鲤春病毒血症病毒焦磷酸测序检测技术的建立

Development of Pyrosequencing Method for Detecting Spring Viremia of Carp Virus

通过收集鲤春病毒血症病毒基因序列,设计了鲤春病毒血症病毒最为保守的指纹序列,以及测序引物,建立了鲤春病毒血症病毒焦磷酸测序检测方法。对所构建的鲤春病毒血症病毒焦磷酸检测方法进行特异性试验和灵敏度检测,试验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批次进行鲤春病毒血症病毒检测。结果显示,焦磷酸测序检测方法可以有效的检出常规RT-PCR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。

By collecting the sequences of spring viremia of carp virus（SVCV）,the fingerprint sequences and the sequencing primers of the virus were designed and the pyrosequencing method for detection of SVCV was established successfully.The specificity and sensitivity of the method is well,which can specifically identify the objective virus in the eight fish viruses.The method has highly sensitivity and the minimum nucleic acid detection limit of 10pg/μL.The method was verified by the detection of SVCV in 80 batches of the samples collected from domestic and imported fishes.The results indicate that the pyrosequencing method can effectively detect the false negative and weakly positive samples,while the traditional method RT-PCR cannot detect them in these situations.The specificity and sensitivity of the method meet the requirement for detection of aquatic animal diseases.