摘要
根据GenBank上公布的犬腺病毒I型(CAV-I)E3基因序列,设计了3对特异性环介导等温扩增(LAMP)引物,以CAV-I DNA为模板,通过条件优化建立了犬传染性肝炎LAMP快速检测方法,该方法能够在63℃恒温下30min内实现目的核酸的大量扩增,在可见光下即可直接观察扩增产物荧光颜色变化来判定结果。特异性和灵敏度试验表明,该LAMP检测方法只对CAV-I有特异性扩增,对其他无关核酸无扩增。与常规PCR检测相比,LAMP的检测灵敏度较好,最低检出限为10-7TCID50,是PCR检测方法的100倍。临床样品检测结果表明,LAMP阳性检出率为36%,与病毒分离鉴定的结果符合率达96.8%,与PCR检测的结果符合率达100%,且快速简便,成本低,适用于基层实验室的快速疾病诊断。
To develop a rapid method for the detection of infectious canine hepatitis,LAMP was established using three pairs of speciifc primers for the E3 gene of CAV-I according to the sequences in GenBank. The assay was optimized to obtain a big amount of ampliifcation products at 63℃for 30min and the ampliifcation products could be observed by the naked eyes. It was shown that the LAMP was able to speciifcally amplify CAV-I DNA,without cross-reaction with other irrelevant nucleotides and it was 100 times more sensitive than PCR assay with a lowest detection limit value of 10-7TCID50/0.1mL. The detection of clinical samples showed that the positive rate of LAMP was 43%. In addition,the agreement rate between LAMP and the virus isolation was 96.8%but between LAMP and PCR was up to 100%.LAMP was more sensitive and simple and could be applied to rapid diagnosis of CAV-I.
出处
《中国动物检疫》
CAS
2014年第8期73-76,共4页
China Animal Health Inspection
基金
国家质检总局项目科技计划项目(2012IK015)
