Yang Jing, Chen Hao, Wang Zhenzhong, Yu Xianglong, Niu Xiaoyu, Tang Yi, Diao Youxiang
College of Animal Science and Technology, Shandong Agricultural University, Tai'an, China.
Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Tai'an, China.
Front Microbiol. 2017 Dec 12;8:2472. doi: 10.3389/fmicb.2017.02472. eCollection 2017.
An infectious disease characterized with short bills and protruding tongues has attacked to meat ducks in China since March 2015, which has caused ducks poor growth and enormous economic losses to duck industry of China. It was eventually proved to be caused by parvovirus after pathogen isolation and identification. As the genomic sequence analysis showed, this pathogen shared 90.8-94.6% of nucleotide identity with goose parvovirus (GPV), and it was called duck-origin novel goose parvovirus (N-GPV). In this study, a quantitative loop-mediated isothermal amplification (qLAMP) assay was developed for the rapid diagnosis of N-GPV. A set of four specific primers, two inner and two outer, were designed targeting at VP3 gene, which could be completed within 60 min at 65°C in water bath or on a real-time PCR instrument for quantitative analysis. Specificity test of LAMP assay showed that there was no cross-reactivity between N-GPV and other duck pathogens, and the detection limit of qLAMP assay was 1.0 × 10 copies/μL. The repeatability of this method was confirmed by inter-assay and intra-assay tests with variability ranging from 0.74 to 2.25%. The results have indicated that the qLAMP assay was a simple, rapid, accurate, sensitive, and specific method for detecting N-GPV, especially on field detection.
自2015年3月以来，一种以喙短和舌头突出为特征的传染病在中国侵袭肉鸭，导致鸭生长不良，给中国鸭产业造成巨大经济损失。经病原体分离鉴定，最终证明是由细小病毒引起的。基因组序列分析表明，该病原体与鹅细小病毒（GPV）的核苷酸同一性为90.8-94.6%，因此被称为鸭源新型鹅细小病毒（N-GPV）。在本研究中，开发了一种定量环介导等温扩增（qLAMP）检测方法用于快速诊断N-GPV。针对VP3基因设计了一组四条特异性引物，两条内引物和两条外引物，该反应可在65℃水浴中60分钟内完成，也可在实时PCR仪器上进行定量分析。LAMP检测的特异性试验表明，N-GPV与其他鸭病原体之间无交叉反应，qLAMP检测的检测限为1.0×10拷贝/μL。通过批间和批内试验证实了该方法的重复性，变异范围为0.74至2.25%。结果表明，qLAMP检测是一种检测N-GPV的简单、快速、准确、灵敏和特异的方法，尤其适用于现场检测。
