猪圆环病毒3型LAMP检测方法的建立与应用

doi:10.11843/j.issn.0366-6964.2018.06.025

畜牧兽医学报 ›› 2018, Vol. 49 ›› Issue (6): 1314-1319.doi: 10.11843/j.issn.0366-6964.2018.06.025

猪圆环病毒3型LAMP检测方法的建立与应用

姜辰龙¹, 严秀文¹, 张日腾², 李勇², 姜平², 白娟^2*

1. 南京农业大学生命科学学院, 南京 210095;
2. 南京农业大学动物医学院农业部动物细菌学重点实验室, 南京 210095

收稿日期:2017-10-20 出版日期:2018-06-23 发布日期:2018-06-22
通讯作者: 白娟,副教授,硕士生导师,E-mail:baijuan@njau.edu.cn
作者简介:姜辰龙(1993-),男,江苏南通人,硕士,主要从事分子生物学研究
基金资助:
国家自然科学基金（31672565；31502082）；中央高校基本科研业务费专项资金资助项目（KJQN201616）；国家生猪现代产业体系建设专项（CARS-36）

Establishment and Application of LAMP Method for Detection of Porcine Circovirus Type 3

JIANG Chen-long¹, YAN Xiu-wen¹, ZHANG Ri-teng², LI Yong², JIANG Ping², BAI Juan^2*

1. Key Laboratory of Zoology of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China;
2. Key Laboratory of Animal Bacteriology of Ministry of Agriculture, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China

Received:2017-10-20 Online:2018-06-23 Published:2018-06-22

摘要/Abstract

摘要：

为快速诊断和检测猪圆环病毒3型（PCV3），本研究根据其ORF2基因保守序列，设计特异性引物，并通过Bst DNA聚合酶、引物最佳浓度比例、Mg²⁺、dNTPs和甜菜碱浓度及反应条件优化，建立了环介导等温核酸扩增（LAMP）检测方法。结果显示，59℃恒温扩增36 min即可出现梯形条带，且产物亦可通过SYBR Green Ⅰ染色进行判断。该方法最低检测限为1.0×10¹copies·μL^-1，与猪繁殖与呼吸综合征病毒（PRRSV）、猪伪狂犬病毒（PRV）、猪瘟病毒（CSFV）及猪圆环病毒1型和2型（PCV1、PCV2）等均无交叉反应。检测68份呼吸道病猪淋巴结样品，PCV3阳性率占30.9%，与PCR检测结果符合率为95.6%（65/68）。相对传统PCR方法而言，该方法敏感特异，简便快速，可用于PCV3检测和临床诊断。

关键词: 猪圆环病毒3型, RT-LAMP, 检测

Abstract:

In order to diagnose and detect porcine circovirus type 3 (PCV3), in this research, specific primers were designed according to the conserved sequence of ORF2 gene. By optimizing the concentration of Bst DNA polymerase, the ratio of primers, Mg²⁺, dNTPs, betaine and reaction condition, the loop-mediated isothermal nucleic acid amplification (LAMP) was established. The results manifested that ladder strips were observed after 36min with a constant temperature, and the product could be judged by SYBR Ⅰ staining. The detection limit of this way was 1.0×10¹ copies·μL^-1 which had no cross reaction with porcine reproductive and respiratory syndrome virus (PRRSV), porcine pseudorabies virus (PRV), classical swine fever virus (CSFV), porcine circovirus type 1 (PCV1) and type 2 (PCV2). From 68 lymph nodes of diseased pigs in respiratory system, the positive rate of 30.9% tested by LAMP had a high coincidence rate of 95.6% (65/68) with that by PCR detection. Compared with traditional technology, this method is more sensitive, simpler and can be used for detection and clinical diagnosis of PCV3.

Key words: PCV3, RT-LAMP, detection

中图分类号:

S852.659.2

姜辰龙, 严秀文, 张日腾, 李勇, 姜平, 白娟. 猪圆环病毒3型LAMP检测方法的建立与应用[J]. 畜牧兽医学报, 2018, 49(6): 1314-1319.

JIANG Chen-long, YAN Xiu-wen, ZHANG Ri-teng, LI Yong, JIANG Ping, BAI Juan. Establishment and Application of LAMP Method for Detection of Porcine Circovirus Type 3[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(6): 1314-1319.

参考文献

[1] GRAU-ROMA L,FRAILE L,SEGALES J.Recent advances in the epidemiology,diagnosis and control of diseases caused by porcine circovirus type 2[J].Vet J,2011,187(1):23-32.
[2] ALARCON P,RUSHTON J,WIELAND B. Cost of post-weaning multi-systemic wasting syndrome and porcine circovirus type-2 subclinical infection in England-an economic disease model[J]. Prev Vet Med, 2013, 110(2):88-102.
[3] FINSTERBUSCH T,MANKERTZ A.Porcine circoviruses-Small but powerful[J].Virus Res, 2009, 143(2):177-183.
[4] PHAN T G,GIANNITTI F,ROSSOW S,et al.Detection of a novel circovirus PCV3 in pigs with cardiac and multi-systemic inflammation[J].Virol J, 2016, 13:184.
[5] PALINSKI R,PIÑEYRO P,SHANG P C,et al.A novel porcine circovirus distantly related to known circoviruses is associated with porcine dermatitis and nephropathy syndrome and reproductive failure[J].J Virol, 2016, 91(1):e01879-16.
[6] KU X,CHEN F,LI P,et al.Identification and genetic characterization of porcine circovirus type 3 in China[J].Transbound Emerg Dis, 2017, 64(3):703-708.
[7] ZHENG S,WU X,ZHANG L,et al.The occurrence of porcine circovirus 3 without clinical infection signs in Shandong Province[J].Transbound Emerg Dis, 2017, 64(5):1337-1341.
[8] ZHANG C F,CUI S J,ZHU C.Loop-mediated isothermal amplification for rapid detection and differentiation of wild-type pseudorabies and gene-deleted virus vaccines[J].J Virol Methods, 2010, 169(1):239-243.
[9] 陈伯祥, 杨明, 常亮,等.环介导等温扩增技术在动物传染病诊断中的应用进展[J].动物医学进展, 2012, 33(4):90-96.
CHEN B X,YANG M,CHANG L, et al. Progress on loop-mediated isothermal ampplification in diagnosis of animal infectious disease[J].Progress in Veterinary Medicine, 2012, 33(4):90-96.(in Chinese)
[10] KIM H R,PARK Y R,LIM D R, et al. Multiplex real-time polymerase chain reaction for the differential detection of porcine circovirus 2 and 3[J].J Virol Methods, 2017, 250:11-16.
[11] WANG J C,ZHANG Y N,WANG J F,et al.Development of a TaqMan-based real-time PCR assay for the specific detection of porcine circovirus 3[J].J Virol Methods, 2017, 248:177-180.
[12] 湛洋,王东亮,王乃东,等.猪圆环病毒3型检测及其Cap结构序列和抗原性预测分析[J].畜牧兽医学报,2017,48(6):1076-1084.
ZHAN Y,WANG D L,WANG N D,et al.Survey on detection and analyses of cap antigenicity prediction of porcine circovirus type 3 isolated from partial provinces of Southern China[J].Acta Veterinaria et Zootechnica Sinica,2017,48(6):1076-1084.(in Chinese)
[13] NOTOMI T,OKAYAMA H,MASUBUCHI H,et al.Loop-mediated isothermal amplification of DNA[J].Nucleic Acids Res,2000,28(12):e63.
[14] 黄火清, 郁昂. 环介导等温扩增技术的研究进展[J].生物技术,2012,22(3):90-94.
HUANG H Q,YU A.Advances of loop-mediated isothermal amplification[J].Biotechnology, 2012, 22(3):90-94.(in Chinese)
[15] 戴婷婷,陆辰晨,郑小波.环介导等温扩增技术在病原物检测上的应用研究进展[J].南京农业大学学报, 2015, 38(5):695-703.
DAI T T,LU C C,ZHENG X B.Application research progress of loop-mediated isothermal amplification in the pathogenic microorganism[J].Journal of Nanjing Agricultural University, 2015, 38(5):695-703.(in Chinese)
[16] 庞笑语,王建昌,韩庆安,等.河北省猪群中检出猪圆环病毒3型[J].中国动物检疫, 2017, 34(11):70-73.
PANG X Y,WANG J C,HAN Q A, et al. Porcine circovirus type 3 was detected from swine herds in Hebei province[J].China Animal Health Inspection, 2017, 34(11):70-73.(in Chinese)

猪圆环病毒3型LAMP检测方法的建立与应用

Establishment and Application of LAMP Method for Detection of Porcine Circovirus Type 3

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	王素华, 帅江冰, 李舟, 袁淑辉, 吴绍强, 吕继洲, 赵治国. 炭疽杆菌双重荧光定量PCR检测技术的建立和应用[J]. 畜牧兽医学报, 2019, 50(8): 1658-1665.
[2]	王素华, 帅江冰, 李舟, 袁淑辉, 吴绍强, 吕继洲, 赵治国. 炭疽杆菌双重荧光定量PCR检测技术的建立和应用[J]. 畜牧兽医学报, 2019, 50(8): 1658-1665.
[3]	俞演, 古玉, 王承东, 胡静, 杨瑷宁, 杨乔, 李德生, 张和民, 邓林华, 凌珊珊, 左之才, 彭广能, 钟志军, 马晓平. 石膏样小孢子菌感染小鼠的Dectin-1、TLR-2和TLR-4及细胞因子动态变化[J]. 畜牧兽医学报, 2019, 50(7): 1449-1457.
[4]	俞演, 古玉, 王承东, 胡静, 杨瑷宁, 杨乔, 李德生, 张和民, 邓林华, 凌珊珊, 左之才, 彭广能, 钟志军, 马晓平. 石膏样小孢子菌感染小鼠的Dectin-1、TLR-2和TLR-4及细胞因子动态变化[J]. 畜牧兽医学报, 2019, 50(7): 1449-1457.
[5]	郑兰兰, 朱静静, 王盼, 舒燕, 梁青青, 李炳晓, 王超群, 魏战勇. 猪δ冠状病毒TaqMan荧光定量RT-PCR检测方法的建立与初步应用[J]. 畜牧兽医学报, 2019, 50(6): 1261-1267.
[6]	秦彤, 周灵, 由欣月, 梁琳, 史利军, 张建伟, 李金祥, 崔尚金. 犬细小病毒高效纳米PCR检测方法的建立及初步应用[J]. 畜牧兽医学报, 2019, 50(6): 1268-1274.
[7]	范慧, 李亮, 姜平, 王先炜, 李玉峰, 白娟. 塞内卡病毒A的RT-PCR检测方法的建立和应用[J]. 畜牧兽医学报, 2019, 50(6): 1312-1318.
[8]	郑兰兰, 朱静静, 王盼, 舒燕, 梁青青, 李炳晓, 王超群, 魏战勇. 猪δ冠状病毒TaqMan荧光定量RT-PCR检测方法的建立与初步应用[J]. 畜牧兽医学报, 2019, 50(6): 1261-1267.
[9]	秦彤, 周灵, 由欣月, 梁琳, 史利军, 张建伟, 李金祥, 崔尚金. 犬细小病毒高效纳米PCR检测方法的建立及初步应用[J]. 畜牧兽医学报, 2019, 50(6): 1268-1274.
[10]	范慧, 李亮, 姜平, 王先炜, 李玉峰, 白娟. 塞内卡病毒A的RT-PCR检测方法的建立和应用[J]. 畜牧兽医学报, 2019, 50(6): 1312-1318.
[11]	郭紫晶, 何琪富, 汤承, 张斌, 岳华. 检测纽布病毒的Real-time RT-PCR方法的建立与应用[J]. 畜牧兽医学报, 2019, 50(4): 893-900.
[12]	郭紫晶, 何琪富, 汤承, 张斌, 岳华. 检测纽布病毒的Real-time RT-PCR方法的建立与应用[J]. 畜牧兽医学报, 2019, 50(4): 893-900.
[13]	王俊伟, 陈芳洲, 库旭钢, 李畅, 何启盖. 基于PCV3-Cap蛋白间接ELISA检测方法的建立及临床应用[J]. 畜牧兽医学报, 2019, 50(2): 454-460.
[14]	王俊伟, 陈芳洲, 库旭钢, 李畅, 何启盖. 基于PCV3-Cap蛋白间接ELISA检测方法的建立及临床应用[J]. 畜牧兽医学报, 2019, 50(2): 454-460.
[15]	卢曾奎, 张利平, 李青, 刘恩民, 杜立新, 储明星, 魏彩虹. mRNA中N⁶-甲基腺苷修饰及其在动物中的研究进展[J]. 畜牧兽医学报, 2019, 50(1): 1-13.