LAMP快速检测犬传染性肝炎方法的建立及应用

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。

Establishment and Application of LAMP for the Rapid Detection of Infectious Canine Hepatitis

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.