水貂阿留申病毒纳米PCR检测方法的建立与初步应用

The Establishment and Application of Nanoparticle PCR Molecular Assay for Detection of Aleutian Mink Disease Virus

为建立便捷、灵敏的水貂阿留申病病毒（AMDV）纳米PCR检测方法,根据AMDV NS1基因的保守序列设计一对特异性引物,对纳米PCR反应的退火温度、胶体金浓度进行了优化;测序检测扩增基因是否发生变异;对特异性和灵敏度进行了评估。结果表明,此纳米PCR的最佳退火温度为51℃,普通PCR为55℃;加入胶体金最佳浓度在0.2~0.8nmol·L-1,纳米PCR与普通PCR产物测序相似性大于99%。特异性检测表明该纳米PCR方法只扩增AMDV而不能扩增犬瘟热病毒和水貂肠炎细小病毒。在粪尿样品检测中该纳米PCR敏感性比普通PCR高10倍。检测40份临床样品,结果表明该方法比对流免疫电泳检测敏感性提高,从而为用粪、尿等低病毒样品检测水貂阿留申病提供了一种新的有效方法。

Nanoparticle-assisted polymerase chain reaction（nanoPCR）is a novel method for the rapid amplification of DNA.This study was aimed to establish a simple and sensitive nanoPCR assay for detection of Aleutian mink disease virus（AMDV）,primers were designed based on the conserved region of the NS1 gene sequences available in GenBank to amplify a 365 bp fragment.The optimized annealing temperature and the concentration of gold nanoparticles were studied.Whether or not the DNA replication fidelity compromised in nanoparticla-assisted PCR was detected by sequencing nanoPCR products.A nanoPCR assay was developed and its sensitivity and specificity were investigated.Under the optimized conditions of nanoPCR assay,annealing temperature was 51℃ but that of conventional PCR was 55℃.The best concentration of gold nanoparticles was in the range of 0.2-0.8nmol·L-1.The 99% homology between the products obtained with the nanoPCR amplification and the conventional PCR showed that highly levels of DNA replication fidelity in this nanoPCR.In addition,the other mink viruses detected by the nanoPCR were all negative.Under the optimized conditions of the AMDV nanoPCR assay,the nanoPCR assay was 10-fold more sensitive than a conventional PCR assay in urine and feces sam-ples.The lower detection limit of the nanoPCR assay was about 4.0×102 copies.Furthermore,a total of 40 clinical samples were detected by this assay.The results showed that the nanoPCR is sensitive than counter immunoelectrophoresis（CIEP）.The nanoPCR assay developed in this study can be applied widely in clinical diagnosis the urine and feces and field surveillance of AMDV-infection.