猪圆环病毒2型交叉引物恒温扩增检测方法的建立

doi:10.11843/j.issn.0366-6964.2016.09.020

Abstract

Abstract:

This experiment was conducted to establish a simple，sensitive and rapid detection method for the biosecurity control of the porcine circovirus 2 (PCV2) diseases.The Cap gene of PCV2 was used as the signature sequence for cross amplification primers design and the constructed Cap gene plasmid DNA was used as the standard template.Amplification temperature，time and constituents of the reaction，including concentration of primers，MgSO4，dNTP，Betaine and Bst DNA polymerase were systematically optimized.Used the nucleic acid test strip detect the amplified products，compared the sensitivity，specificity and consistency of CPA，PCR and ELISA by 127 standard clinical samples.The detection results showed that the cross amplification could be realized at 58 ℃ within 50 min，could specifically detect PCV2 with a sensitivity that was about 10 times higher than conventional PCR method.The detection limit of recombinant plasmid was 7.6 copies•μL^-1 and the assay gave no amplification from PCV1，PRV，CSFV，PRRSV，PEDV，TGEV and RV.The final optimal CPA condition established for rapid detection of PCV2 was as follows：0.5 μmol•L^-1 each of F3 and B3，1.0 μmol•L^-1 of CPR，0.7 μmol•L^-1 of DF5F，0.9 μmol•L^-1 of DF5B，0.075 mol•L^-1 Betaine，3 mmol•L^-1 MgSO₄， 0.4 mmol•L^-1 dNTPs，9.6 U Bst DNA polymerase，2 μL 10×ThermoPol Reaction Buffer，4 μL templates and sterilized double-distilled water in 20 μL volumes.The sensitivity，specificity and consistency were 78.08% to 94.52%，87.03% to 92.59% and 84.25% to 91.34% among the three methods，respectively.Receiver operating characteristic curve showed that CPA was the best detection method. PCV2 cross priming amplification method does not require expensive equipment and skilled technicians，the visual detection could provide an intuitive and objective decision for people，and could also effectively avoid the aerosol pollution，which could be used in the on-site diagnosis for effective prevention and control of porcine circovirus 2 diseases.

CLC Number:

S852.659.2

YUAN Dan-yi，JIN Ya-nan，LI Ling，HE Qian，HU Lin，LI Xiao-qi，SUN Zhi-yong. Establishment of Cross Priming Isothermal Amplification for Porcine Circovirus 2 Detection[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(9): 1905-1913.

0
/ / Recommend

Add to citation manager EndNote|Reference Manager|ProCite|BibTeX|RefWorks

URL: https://www.xmsyxb.com/EN/10.11843/j.issn.0366-6964.2016.09.020

https://www.xmsyxb.com/EN/Y2016/V47/I9/1905

References

［1］SEGALÉS J，SITJAR M，DOMINGO M，et al.First report of post -weaning multisystemic wasting syndrome in pigs in Spain［J］. Vet Rec，1997，141(23)：600-601.
［2］MANKERTZ A，DOMINGO M，FOLCH J M，et al.Characterisation of PCV-2 isolates from Spain，Germany and France［J］. Virus Res，2000，66（1）：65-77.
［3］GARKAVENKO O，ELLIOTT R B，CROXSON M C.Identification of pig circovirus type 2 in new zealand pigs［J］.Transplant Proc，2005，37：506-509.
［4］芦银华，谈国蕾，陈德胜，等.猪圆环病毒多重PCR检测方法的建立及初步应用［J］.农业生物技术学报，2002，10(4)：415-416.
LU Y H，TAN G L，CHEN D S，et al.The development and preliminary application of multiplex PCR for detection of PCV［J］.Chinese Journal of Agricultural Biotechnology，2002，10(4)：415-416.(in Chinese)
［5］史利军，任林柱，李刚.猪圆环病毒2型免疫抑制激励研究进展［J］.动物医学进展，2009，30(3)：74-77.
SHI L J，REN L Z，LI G..Progress on immunosuppression mechanism of PCV 2［J］.Progress in Veterinary Medicine，2009，30(3)：74-77.(in Chinese)
［6］郝立沙，张璐，佟杰，等.猪圆环病毒Ⅱ型TaqMan荧光定量PCR检测方法的建立及应用［J］.黑龙江畜牧兽医，2015，06：168-171.
HAO L S，ZHANG L，TONG J，et al.Establishment and application of TaqMan fluorescent probe-based quantitative PCR assay for detection of porcine circovirus type 2［J］.Heilongjiang Animal Science and Veterinary Medicine，2015，06：168-171.(in Chinese)
［7］杨泽晓，侯义宏，曾辉，等.猪圆环病毒2型LAMP检测方法的建立与应用［J］.中国兽医科学，2014，44(2)：152-158.
YANG Z X，HOU Y H，ZENG H，et al.Development and application of LAMP method for detection of porcine circovirus type 2［J］.Chinese Veterinary Science，2014，44(2)：152-158.(in Chinese)
［8］王璐璐.交叉引物扩增技术快速检测肠出血性大肠杆菌的研究［D］.长春：吉林大学，2013.
WANG L L.Study on development of cross priming amplification method for rapid detection of EHEC［D］.Changchun：Jilin University，2013.(in Chinese)
［9］MAHÉ D，BLANCHARD P，TRUONG C，et al.Differential recognition of ORF 2 protein from type 1 and type 2 porcine circoviruses and identification of immunorelevant epitopes［J］.J Gen Virol，2000，81(7)：1815-1824.
［10］李聪慧，徐进，刘文枝，等.锦鲤疱疹病毒单交叉引物等温扩增检测方法的建立［J］.水产学报，2015，39(9)：1422-1431.
LI C H，XU J，LIU W Z，et al.Establishment of single cross priming isothermal amplification for Koi herpesvirus detection［J］.Journal of Fisheries of China，2015，39(9)：1422-1431.(in Chinese)
［11］翟聪聪，黄新，朱水芳，等.一种交叉引物扩增技术检测转基因水稻品系Bt63方法的建立［J］.植物检疫，2014，28(1)：61-66.
ZHAI C C，HUANG X，ZHU S F，et al.Cross priming amplifi cation method for detection of the transgenic rice Bt 63［J］.Plant Quarantine，2014，28(1)：61-66.(in Chinese)
［12］MARSHALL P L，KING J L，BUDOWLE B，et al.Utility of amplification enhancers in low copy number DNA analysis［J］.Int J Legal Med，2015，129(1)：43-52.
［13］XU Y M，LIU X L，MA J.Simple，specific，sensitive and rapid loop-mediated method for detecting Yersinia enterocolitica［J］.Southeast Asian J Trop Med Public Health，2014，45(3)：670-679.
［14］Hwang J，Suh S S，Park M，et al.Detection of coat protein gene of nervous necrosis virus using loop-mediated isothermal amplification［J］.Asian Pac J Trop Med，2016，9(3)：235-240.
［15］胡瑞丽，倪建平，赵笑.猪圆环病毒2型LAMP可视化快速检测方法的研究［J］.上海农业学报，2016，32(1)：23-28.
HU R L，NI J P，ZHAO X.Visual rapid detection of porcine circovirus type 2 by a loop-mediated isothermal amplification assay［J］.Acta Agriculturae Shanghai，2016，32(1)：23-28.(in Chinese)

[1]	FAN Jinquan, ZHANG Yuhang, TANG Wuyang, ZHAO Xinyu, LI Pishun, ZHENG Xiaofeng. Inhibitory Effect of Decitabine on Porcine Circovirus Type 2 in vitro [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 5134-5142.
[2]	SHANG Jinyuan, YAN Manping, YE Jingfei, CHENG Yuening, WANG Zhenjun, FENG Erkai, WANG Chunxia, ZHAO Yan, ZHU Xianpeng, LIAO Yuanjun, LUO Guoliang. Research Progress on Virus Infection and Its Anti-tumor Mechanisms of Protoparvovirus [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(11): 4551-4559.
[3]	LI Chang, ZHOU Ping, WEN Ping, YANG Keli, LIU Wei, GUO Rui, LIU Zewen, YUAN Fangyan, GAO Ting, SONG Haofei, TIAN Yongxiang, ZHOU Danna. Investigation of the Genome-wide Sequence and Immunogenicity of Capsid Protein in Mice for a Porcine Circovirus 2d Genotype Strain [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(10): 4289-4299.
[4]	KU Xugang, YU Xuexiang, SUN Qi, LI Panpan, ZHANG Mengjia, LUO Rui, QIAN Ping, HE Qigai. Isolation and Pathogenicity Analysis of Porcine Circovirus Type 3 [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(4): 1568-1578.
[5]	JIA Hanxiao, YUAN Honggen, LI Zhen, GUAN Shuaiyin, ZHANG Jinhua, SONG Yunfeng. Screening of Proteins Interacting with PCV2 Replication Initiation Region and Study on the Regulation of Virus Replication by PARP1 Protein [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(10): 3511-3521.
[6]	CHENG Baoyu, LI Zihe, CUI Yanlei, LI Jiahui, YANG Xinwei, YU Yongle, YANG Haiyan, SHAN Hu, ZHANG Chuanmei. Genetic Evolution of Feline Parvovirus and Pathogenicity of an Isolated Strains [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(9): 3121-3131.
[7]	HE Qing, LI Zhoumian, WANG Huan, LI Dantong, TAN Qinghui, ZOU Yawen, SU Jianming, DENG Zhibang, YANG Yi, WANG Naidong. Detection and Genetic Evolution of Porcine Circovirus Type 3 in Swine Aborted Fetus [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(5): 1527-1535.
[8]	ZHOU Qun, YANG Yuan, SONG Xin, HE Xinyi, CAO Hui, ZHANG Bin. Detection of Feline Parvovirus and Mutation Analysis of VP2 Gene in Chengdu from 2017 to 2021 [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(4): 1303-1309.
[9]	WANG Mingrui, WU Junhua, WANG Fei, SHAO Hongxia, QIAN Kun, YE Jianqiang, QIN Aijian. Development and Epitope Identification of a Monoclonal Antibody against VP2 Protein of Chicken Infectious Anemia Virus [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(2): 597-606.
[10]	LI Shaohan, YOU Xinyue, FAN Junwen, XU Yi, HAO Yunfeng, CUI Shangjin, QIN Tong. Sequence Analysis of the Complete VP2 and NS1 Genes of 14 Canine Parvovirus Isolates in Beijing [J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(1): 262-267.
[11]	WANG Shubo, XU Yigang, CHEN Qiuyan, MEI Zhuyuan, CUI Wen, JIANG Yanping, ZHOU Han, WANG Li, QIAO Xinyuan, LI Yijing, TANG Lijie. Analysis of Immune Response Induced by Recombinant Lactobacillus reuteri Expressing Cap Protein of Porcine Circovirus Type 2 in Mice [J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(9): 2238-2249.
[12]	CHEN Hongbo, DUAN Dianning, HU Yao, YANG Runze, HONG Qixiang, LI Yu, QIU Longxin, YANG Xiaoyan. Identification of Exosomes in Porcine Circovirus Type 2 Infected PK-15 Cells and Its Effects on Cell Proliferation and Apoptosis [J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(7): 1688-1698.
[13]	WU Yanhong, ZHAO Yongqiang, WEI Tao, ZHANG Xiuting, CONG Li, YUE Zhigang, XIAO Jiamei, SHAO Xiqun. Cloning and Epitopes Analysis of VP2 and NS1 Proteins of Raccoon Dog and Arctic Fox Amdoparvovirus [J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(6): 1399-1407.
[14]	QIN Tong, ZHOU Ling, YOU Xinyue, LIANG Lin, SHI Lijun, ZHANG Jianwei, LI Jinxiang, CUI Shangjin. Establishment and Application of Nano PCR Assay for Detection of the Canine Parvovirus [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(6): 1268-1274.
[15]	WU Leyi, WU Sufang, CHE Ying, ZHANG Qiang, LI Peng, BIAN Guanglin, WANG Xuannian. The Establishment of CHO-K1 Cell Line Stably Expressing High Level PCV2-Cap and Immunogenicity Analysis [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(5): 1056-1063.

Establishment of Cross Priming Isothermal Amplification for Porcine Circovirus 2 Detection

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments