一种鉴别尼帕病毒和高致病性猪繁殖与呼吸综合征病毒二重荧光RT-PCR检测方法的建立

doi:10.16431/j.cnki.1671-7236.2016.02.009

Abstract

Abstract: To establish a rapid,sensitive and specific assay for the differential detection of Nipah virus (NiV) and highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV),a duplex Real-time RT-PCR was developed with specific primers and probes targeting to the special sequences of NiV M gene and HP-PRRSV nsp2 gene by optimization of reaction conditions.The performance of the assay was linear ranging from 4.6×10¹ to 4.6×10⁷ copies/μL for RNA standard control of NiV M (NiV-M-RNA) and from 4.1×10¹ to 4.1×10⁸ copies/μL for RNA standard control of HP-PRRSV nsp2 (HP-PRRSV-nsp2-RNA),and detection limits of the assay was 46 copies for the NiV-M-RNA and 4.1 copies for the HP-PRRSV-nsp2-RNA,respectively.The coefficients of variation (CVs) of both inter-assay and intra-assay repeatability were less than 2.0%,showing good repeatability.The assay was able to specifically detect NiV and HP-PRRSV simultaneously without cross-reaction with classical swine fever virus (CSFV),porcine epidemic diarrhea virus (PEDV),swine influenza virus (SIV),porcine parvovirus (PPV),pseudorabies virus (PRV) and porcine circovirus type 2 (PCV2).Of the 236 samples from pigs for both NiV and HP-PRRSV detection by the established assay,all the samples were negative for NiV,8 samples were HP-PRRSV positive.In conclusion,this assay offers a useful approach for the differential detection of NiV and HP-PRRSV in clinical specimens from the pigs.

Key words: Nipha virus; highly pathogenic porcine reproductive and respiratory syndrome virus; TaqMan-MGB probe; duplex Real-time RT-PCR

CLC Number:

WANG Jian-hua, DONG Zhi-zhen, WANG Yu-ling, XIAO Yan, ZHAO Xiang-ping. Establishment of a Duplex Real-time RT-PCR Assay for the Differential Detection of Nipha Virus and Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus[J]. , 2016, 43(2): 348-355.

References

[1] Chua K B,Bellini W J,Rota P A,et al.Nipah virus:A recently emergent deadly paramyxovirus[J].Science,2000,288:1432-1435.
[2] Harcourt B H,Tamin A,Ksiazek T G,et al. Molecular characterization of Nipah virus,a newly emergent paramyxovirus[J]. Virology,2000,271:334-349.
[3] Chua K B,Koh C L,Hooi P S,et al.Isolation of Nipah virus from Malaysian Island flying-foxes[J].Microbes Infect,2002,4(2):145-151.
[4] 陈继明,王志亮,赵永刚,等.我国尼帕病毒病风险与防控措施初步分析[J].中国动物检疫,2005,1:51-53.
[5] Li Y,Wang J M,Andrew C,et al. Antibodies to Nipah or Nipah-like viruses in bats,China[J].Emerg Infect Dis,2008,14(12):1974-1976.
[6] Chua K B,Goh K J,Wong K T,et al.Fatal encephalitis due to Nipah virus among pig farmers in Malaysia[J].Lancet,1999,354:1257-1259.
[7] Tian K,Yu X,Zhao T,et al.Emergence of fatal PRRSV variants:Unparalleled outbreaks of atypical PRRS in China and molecular dissection of the unique hallmark[J].PLoS One,2007,2(6):e526.
[8] Kappes M A,Faaberg K S.PRRSV structure,replication and recombination:Origin of phenotype and genotype diversity[J]. Virology,2015,479-480:475-486.
[9] Yu X,Chen N,Wang L,et al.New genomic characteristics of highly pathogenic porcine reproductive and respiratory syndrome viruses do not lead to significant changes in pathogenicity[J].Vet Microbiol,2012,158(3-4):291-299.
[10] 王艳丰,张丁华,曹智高,等.高致病性猪繁殖与呼吸综合征协同感染及防制研究进展[J].中国畜牧兽医,2014,41(5):235-239.
[11] Bell M,Azad A K,Islam M R,et al.Duplex nested RT-PCR for detection of Nipah virus RNA from urine specimens of bats[J].J Virol Methods,2007,141(1):97-101.
[12] Guillaume V,Lefeuvre A,Faure C,et al.Specific detection of Nipah virus using Real-time RT-PCR (TaqMan)[J].J Virol Methods,2004,120(2):229-237.
[13] Yang K,Li Y,Duan Z,et al.A one-step RT-PCR assay to detect and discriminate porcine reproductive and respiratory syndrome viruses in clinical specimens[J].Gene,2013,531(2):199-204.
[14] 马志亮,孙亭亭,杨洁,等.猪繁殖与呼吸综合征活疫苗中病毒含量荧光定量PCR测定方法的建立及初步应用[J].中国畜牧兽医,2014,41(5):71-76.

Establishment of a Duplex Real-time RT-PCR Assay for the Differential Detection of Nipha Virus and Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 10

Recommended Articles

Metrics

Comments

[1]	WANG Bo, WANG Hui-yu, ZHAO Bao-hua, LUO Jing, WANG Cheng-min, HE Hong-xuan, HAN Xue-qing. Development and Application of Duplex Real-time RT-PCR Assay for Detection of H1 and H3 Subtype Swine Influenza Viruses [J]. , 2017, 44(10): 3035-3041.
[2]	LUO Si-si, XIE Zhi-xun, XIE Zhi-qin, DENG Xian-wen, LIU Jia-bo, XIE Li-ji, HUANG Li, HUANG Jiao-ling, ZENG Ting-ting, ZHANG Yan-fang, WANG Sheng. Establishment of a Duplex Real-time RT-PCR for Detection of H6N1 Subtype Avian Influenza Virus [J]. , 2016, 43(2): 326-332.
[3]	LUO Cheng-bo, CUI Min, KONG Ling-ping, CHENG Zhen-tao, ZHOU Bi-jun, MAO Li-hong. Molecular Characteristics Analysis of Two Strains of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus [J]. , 2014, 41(8): 50-56.
[4]	CHENG Bang-zhao，LIU Hua. Application of Armored Virus as Internal Control in the Detection of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus with Real-time RT-PCR [J]. , 2014, 41(7): 85-90.
[5]	ZHANG Xue-mei, ZHANG Yan-yan, JIANG Ying, HAO Mao-xian, ZOU Min-fa, WANG Qin-fu. Effect of Vaccination with Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Attenuated Live Vaccine at Gestation on Immunity in their Piglets [J]. , 2014, 41(7): 230-234.
[6]	ZHAO Wen-hua, YANG Shi-biao, GAO Hua-feng, WANG Jin-ping. Development of the Duplex Real-time RT-PCR Assay with TaqMan Probe of N-ITR Genes for Detection of Peste des Petits Ruminants Virus and Goat Pox Virus [J]. , 2014, 41(3): 65-71.
[7]	ZHANG Yan-fang, XIE Zhi-xun, XIE Li-ji, LIU Jia-bo, FAN Qing, PANG Yao-shan, LUO Si-si, DENG Xian-wen, XIE Zhi-qin. Development of Duplex Real-time RT-PCR Assay for Detection of Duck Tembusu Virus and Duck Plague Virus [J]. , 2014, 41(3): 77-82.
[8]	AN Chun-xia, YAN Ruo-qian, WU Zhi-ming, ZHAO Ming-jun, ZHAO Xue-li, WANG Dong-fang, LIU Shu-min. Establishment and Application of Duplex RT-PCR Assay for Detection of Highly and Lowly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus [J]. , 2013, (5): 66-70.
[9]	LIANG Yao-feng;GUO Xiao-feng;SONG Li;LIU Yang;. Establishment of TaqMan-MGB Fluorescence Quantitative RT-PCR Assay for Detection of Avian Infectious Bronchitis Virus H52 Strain [J]. , 2012, 39(4): 169-173.
[10]	QIU Shen-ben; SU Dan-ping; HUANG Ai-fang; LUO Ying-xia;ZHAO Zhi-quan; HE Dong-sheng. Isolation, Identification and Homology Analysis of Non-structural ProteinNsp2 Gene of High Pathogenic Porcine Reproductive and Respiratory Syndrome GD07b Strain [J]. , 2010, 37(3): 91-93.