牛病毒性腹泻病毒链特异性SYBR Green荧光定量PCR方法的建立及应用

doi:10.11843/j.issn.0366-6964.2019.10.014

Abstract

Abstract: To quantitatively detect the positive and negative strand RNAs of bovine viral diarrhea virus (BVDV) during intracellular replication, the specific reverse transcription primers with non-viral nucleotide sequences for distinguishing the positive and negative strand RNAs of BVDV from each other and primers for real-time PCR were designed using DNAStar and Primer express 3.0 software. Primer pairs composed of real-time PCR primers and non-viral nucleotide sequences were used for the establishment of strand specific reverse transcription real-time quantitative PCR (ssRT-qPCR). The sensitivity, reproducibility and specificity of the ssRT-qPCR were evaluated. The dynamic changes of the positive and negative strand RNAs of BVDV during intracellular replication were described by the established ssRT-qPCR. Results were as follows:The standard curve of ss(+)RT-qPCR and ss(-)RT-qPCR had good linear relationships in the range of 10²-10⁷ copies of template with the linear correlation coefficients up to 0.998 1 and 0.995 3, respectively. Sensitivity test showed that the lowest detection limit of ss(+)RT-qPCR was 10 copies of template, while that of ss(-)RT-qPCR was 100 copies of template. The ssRT-qPCR was reproducibility with less than 1% of the coefficient of valuation. There were no cross-reactions with bovine parainfluenza virus type 3, infectious bovine rhinotracheitis virus, and bovine respiratory syncytial virus. The dynamic changes of the positive and negative strand RNAs of BVDV during replication in MDBK cells infected with different multiplicity of infection were analyzed using ssRT-qPCR. The results showed that the positive and negative strand RNAs of BVDV increased firstly, then decreased, and then gradually increased in MDBK cells inoculated with BVDV at 10 and 0.1 MOI. Meanwhile, the dynamic change of positive and negative strand RNAs of BVDV was different in MDBK cells infected with BVDV at 1 MOI which the overall upward trend. The positive and negative strand RNAs of BVDV eventually reached a plateau in MDBK cells inoculated with BVDV at 10 and 1 MOI after 36 hours. However, the positive and negative strand RNAs of BVDV eventually reached a plateau in MDBK cells inoculated with BVDV at 0.1 MOI after 24 hours. The applicability of ssRT-qPCR was further validated by BVDV chain specificity test. ssRT-qPCR of BVDV was established in this study and the dynamic changes of positive and negative strand RNAs of BVDV during intracellular replication were described. This study provided a research tool for the researches of the antiviral mechanisms of host proteins and replication and regulation mechanisms of BVDV.

CLC Number:

S852.659.6

LIU Cun, DENG Yong, LIANG Lin, LI Jinxiang, ZHANG Yanming, CUI Shangjin. Establishment and Application of Strand Specific SYBR Green Real-time PCR for Detection of Bovine Viral Diarrhea Virus[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(10): 2079-2087.

0
/ / Recommend

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

URL: https://www.xmsyxb.com/EN/10.11843/j.issn.0366-6964.2019.10.014

https://www.xmsyxb.com/EN/Y2019/V50/I10/2079

References 30

[1]	BROCK K V. The persistence of bovine viral diarrhea virus[J]. Biologicals, 2003, 31(2):133-135.
[2]	RIDPATH J F, NEILL J D, VILCEK S, et al. Multiple outbreaks of severe acute BVDV in North America occurring between 1993 and 1995 linked to the same BVDV2 strain[J]. Vet Microbiol, 2006, 114(3-4):196-204.
[3]	LACKNER T, MÜLLER A, PANKRAZ A, et al. Temporal modulation of an autoprotease is crucial for replication and pathogenicity of an RNA virus[J]. J Virol, 2004, 78(19):10765-10775.
[4]	FIRTH A E, ATKINS J F. A conserved predicted pseudoknot in the NS2A-encoding sequence of West Nile and Japanese encephalitis flaviviruses suggests NS1' may derive from ribosomal frameshifting[J]. Virol J, 2009, 6(1):14.
[5]	SCHWEIZER M, PETERHANS E. Pestiviruses[J]. Annu Rev Anim Biosci, 2014, 2:141-163.
[6]	GOYAL S M, RIDPATH J F. Bovine viral diarrhea virus:diagnosis, management, and control[M]. Ames:Wiley Blackwell 2005.
[7]	GINZINGER D G. Gene quantification using real-time quantitative PCR:an emerging technology hits the mainstream[J]. Exp Hematol, 2002, 30(6):503-512.
[8]	王洪梅, 贾春涛,胡桂学,等. 口蹄疫病毒链特异性荧光定量RT-PCR检测方法的建立[J]. 黑龙江畜牧兽医, 2015(11):159-163.
	WANG H M, JIA C T, HU G X, et al. Establishment of strand-specific reverse transcription quantitative PCR (ssRT-qPCR) assay for the detection of Foot-and-mouth disease virus[J]. Heilongjiang Animal Science and Veterinary Medicine, 2015(11):159-163. (in Chinese)
[9]	VASHIST S, URENA L, GOODFELLOW I. Development of a strand specific real-time RT-qPCR assay for the detection and quantitation of murine norovirus RNA[J]. J Virol Methods, 2012, 184(1-2):69-76.
[10]	顾潮江, 李勇,张玮莹,等. 口蹄疫病毒链特异性实时荧光定量RT-PCR技术的建立及应用[C]//第二届中国青年学者微生物遗传学学术研讨会论文集. 武汉:中国遗传学会微生物遗传学专业委员会, 2006:1.
	GU C J, LI Y, ZHANG W Y, et al. Establishment and application of strand-specific reverse transcription quantitative PCR assay for Foot-and-mouth disease virus[C]//Professional Committee of Microbial Genetics, Chinese Society of Genetics, the Second Chinese Young Scholars Symposium on Microbial Genetics. Wuhan:Professional Committee of Microbial Genetics, 2006:1.(in Chinese)
[11]	FENG L C, LINTULA S I, HO T H T, et al. Technique for strand-specific gene-expression analysis and monitoring of primer-independent cDNA synthesis in reverse transcription[J]. BioTechniques, 2012, 52(4):263-270.
[12]	RICHARDSON J, MOLINA-CRUZ A, SALAZAR M I, et al. Quantitative analysis of dengue-2 virus RNA during the extrinsic incubation period in individual Aedes[J]. Amer J Trop Med Hyg, 2006, 74(1):132-141.
[13]	HAIST K, ZIEGLER C, BOTTEN J. Strand-specific quantitative reverse transcription-polymerase Chain reaction assay for measurement of arenavirus genomic and antigenomic RNAs[J]. PLoS One, 2015, 10(5):e0120043.
[14]	KOMURIAN-PRADEL F, PERRET M, DEIMAN B, et al. Strand specific quantitative real-time PCR to study replication of hepatitis C virus genome[J]. J Virol Methods, 2004, 116(1):103-106.
[15]	LAGAYE S, BRUN S, GASTON J, et al. Anti-hepatitis C virus potency of a new autophagy inhibitor using human liver slices model[J]. World J Hepatol, 2016, 8(21):902-914.
[16]	刘存,崔尚金. 牛病毒性腹泻病毒SYBR Green荧光定量PCR方法的建立及其应用[J]. 现代畜牧兽医, 2018(12):9-13.
	LIU C, CUI S J. Establishment and application of a SYBR Green real-time PCR assay for detection of bovine viral diarrhea virus[J]. Modern Journal of Animal Husbandry and Veterinary Medicine, 2018(12):9-13. (in Chinese)
[17]	LESTER S N, LI K. Toll-like receptors in antiviral innate immunity[J]. J Mol Biol, 2014, 426(6):1246-1264.
[18]	KUMAR H, KAWAI T, AKIRA S. Pathogen recognition by the innate immune system[J]. Int Rev Immunol, 2011, 30(1):16-34.
[19]	YONEYAMA M, ONOMOTO K, JOGI M, et al. Viral RNA detection by RIG-I-like receptors[J]. Curr Opin Immunol, 2015, 32:48-53
[20]	HOLL E K, ALLEN I C, MARTINEZ J. Holding the inflammatory system in check:TLRs and NLRs[J]. Mediators Inflamm, 2016, 2016:8156816.
[21]	NAN Y C, NAN G X, ZHANG Y J. Interferon induction by RNA viruses and antagonism by viral pathogens[J]. Viruses, 2014, 6(12):4999-5027.
[22]	杨超,陈舜,汪铭书,等. OAS蛋白结构与抗病毒机制关系的研究进展[J]. 中国预防兽医学报, 2017, 39(8):682-685.
	YANG C, CHEN S, WANG M S, et al. Advances in the study of the relationship between the structure of OAS Protein and its Antiviral Mechanisms[J]. Chinese Journal of Preventive Veterinary Medicine, 2017, 39(8):682-685. (in Chinese)
[23]	SEN G C, PETERS G A. Viral stress-inducible genes[J]. Adv Virus Res, 2007, 70:233-263.
[24]	BERGMANN M, GARCIA-SASTRE A, CARNERO E, et al. Influenza Virus NS1 protein counteracts PKR-mediated inhibition of replication[J]. J Virol, 2000, 74(13):6203-6206.
[25]	DEB A, ZAMANIAN-DARYOUSH M, XU Z, et al. Protein kinase PKR is required for platelet-derived growth factor signaling of c-fos gene expression via Erks and Stat3[J]. EMBO J, 2001, 20(10):2487-2496.
[26]	SEN A, PRUIJSSERS A J, DERMODY T S, et al. The early interferon response to rotavirus is regulated by PKR and depends on MAVS/IPS-1, RIG-I, MDA-5, and IRF3[J]. J Virol, 2011, 85(8):3717-3732.
[27]	RAFIQUE S, IDREES M, ILYAS M, et al. Positional effect of phosphorylation sites 266 and 267 in the cytoplasmic domain of the E2 protein of hepatitis C virus 3a genotype:interferon Resistance analysis via Sequence Alignment[J]. Virol J, 2011, 8(1):204.
[28]	SAWAI H, HIRANO A, MORI H, et al. Synthesis, characterization, and biological properties of 8-azido-and 8-amino-substituted 2', 5'-oligoadenylates[J]. J Med Chem, 2003, 46(23):4926-4932.
[29]	MORIN B, RABAH N, BORETTO-SOLER J, et al. High yield synthesis, purification and characterisation of the RNase L activators 5'-triphosphate 2'-5'-oligoadenylates[J]. Antiviral Res, 2010, 87(3):345-352.
[30]	SHIBATA S, KAKUTA S, HAMADA K, et al. Cloning of a novel 2', 5'-oligoadenylate synthetase-like molecule, Oasl5 in mice[J]. Gene, 2001, 271(2):261-271.

Establishment and Application of Strand Specific SYBR Green Real-time PCR for Detection of Bovine Viral Diarrhea Virus

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References 30

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	HUANG Jin, LI Siyuan, MAO Li, CAI Xuhang, XIE Lingling, WANG Fu, ZHOU Hua, LI Jizong, LI Bin. Eukaryotic Expression of Bovine Coronavirus S1 Protein and Establishment and Application of Indirect ELISA [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 2050-2060.
[2]	MA Rumeng, ZHAO Yuliang, MA Mingshuang, GUO Guihai, LIU Xinzi, LI Jiaxuan, CUI Wen, JIANG Yanping, SHAN Zhifu, ZHOU Han, WANG Li, QIAO Xinyuan, TANG Lijie, WANG Xiaona, LI Yijing. Comparative Study on the Immune Response Induced by the Different Porcine Receptor Bacteria with Expressing the Protective Antigen S1 of Porcine Epidemic Diarrhea Virus [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 2090-2099.
[3]	XU Hong, SHANG Hongqi, ZHANG Xue, QIAN Jiali, WANG Chuanhong, SONG Xu, BAO Meiying, LIU Shiyu, ZHANG Gege, GUO Rongli, ZHAO Yongxiang, FAN Baochao, LI Bin. Inhibition Effect of C8orf4 Gene Encoding Protein on in vitro Replication of Porcine Epidemic Diarrhea Virus [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 2100-2108.
[4]	XIONG Ting, HE Xianming, ZHAO Xiya, ZHUANG Tingting, HUANG Meizhen, LIANG Shijin, YU Chuanzhao, LIANG Xuejing, CHEN Ruiai. Whole Genome Analysis of Three Predominant Epidemic Strains of Chicken Infectious Bronchitis Virus and Their Pathogenicity [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 2109-2122.
[5]	WANG Jing, ZHANG Shujuan, HU Xia, LIU Xiangyang, ZHANG Xingcui, SONG Zhenhui. CD44 Regulates Na⁺/H⁺ Exchanger 3 Activity by Influencing Porcine Epidemic Diarrhea Virus Replication [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 2176-2185.
[6]	LIN Lili, ZHANG Mengdi, ZHU Linlin, MA Hailong, SUN Qi, HE Qigai, ZHANG Mengjia, LI Wentao. Establishment of Neutralizing Antibody Detection Method based on Recombinant Fluorescent Virus of Porcine Epidemic Diarrhea Virus GⅡb Strain [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(4): 1649-1660.
[7]	JING Yang, WANG Yumiao, LI Yang, CHANG Hui, MA Zhiqian, LI Zhiwei, XIAO Shuqi. Construction of MARC-145^ORF6 Cell Line Stably Overexpressing PRRSV M Protein and Its Effect on PRRSV Proliferation [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(3): 1159-1169.
[8]	YU Qisheng, ZHU Qing, ZHOU Qun, SONG Xin, ZHANG Jiaqi, CHEN Taoyun, XU Lin, ZHANG Chaohui, ZHANG Bin. Expression of BCoV Spike Protein by Baculovirus Expression System and Its Immunogenicity in Mice [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(2): 640-648.
[9]	ZHANG Jinpeng, CHEN Cuiteng, LIN Lin, FU Huanru, LI Zhaolong, JIANG Bin, HUANG Yu, WAN Chunhe. Establishment of a Real-time Fluorescent RT-PCR Assay for Detection of Pigeon Megrivirus [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(2): 860-866.
[10]	SONG Wenyan, ZHANG Hanwen, WU Aodi, ZHANG Liyan, LIU Zhao, YE Tongtong, CHEN Chuangfu, SHENG Jinliang. Screening of Nanobodies against Porcine Reproductive and Respiratory Syndrome Virus GP5 Protein and Exploration of Their Inhibitory Effect on Virus Replication [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 258-270.
[11]	WANG Ying, LI Jiakang, ZENG Yue, SHI Kaituo, WEI Liting, PENG Jiajia, LI Qiuyan, CAO Longlong, ZHOU Dengyuan. Development of Monoclonal Antibodies against Feline Calicivirus [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 401-405.
[12]	LI Huihui, DONG Keer, LIU Xinbo, ZHANG Chunxiao, MA Chao, CHEN Liping, ZHONG Qi, YAO Gang, MA Xuelian. Establishment and Application of Rapid Detection Method for Bovine Norovirus and Bovine Rotavirus Dual RAA-LFD [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 406-412.
[13]	ZHOU Jianhao, WANG Dongfang, LIU Ying, WANG Shujuan, MA Zhenyuan, XIE Caihua, ZHAO Xueli, YANG Haibo, FENG Guidan, KANG Taisheng, HU Yufeng, LI Bowen, YAN Ruoqian. Establishment and Preliminary Application of a Quantitative Droplet Digital PCR Assay for Porcine Epidemic Diarrhea Virus [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 413-418.
[14]	ZHANG Jiaqi, JIA Xining, ZHOU Qun, SONG Xin, ZHU Chenxi, LI Gege, ZHANG Bin. Construction of BVDV-1 Virus-like Particles based on E2 Protein and Evaluation of Its Immune Efficacy in Mice [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 5143-5153.
[15]	ZHOU Likun, DONG Xinyuan, LI Jiahui, CUI Zhiying, ZHAO Shijie, XU Jie, CHEN Jing, ZHANG Yina, XIA Ping’an. Regulatory Role of MID2 on Porcine Reproductive and Respiratory Syndrome Virus Replication [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(11): 4735-4744.