摘要
为建立一种利用逆转录环介导等温核酸扩增(RT-LAMP)快速检测A型塞内卡病毒(SVA)的方法,从SVA细胞培养液中提取总RNA,根据SVA VP1基因序列,设计一套对应目的片段6个区域的4条特异性引物进行核酸扩增,建立RT-LAMP检测方法。利用建立的RT-LAMP检测方法对口蹄疫病毒、猪繁殖与呼吸综合征病毒、猪瘟病毒、猪伪狂犬病病毒与塞内卡病毒进行特异性试验。将SVA VP1质粒10倍梯度稀释液作为模板(1×10~0~1×10~7拷贝)进行RT-LAMP和RT-PCR反应,测试RT-LAMP检测方法的灵敏性。采集疑似发病猪的鼻腔拭子和水疱液共126份样品,应用RT-LAMP和RT-PCR检测,加入SYBR GreenⅠ进行可视化鉴定。结果表明,与传统RT-PCR相比,RT-LAMP检测方法灵敏度高1 000倍,且与其他病毒无交叉反应,具有高度敏感性和特异性。临床样品检测结果显示,鼻腔拭子样本SVA阳性率为29.6%,水疱液样本SVA阳性率为100.0%,2种方法检测结果一致,符合率为100%。综上所述,本研究建立的RT-LAMP检测方法准确、简便、经济有效,尤其适用于现场和基层实验室对SVA的快速诊断。
The objective of this study is to establish a method for detecting Senecavirus A(SVA) simply and quickly by reverse transcription-loop mediated isothermal amplification (RT-LAMP) technology. A set of four primers specific to six regions within the SVA VP1 gene was designed for the RT-LAMP assay using total RNA extracted from SVA cell culture. To evaluate the specificity of the RT-LAMP assay, viruses related to foot-and-mouth disease virus (FMDV) or known of porcine common virus, including pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), were tested together with SVA. To assess the detection sensitivity, 10 fold serial dilutions of RNA template (1×10 0 -1×10 7 copies) were used in RT-PCR and RT-LAMP. To evaluate the application value of this method, a total of 126 nasal swab and vesicular fluid samples were collected from suspected sick pigs to detect by RT-LAMP and RT-PCR, and SYBR GreenⅠwas added to visualize the detection. The results showed that the sensitivity of RT-LAMP detection method was 1 000 times higher than that of traditional RT-PCR. And there was no cross reaction with other viruses, so it had high sensitivity and specificity. The results of clinical samples detection showed that the positive rates of SVA in nasal swab samples and vesicular fluid samples were 29.6% and 100.0%, respectively. The results of the two methods were consistent and the coincidence rate was 100%. In conclusion, the RT-LAMP assay established in this study is rapid, simple and cost-effective, especially suitable for rapid diagnosis of SVA in the field and basic laboratory.
作者
杨钰楚
王晨曦
周雪珂
赵军
徐志文
朱玲
YANG Yu-chu;WANG Chen-xi;ZHOU Xue-ke;ZHAO Jun;XU Zhi-wen;ZHU Ling(College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China;College of Life Sciences, Sichuan University, Chengdu 611130, China;Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu 611130, China)
出处
《江苏农业学报》
CSCD
北大核心
2019年第4期868-873,共6页
Jiangsu Journal of Agricultural Sciences
基金
四川省科技支撑计划项目(2015NZ0072)
“十三五”育种攻关计划课题(2016NY20052)
关键词
A型塞内卡病毒
RT-LAMP
快速检测
Senecavirus A
reverse transcription-loop mediated isothermal amplification (RT-LAMP)
rapid detection
