猪圆环病毒2型新疆株全基因组的滚环扩增与序列分析

Rolling-circle Amplification and Sequence Analysis of PCV2 Genomes of Xinjiang Strains

摘要:

滚环扩增技术是一种体外恒温DNA扩增方法, 特异性好, 敏感性强, 已广泛应用于微生物学领域。首先采用鉴别PCR对来自新疆某地区的5份猪病料进行猪圆环病毒2型的检测, 接着对检出的2份猪圆环病毒2型阳性DNA样品进行滚环扩增。滚环扩增产物经单一限制性内切酶(SacⅡ)酶切及琼脂糖凝胶电泳鉴定, 结果显示2份样品都出现了猪圆环病毒2型基因组大小的条带。对目的条带进行回收、克隆与测序, 结果表明2株新疆株猪圆环病毒2型的全基因组大小皆为1768 bp。遗传进化分析显示2株的基因型为PCV2a和PCV2e。

Abstract:

Rolling-circle amplification (RCA) is an isothermal DNA amplification assay in vitro, which has good specificity and sensitivity and has been extensively applied to microbiological investigations. In this study, five diseased swine samples from certain region of Xinjiang were first detected by differential PCR for porcine circovirus type 2 (PCV2), and then RCA was utilized for amplification of the full-length genomic DNA of two positive samples. Verification with Sac II resticition enzymatic mapping showed that PCV2 specific genome-size bands appeared on agarose gel. The targeted bands were purified and cloned. With the aid of sequencing, the genomes of two viral strains were obtained and proved to be 1768 bp in length. Moreover, the phylogenetic analysis indicated that they were divided into two different genotypes (PCV2a and PCV2e).