[1]杨钰楚,王晨曦,周雪珂,等.A型塞内卡病毒RT-LAMP检测方法的建立及应用[J].江苏农业学报,2019,(04):868-873.[doi:doi:10.3969/j.issn.1000-4440.2019.04.017]
　YANG Yu chu,WANG Chen xi,ZHOU Xue ke,et al.Development and application of a reverse transcriptionloop mediated isothermal amplification (RTLAMP) assay for detection of Senecavirus A(SVA)[J].,2019,(04):868-873.[doi:doi:10.3969/j.issn.1000-4440.2019.04.017]
点击复制

A型塞内卡病毒RT-LAMP检测方法的建立及应用()
分享到：

江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2019年04期
页码:
868-873
栏目:
畜牧兽医·水产养殖
出版日期:
2019-08-31

文章信息/Info

Title:
Development and application of a reverse transcriptionloop mediated isothermal amplification (RTLAMP) assay for detection of Senecavirus A(SVA)
作者:
杨钰楚1; 王晨曦1; 周雪珂2; 赵军3; 徐志文1; 2; 朱玲1; 2
（1.四川农业大学动物医学院，四川成都611130；2.四川大学生命科学学院，四川成都611130；3.四川农业大学动物疫病与人类健康四川省重点实验室，四川成都611130）
Author(s):
YANG Yuchu1; WANG Chenxi1; ZHOU Xueke2; ZHAO Jun3; XU Zhiwen1; 2; ZHU Ling1; 2
（1.College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130， China； 2.College of Life Sciences, Sichuan University, Chengdu 611130, China； 3.Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu 611130， China)
关键词:
A型塞内卡病毒; RTLAMP; 快速检测
Keywords:
Senecavirus A; reverse transcriptionloop mediated isothermal amplification (RTLAMP); rapid detection
分类号:
Q9394
DOI:
doi:10.3969/j.issn.1000-4440.2019.04.017
文献标志码:
A
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（1×100～1×107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。
Abstract:
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 (1×1001×107copies) 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.

参考文献/References:

[1]BLOMSTRM A L, HAKHVERDYAN M, REIDS M, et al. A onestep reverse transcriptase loopmediated isothermal amplification assay for simple and rapid detection of swine vesicular disease virus[J]. J Virol Methods, 2008, 147: 188.
[2]BRACHTA J, O′HEARNE S, FABIAN W A, et al. Realtime reverse transcription PCR assay for detection of senecavirusA in swine vesicular diagnostic specimens[J]. PLoS ONE, 2016, 11: e0146211.
[3]BURKE MJ. Oncolytic seneca valley virus: past perspectives and future directions[J]. Oncolytic Virotherapy, 2016, 5: 81.
[4]CANNING P, CANON A, BATES J L, et al. Neonatal mortality, vesicular lesions and lameness associated with Senecavirus A in a U.S. sow farm[J]. Transbound Emerg Dis, 2016, 63: 373-78.
[5]FERONATO C, LEME R A, DINIZ J A, et al.Development and evaluation of a nestedPCR assay for Senecavirus A diagnosis[J]. Trop Anim Health Pro, 2017, 50: 1-8.
[6]FOWLE R, VERONICA L, RUSSELL H RANSBURGH, et al.Development of a novel realtime RTPCR assay to detect Seneca Valley virus associated with emerging cases of vesicular disease in pigs[J]. J Virol Methods, 2017, 239: 34.
[7]GIMENEZLIROLA G L, RADEMACHER C, LINHARES D,et al. Serological and molecular detection of Senecavirus A associated with an outbreak of Swine idiopathic vesicular disease and neonatal mortality[J]. J Clin Microbiol,2016, 54: 2082.
[8]GUOB Q, PIEYROP E, RADEMACHERC J, et al. Novel Senecavirus A in swine with vesicular disease, United States, july2015[J]. Emerg Infect Dis, 2016, 22(7): 1325-1327.
[9]AGNOL A M, OTONEL R A A, LEME R A,et al. A TaqManbased qRTPCR assay for Senecavirus A detection in tissue samples of neonatal piglets[J]. Mol Cell Probes, 2017, 33: 28-31.
[10]HALES L M, KNOWLES N L, REDDY P S, et al. Complete genome sequence analysis of Seneca Valley virus001, a novel oncolytic picornavirus[J]. J Gen Virol, 2008, 89: 1265.
[11]MORTON C L, HOUGHTON P J, KOLB E A, et al. Initial testing of the replication competent Seneca Valley virus (NTX010) by the pediatric preclinical testing program[J]. Pediatric Blood & Cancer, 2010, 55: 295.
[12]NOTOMI T. Loopmediated isothermal amplification of DNA[J]. Nucleic Acids Res, 2000, 28: 63.
[13]POIRIER J T, DOBROMILSKAYA I, MORIARTY WF,et al. Selective tropism of seneca valley virus for variant subtype small cell lung cancer[J]. J Natl Cancer I, 2013, 105: 1059.
[14]REDDY P S, BURROUGHS K D,HALES L M, et al. Seneca Valley virus, a systemically deliverable oncolytic picornavirus, and the treatment of neuroendocrine cancers[J]. J Natl Cancer I, 2007,99: 1623.
[15]RUDINC M, POIRIER J T, SENZER N N, et al. Phase I clinical study of Seneca Valley Virus (SVV001), a replicationcompetent picornavirus, in advanced solid tumors with neuroendocrine features[J]. Clinical Cancer Research An Official Journal of the American Association for Cancer Research,2011, 17: 888.
[16]SAENGCHUTO K, STOTTC J, WEGNER M, et al. The fulllength genome characterization, genetic diversity and evolutionary analyses of Senecavirus A isolated in Thailand in 2016[J]. Infection, Genetics and Evolution, 2018, 64: 32-45.
[17]VANNUCCI F A, LINHARES D C L, Barcellos D E S N, et al. Identification and complete genome of Seneca Valley Virus in vesicular fluid and sera of pigs affected with idiopathic vesicular disease, Brazil[J]. Transbound Emerg Dis, 2015, 62: 589-593.
[18]WU Q, ZHAO X, BAI Y, et al. The first identification and complete genome of Senecavirus A affecting pig with idiopathic vesicular disease in China[J]. Transbound Emerg Dis,2017, 64: 1633.
[19]YU L, BAXTER P A, ZHAO X, et al. A single intravenous injection of oncolytic picornavirus SVV001 eliminates medulloblastomas in primary tumorbased orthotopic xenograft mouse models[J]. Neurooncology, 2011, 13: 14-27.
[20]ZHAO X, WU Q, BAI Y, et al. Phylogenetic and genome analysis of seven SenecavirusA isolates in China[J]. Transbound Emerg Dis, 2017, 64: 2075-2082.
[21]SAITO R, MISAWA Y, MORIYA K, et al. Development and evaluation of a loopmediated isothermal amplification assay for rapid deteclion of Mycoplasma pneumoniae[J]. J Med Microbiol, 2005, 54(11)：1037-1041.
[22]POON L L, LEUNG C S, TASHIRO M, et al. Rapid detection of the severe acute respiratory syndrome (SARS) coronavirus by a loopmediated isot hermal amplification assay[J]. Clinical Chemistry, 2004, 50(6): 1050-1052.

备注/Memo

备注/Memo:
收稿日期：2019-05-15 基金项目：四川省科技支撑计划项目（2015NZ0072）；“十三五”育种攻关计划课题(2016NY20052) 作者简介： 杨钰楚(1998-)，女，成都人，本科，主要研究方向为猪病病原分子诊断技术。王晨曦、周雪珂为共同第一作者。 通讯作者：朱玲，（E-mail）abtczl72@126.com
更新日期/Last Update: 2019-08-31