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      2. 流行性出血病病毒血清分型RT-PCR方法的建立及應用
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        國家重點研發計劃項目 (2016YFD0500908;2017YFC1200505);云南省中青年學術和技術帶頭人后備人才培養項目(2017HB055)


        Development and utilization of RT-PCR assay for determining serotypes of epizootic haemorrhagic disease virus
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          摘要:

          以決定流行性出血病病毒(EHDV)血清型的第二基因節段(Seg-2)為檢測靶基因,設計7對特異性引物,建立用于鑒定EHDV血清型的RT-PCR檢測方法,并通過對擴增產物的測序,進一步了解病毒Seg-2的遺傳特性。特異性試驗結果顯示,該檢測方法可準確鑒定不同血清型的EHDV毒株,與藍舌病病毒、中山病病毒、阿卡斑病毒均無交叉反應;靈敏度試驗結果顯示,對不同血清型EHDV核酸的檢測下限均可達102拷貝;對我國不同時間與不同地域分離的EHDV毒株進行血清型RT-PCR鑒定,結果顯示分離的31株EHDV分屬EHDV-1、-5、-6、-7與-10型等5種血清型,與血清中和試驗的鑒定結果完全吻合。以上結果表明,該方法具有良好的特異性和較高的靈敏度,可快速準確地鑒定EHDV毒株的血清型。

          Abstract:

          Epidemic haemorrhagic disease virus (EHDV) is an arbovirus that seriously endangers ruminants. Currently,nine serotypes of EHDV have been discovered worldwide,and five of them are prevalent in China. Neutralization test is the gold standard for serotype identification of EHDV,which has the shortcomings of time-consuming,laborious and long experimental period. Therefore,establishing an accurate and rapid serotyping method for EHDV is of great significance for the prevention and control of this disease. In this study,the second gene segment (Seg-2),which determines the serotype of epizootic haemorrhagic disease virus (EHDV),was used as the target gene to establish a one-step RT-PCR method for serotype-specific detection of EHDV. The results of specificity test showed that the method could accurately identify different serotypes of EHDV,and had no cross-reaction with bluetongue virus,chuzan Virus and akabane virus. The results of sensitivity test showed that the detection limit of the EHDV nucleic acid for different serotypes could reach 102 copies. EHDV strains isolated from different times and different regions in China were identified by this one-step RT-PCR and the results showed that the 31 EHDV isolates belonged to five serotypes,inculding EHDV-1,-5,-6,-7 and -10. The results of the RT-PCR were completely consistent with the results of the serum neutralization test. The above results showed that the method established in this study had good specificity and high sensitivity,and could quickly and accurately identify the serotype of EHDV strains.

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        楊振興,李占鴻,宋子昂,李卓然,朱建波,廖德芳,楊恒.流行性出血病病毒血清分型RT-PCR方法的建立及應用[J].華中農業大學學報,2020,39(4):85-92

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        • 收稿日期:2019-09-04
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        • 在線發布日期: 2020-07-30
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