used for rapid diagnosis and epidemiology investigation on SCRV and ISKNV of Mandarin fish. |
| Key words: Siniperca chuatsi rhabdovirus infectious spleen and kidney necrosis virus duplex PCR virus detection |
