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Sci Rep:农科院特产所王凤雪研究组发表动物病毒快速诊断研究成果

摘要 : 近日, 国际学术权威刊物自然出版集团旗下子刊《Scientific Reports》在线发表了中国农业科学院特产研究所王凤雪研究员领衔的“特种动物病原与免疫科技创新团队”在动物病毒快速诊断方法研究突破进展文章。

 近日, 国际学术权威刊物自然出版集团旗下子刊《Scientific Reports》在线发表了中国农业科学院特产研究所王凤雪研究员领衔的“特种动物病原与免疫科技创新团队”在动物病毒快速诊断方法研究突破进展文章,研究题为“Reverse Transcription Cross-Priming Amplification-Nucleic Acid Test Strip for Rapid Detection of Porcine Epidemic Diarrhea Virus”文章提出交叉引物扩增(Cross-Priming Amplification)检测方法,该检测方法快速、灵敏且摆脱了试验仪器和条件的束缚,可以直接用于现场检测。

研究成果对危害我国规模化养殖中的重大病毒性传染病新型快速的诊断方法进行了深入研究。研究报告以国内外近年来危害养猪业较为严重的猪传染性胃肠炎为突破口,该病是一种高度接触性消化道传染病,以发病猪呕吐、水样腹泻和脱水为特征。OIE将其列为B类动物疫病。临床特征为腹泻、呕吐和脱水;主要发生在气温低的冬季和春季,成年猪和仔猪均可感染、以仔猪感染发病最为严重。5日龄内的仔猪染病,死亡率95%~100%,尤其在集约化养猪场,若预防和防治不力,会造成重大损失。

通常对发生腹泻的病猪确诊需根据流行病学、症状和病变进行判定作出诊断。且需与传染性腹泻、轮状病毒和仔猪大肠杆菌病相区别,确诊需要分离病毒或接种新生仔猪,对于生产实际当中很难普及。该团队建立的CPA诊断方法,灵敏度与RT-PCR相当;只需2-3个小时便可以判读结果;不需要离心机、PCR仪器和电泳槽等实验室仪器,即可完成检测。该研究报告中建立的针对猪传染性胃肠炎病毒核酸特异性的诊断方法,为现场快速准确的诊断该病提供了很好的解决方法。

王凤雪一直致力于病毒性传染病的新型疫苗和诊断方法的研究工作,先后建立了鉴别检测猪蓝耳病和犬瘟热疫苗免疫和野毒感染的PCR区分方法; 筛选得到了针对高致病性蓝耳病TJM疫苗株,天然缺失的非结构蛋白(NSP2)的单克隆抗体,为从血清学方面入手建立可用于免疫清群净化该病毒提供了有力的技术支撑。

CPA-NATS and gel electrophoresis results of different components and conditions optimization: (a) external primers, (b) probe, (c) cross primer, (d) dNTP, (e) MgSO4, (f) betaine, (g) Bst DNA polymerase, (h) reaction time of 60?min, (i) reaction time of 75?min, (j) reaction time of 90?min, and (k) agarose gel electrophoresis confirmations of the CPA reaction corresponding to the results on the strips. N, negative control.

原文链接:

Reverse Transcription Cross-Priming Amplification–Nucleic Acid Test Strip for Rapid Detection of Porcine Epidemic Diarrhea Virus

原文摘要:

Porcine epidemic diarrhea virus (PEDV) is a highly transmissible coronavirus that causes a severe enteric disease particularly in neonatal piglets. In this study, a rapid method for detecting PEDV was developed based on cross-priming amplification and nucleic acid test strip(CPA-NATS). Five primers specific for the N gene sequence of PEDV were used for the cross-priming amplification. Detection of amplification products based on labeled probe primers was conducted with strip binding antibody of labeled markers. The CPA method was evaluated and compared with a PCR method. The reverse transcription CPA system was further optimized for detecting PEDV RNA in clinical specimens. Results showed that the method was highly specific for the detection of PEDV, and had the same sensitivity as PCR, with detection limit of 10−6 diluted plasmid containing the target gene of PEDV. It was also successfully applied to detecting PEDV in clinical specimens. The reverse transcription CPA-NATS detection system established in this study offers a specific, sensitive, rapid, and simple detection tool for screening PEDV, which can contribute to strategies in the effective control of PEDV in swine.

来源: Scientific Reports 浏览次数:0

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