禽波纳病毒分离鉴定及其恒温扩增检测分析  

Isolation and Rapid Detection of Avian Borna Virus by a Reverse Transcription Loop-mediated Isothermal Amplification Assay for Outbreaks in Psittacine Birds

作　　者：田纯见[1] 王宏[1] 罗琼[1] 林志雄[1] 赵吟[1] 罗长保[1] 鱼海琼[1] 刘志玲[1] 陈茹[1] 唐羿 周小明[1] 常彦磊 吴晓薇[1] 朱道中[1] 

机构地区：[1]广东出入境检验检疫局检验检疫技术中心,广州510623 [2]广州迪澳生物科技有限公司,广州510631

出　　处：《畜牧兽医学报》2012年第11期1847-1854,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：出入境检验检疫科研项目(2009-GDK16);质检公益性行业科研专项(10-65)

摘　　要：利用腺胃扩张症(PDD)患病鹦鹉腺胃RT-PCR阳性病料,接种猪睾丸(ST)传代细胞,分离禽波纳病毒(ABV),建立实时RT-LAMP检测方法。将阳性病料接种ST细胞单层传代,出现细胞圆缩、脱落,ABV基质蛋白(M)基因扩增产物出现预计大小351bp条带,测序后进化树分析显示为ABV5基因型。针对M基因设计ID37、ID30、ID19、ID6和ID1共5组引物,后3组引物RT-LAMP呈阳性反应。利用钙黄绿素建立实时RT-LAMP,分别在36(ID30)、38(ID37)和49(ID19)min出现扩增反应曲线,60min内扩增达到峰值。对各种临床样品检测与RT-PCR结果一致,新城疫等类症病毒未见阳性反应,显示较高的特异性;对细胞培养物检测10-1～10-5为阳性,比较RT-PCR敏感性提高约100倍。RT-LAMP检测方法的建立为PDD防制提供新的检测方法,也是波纳病公共卫生研究有益的参考。In this study an avian bornavirus（ABV） strain was isolated from sick parrots with proventricular dilatation disease（PDD）. The virus grew in swine testicular （ST） cell monolayer with granulating, shrinking, rounding and falling off although classical Borna disease virus strains replicate very efficiently in cultured mammalian cells in which persistent, noncytolytic infections was readily established. Viruses were successfully isolated and demonstrated by reverse transcription-PCR analysis from the proventricular glands of parrot “glass 363” and “color” with confirmed PDD. The 351 bp product of the expected size bands of matrix protein （M） gene was cloned, the sequence and phylogenetic tree analysis showed that the isolated virus belonging to genotype ABV5. Five sets of M gene RT-LAMP primers ID1, ID6, ID19, ID30 and ID37 were designed using DNAStar and PrimerExplorer V5.0 （network） and later three set reactions showed positive color reaction with specific electrophoretic bands. The amplification curves of of real-time RT-LAMP using fluorescent indicator calcein were shown in 36 （ID30）, 38 （ID37） and 49 （ID19） minutes, respectively and an amplification peak in 60 minutes. Meanwhile the three amplification curves of turbidimetric determination were shown in 56, 58 and 65 minutes. According to the results of clinical and related samples detected by RT-LAMP and RT-PCR, proventriculus were the ABV positive while large intestine, small intestine, duodenum, heart, liver, spleen, lung, kidney, gizzard, proventriculus contents , pancreas, crop, brain, and muscle were negative. The virus was positive in 10-1 to 10-5 of ST cell culture material by RT-LAMP detection while it was negative after 10-3 by RT-PCR detection. There was also no RT-LAMP positive response to Newcastle disease, avian flu, bursal disease, leukemia subgroup J, encephalomyelitis. The findings provide new technological tools for the PDD control of parrots and BDV public health research.

关 键 词：禽波纳病毒 腺胃扩张症 环介导逆转录恒温扩增 快速检测 进化树分析 

分 类 号：S852.659.3[农业科学—基础兽医学] S858.315.3[农业科学—兽医学]