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- Title
F亚群禽白血病病毒SYBR Green I荧光定量PCR 检测方法的建立.
- Authors
严立福; 陈 建; 郑晓翠; 李 昱; 余 蕴; 康杨帆; 李海霞; 张雪婷; 程 颂; 曹伟胜
- Abstract
To develop SYBR green I fluorescent quantitative PCR method (qPCR) for the detection of subgroup F avian leukosis virus (ALV-F), we first used the env gene of ALV-F FGD1803 strain as a template to construct a recombinant plasmid pMD18-T-F as standard. The standard cure was established with this standard plasmid, and then the fluorescence qPCR method for detection ALV-F was developed after the optimizing of reaction conditions. The optimized reaction conditions of this method are 10 μmol/L 0.5 μL upstream and downstream primers, and an annealing temperature of 55 ℃. Specificity results showed that this method could only specifically detect ALV-F, and had no cross reaction with ALV-A, ALV-B, ALV-J, ALV-K, ALV-E, REV, MDV, AIV, NDV, IBV and other avian viruses. The sensitivity results showed that the minimum detection limit of this method for plasmid standards was 1.16×102 copies/μL, and the sensitivity is 100 times that of the routine PCR methods. The reproducibility results showed that the coefficient of variation of intra-assay and inter-assay reproducibility was less than 5% with 3 recombinant plasmids used as templates. Thirty (21 ALV-positive and 9-negative) cell cultures of blood plasma samples of colorful pheasant were detected with this method, the routine PCR method, and virus isolation. The results showed that the positive rate of this method was 33.3% (10/30), and the positive rate of the routine PCR method was 20.0% (6/30), and the positive rate of the virus isolation was 13.3% (4/30). The coincidence rate of this method and virus isolation was 80.0%. In this study, a fluorescence quantitative PCR method for the detection of ALV-F was established for the first time in China. All results indicated that this method had a specificity, sensitivity and stability method for the diagnosis of ALV-F from clinical samples.
- Publication
Chinese Journal of Preventive Veterinary Medicine / Zhongguo Yufang Shouyi Xuebao, 2021, Vol 43, Issue 4, p388
- ISSN
1008-0589
- Publication type
Article
- DOI
10.3969/j.issn.1008-0589.202006030