检测施马伦贝格病毒核酸的荧光定量RT-PCR方法

doi:10.11843/j.issn.0366-6964.2014.11.012

Abstract

Abstract:

In the present study，a pair of primers and a TaqMan probe were designed based on the S-segment sequence of Schmallenberg virus (SBV)，and a real-time quantitative reverse transcription-PCR (RT-qPCR) for the detection of SBV nucleic acid was established.After optimization of the reaction system and condition，the method presented a good linear relationship with the RNA template ranging from 10¹ to 10⁷ 50% tissue culture infectious dose (TCID₅₀) SBV.By using SBV nucleic acid-positive samples (n=140)，SBV nucleic acid-negative samples (n=132)，and RNA samples (n=16) of related viruses within the Simbu serogroup，the sensitivity，specificity and reproducibility of the RT-qPCR were validated.All the samples used for validation were prepared and preserved by the Friedrich-Loeffler-Institut (FLI) of Germany.The results demonstrated that the developed RT-qPCR had a similar sensitivity with that of FLI and the concordance rate between the two assays was 96.4% (135/140)，that the RT-qPCR could sensitively detect 1 TCID₅₀ SBV RNA and it had a good reproducibility，and that the RT-qPCR showed no cross-reactivity with related viruses within the Simbu serogroup except for Douglas virus.The RT-qPCR was further used to detect the RNA samples extracted from sheep sera which were collected from a breeding farm located in Inner Mongolia (n=47) and from the sheep imported from Australia (n=47)，respectively.However，no SBV nucleic acid-positive sample was detected.The successful development of SBV RT-qPCR provides us with an efficient detection tool for dealing with Schmallenberg disease.

Key words: Schmallenberg virus (SBV), nucleic acid；primer, TaqMan probe, real-time quantitative reverse transcription-PCR (RT-qPCR)

CLC Number:

S852.659.5

ZHANG Yong-ning，WU Shao-qiang，LYU Ji-zhou，FENG Chun-yan，WANG Cai-xia，DENG Jun-hua，YUAN Xiang-fen，LIN Xiang-mei. A Real-time Quantitative Reverse Transcription-PCR for the Detection of Schmallenberg Virus Nucleic Acid[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(11): 1824-1829.

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[1]	ZHANG Yong-ning，SONG Shan-shan，WU Shao-qiang，LIN Xiang-mei. Expression of the Nucleocapsid Protein of Schmallenberg Virus Using the Bac-to-Bac^® Baculovirus Expression System and Preparation of Monoclonal Antibodies against This Protein [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(6): 1280-1286.
[2]	ZHANG Yong-ning，SONG Shan-shan，WU Shao-qiang，LIN Xiang-mei. Expression of the Nucleocapsid Protein of Schmallenberg Virus Using the Bac-to-Bac^® Baculovirus Expression System and Preparation of Monoclonal Antibodies against This Protein [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(6): 1280-1286.
[3]	ZHANG Yong-ning，WU Shao-qiang，LIN Xiang-mei. The Research Progress of Schmallenberg Disease [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(7): 1029-1037.
[4]	ZHANG Yong-ning，WU Shao-qiang，LIN Xiang-mei. The Research Progress of Schmallenberg Disease [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(7): 1029-1037.
[5]	ZHANG Yong-ning，WU Shao-qiang，LYU Ji-zhou，FENG Chun-yan，WANG Cai-xia，DENG Jun-hua，YUAN Xiang-fen，LIN Xiang-mei. A Real-time Quantitative Reverse Transcription-PCR for the Detection of Schmallenberg Virus Nucleic Acid [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(11): 1824-1829.
[6]	ZHANG Yong-ning, WU Shao-qiang, LYU Ji-zhou, FENG Chun-yan, WANG Cai-xia, YUAN Xiang-fen, DENG Jun-hua, LIN Xiang-mei, WERNIKE Kerstin. Prokaryotic Expression and Purification of the Nucleocapsid Protein of Schmallenberg Virus and Preparation of Its Polyclonal Antibodies [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2013, 44(10): 1629-1636.
[7]	ZHANG Yong-ning, WU Shao-qiang, LYU Ji-zhou, FENG Chun-yan, WANG Cai-xia, YUAN Xiang-fen, DENG Jun-hua, LIN Xiang-mei, WERNIKE Kerstin. Prokaryotic Expression and Purification of the Nucleocapsid Protein of Schmallenberg Virus and Preparation of Its Polyclonal Antibodies [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2013, 44(10): 1629-1636.

A Real-time Quantitative Reverse Transcription-PCR for the Detection of Schmallenberg Virus Nucleic Acid

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