鹅细小病毒强毒PCR检测方法的建立

参照GenBank中登录的鹅细小病毒 (GPV)B株的全基因序列 ,针对GPV的VP3保守基因设计了 1对引物 ,建立了GPV的PCR检测方法。采用该方法检测GPV能够扩增出预期大小约 4 41bp的特异性片段 ,而对鸭瘟病毒 (DPV)、鹅源致病性大肠埃希氏菌 (E .coli)O8和O1、雏鹅新型病毒性肠炎病毒 (NGVEV)呈阴性反应。该法检测GPV核酸的灵敏度可达 0 .4 7pg ;对GPV强毒皮下注射感染雏鹅各器官的检测结果表明 ,感染后 8h即可从心、肝病料中检出病毒DNA ,感染后 2 4h可从延髓、胸腺、胰腺、十二指肠、空肠、盲肠、腔上囊、心、肝、脾、肺、肾、血液、小脑、直肠、肌肉、粪便中检出GPVDNA ,感染 4 8h后可从骨髓中检出病毒DNA ,感染 312h后仍能从十二指肠、空肠、盲肠、心、肝、肾、粪便中检出病毒DNA。病毒分离阳性的可疑病料PCR检测为阳性 ,对临床送检病料的检测结果表明 ,PCR的敏感性显著高于病毒分离。

Development and application of PCR to detecting goose parvovirus

With primers based on the complete nucleotide sequence of goose parvovirus strain B from GenBank,the PCR method for goose parvovirus detection was developed.Using this method the specific fragment about 441 bp in length could be amplified from GPV samples, but not be amplified from duck plague virus, Escherichia coli O (1)and O (8), new-type gosling viral enteritis virus (NGVEV)samples. By this method a minimal sample of 0.47 pg GPV DNA could be detected. The distribution regularity of virulent GPV in artificially-infected gosling body was studied using the PCR. The results were as follows. At hour 8 post-infection(PI), the GPV DNA could be detected from heart and liver. At hour 24 PI, the GPV DNAcould be detected from mudulla oblongata, thymus, pancreas, duodenum,small intestine,appendix,bursa fabricius, heart, liver spleen, lung,kidney, blood, cerebellum, rectum,skeletal muscle and feces; At hour 48 PI, the GPV DNA could be detected from bone marrow. At hour 312 PI, the GPV DNA could still be detected from duodenum, small intestine,appendix,heart,liver,kidney and feces. The results of detecting samples from field infected goslings indicated that when virus isolation was positive,PCR result was also positive, and PCR was more sensitive thanvirus isolation. It was confirmed from the ablove-mentioned results that the PCR is a rapid, specific andsensitivemethod to detect GPV.