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当前位置: 首页 > 文章 > 斑点叉尾鮰病毒焦磷酸测序检测方法的建立 畜牧与兽医 2018 (5) 89-92
Position: Home > Articles > Establishment of pyrosequencing assay for detection of channel catfish virus Animal Husbandry & Veterinary Medicine 2018 (5) 89-92

斑点叉尾鮰病毒焦磷酸测序检测方法的建立

作  者：
王娜;张旻;景宏丽;江育林;吴绍强
关键词：
斑点叉尾鮰病毒;焦磷酸测序;PCR;蛋白激酶基因;检测
摘  要：
旨在建立一种基于PCR的焦磷酸测序检测方法,用于斑点叉尾鮰病毒病的快速检测和确诊。针对斑点叉尾鮰病毒(CCV)蛋白激酶(PK)基因的保守区域设计扩增引物与测序引物,经PCR扩增后对PCR产物进行焦磷酸测序,借助测序结果比对确定是否为CCV核酸序列。通过对反应条件与体系的优化,成功建立了CCV PK基因焦磷酸测序检测方法。结果表明:建立的斑点叉尾鮰病毒焦磷酸测序检测方法扩增基因片段长度为144 bp,能够特异性检测出目的病毒,最低核酸检测限为5×10-6ng/μL,重复测序3次均能准确测出45 bp核酸序列。本研究建立的斑点叉尾鮰病毒焦磷酸测序检测方法特异性强、灵敏度高、重复性好,适合高通量检测且耗时短仅需4 h,为CCV的快速检测提供了一种可行方法。
译  名：
Establishment of pyrosequencing assay for detection of channel catfish virus
作  者：
WANG Na;ZHANG Min;JING Hongli;JIANG Yulin;WU Shaoqiang;Chinese Academy of Inspection and Quarantine;
关键词：
channel catfish virus;;pyrosequencing;;PCR;;protein kinase gene;;detection
摘  要：
The aim of the study was to establish a pyrosequencing assay for rapid detection and identification of channel catfish virus( CCV). The amplification primers and sequencing primer were designed for the conserved region of protein kinase gene of CCV. After PCR reaction,the product was detected with pyrosequencing and identified by genomic alignment. The pyrosequencing assay was established of the PK gene for CCV by optimizing the reaction conditions and system. The results showed that the PCR product was 144 bp and it could specifically identify the target viruses. The minimum detection limit of the nuclei acid was 5×10-6 ng/μL. Pyrosequencing repeated three times accurately determined the nucleotide sequence to be 45 bp long. It was demonstrated that the pyrosequencing assay established here possessed good specificity,high sensitivity and stability in detection of CCV,The assay is applicable to r high throughput and time-consuming detection of CCV. It takes only 4 h.

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