传染性造血器官坏死病毒焦磷酸测序检测技术的建立

根据传染性造血器官坏死病毒(IHNV)基因序列,设计了IHNV最为保守的指纹序列,以及测序引物,建立了IHNV焦磷酸测序检测方法。对所构建的IHNV焦磷酸检测方法进行特异性试验和灵敏度检测。结果表明,所建立的方法特异性好,在8种鱼类病毒中能够特异性检测出目的病毒,检测方法灵敏度高,最低检出核酸量为10pg/μL。对建立的焦磷酸测序检测方法进行了实际应用研究,选取国内采集与进口的鱼类样本共计80批次进行IHNV检测。结果显示,焦磷酸测序检测方法可以有效的检出常规RTPCR不能检出的假阴性样本和弱阳性样本,该方法的灵敏度和特异性可以满足水生动物疫病检测的需要。

Development of Pyrosequencing Method for Detecting Infectious Hematopoietic Necrosis Virus

By collecting the sequences of IHNV,the fingerprint sequences and the sequencing primers of the viruse were designed.The pyrosequencing method for detection of IHNV was established successfully.The method has good specificity and sensitivity,and can specifically identify the objective viruses in the eight fish viruses.The method has high sensitivity and the minimum detection limit of nucleic acid is 10 pg/μL.The method was verified by the detection of IHNV in 80 batches of the samples collected from domestic and imported fishes.The results indicated that the pyrosequencing method can effectively detect the false negative and weakly positive samples,which the traditional method RT-PCR cannot detect.The specificity and sensitivity of the method met the requirement for detection of aquatic animal diseases.