水貂阿留申病毒纳米PCR检测方法的建立与初步应用

doi:10.11843/j.issn.0366-6964.2016.11.017

Abstract

Abstract:

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 0.2-0.8 nmol•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 samples. 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.

CLC Number:

S852.659.2

YANG Rui-mei, ZHANG Chuan-mei, ZHANG Hong-liang, SHAN Hu. The Establishment and Application of Nanoparticle PCR Molecular Assay for Detection of Aleutian Mink Disease Virus[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(11): 2288-2293.

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

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