摘要
为准确特异灵敏地检测猪细小病毒(PPV),建立一种新的LDR-PCR方法。首先在病毒的保守区内设计一对LDR探针,LDR探针两端各连有一段引物对应序列,以连接产物为模板进行PCR,琼脂糖凝胶电泳检测结果。以标准质粒为模板,通过对LDR反应的退火温度、连接酶浓度及探针浓度等反应条件进行优化,确定了LDR最佳的反应体系,并建立了LDR-PCR方法。结果表明,可以特异地检测PPV,与猪繁殖与呼吸障碍综合征病毒(PRRSV)、猪瘟病毒(CSFV)、伪狂犬病毒(PRV)、猪圆环病毒(PCV)、猪传染性胃肠炎病毒(TGEV)、猪流行性腹泻病毒(PEDV)无交叉反应;最低检测限为102个拷贝。利用建立的方法对41例临床样本进行检测,14份样品PPV阳性,与普通PCR检测结果符合率为97.6%。
A new method based on LDR-PCR was developed to detect porcine parvovirus（PPV） that causes swine severe reproductive failure in this study.According to alignment of the virus sequences,a pair of LDR probes for PPV was designed,which were flanked by universal primer sequences on both sides of the probes.After LDR,the ligated product was PCR amplified and detected by agarose gel electrophoresis.Following optimization of reaction system,the LDR-PCR for detection of PPV was successfully established.The method was highly specific,without cross reacting with porcine reproductive and respiratory syndrome virus（PRRSV）,classical swine fever virus（CSFV）,pseudorabies virus（PRV）,porcine circovirus type 1（PCV1） and type 2（PCV2）,transmissible gastroenteritis virus（TGEV）,porcine epidemic diarrhea virus（PEDV）.As few as 102 copies target molecules can be detected by the assay.41 clinical samples,14 samples were detected positive by the LDR-PCR.The results of the LDR-PCR and conventional PCR for PPV showed a concordant rate of 97.6%.
出处
《生物技术通报》
CAS
CSCD
北大核心
2012年第2期165-169,共5页
Biotechnology Bulletin
基金
浙江省科技计划项目(2008C22081)
浙江省自然科学基金项目(Y3090166)
浙江省科技攻关重点项目(2005C22032)
浙江省生物医学重中之重学科开放基金项目(SWYX0908)
