Establishment of Multiplex PCR Detection Method for Three Kinds of Main DNA Viruses in Chicken
LI Xuewu
LIU Yuan
WANG Li
DENG Ruiguang
ZHAO Dong
YANG Jifei
CHAI Shujun
Abstract：Specific primers were designed upon the conservative sequences of Marek's disease virus (MDV),infectious laryngotracheitis virus (ILTV),and fowlpox virus (FPV) genomic nucleotide sequences from registered in GenBank.Three pairs of specific primers for each virus were designed,a total of 9 pairs of primers for three viruses(MDV,ILTV,FPV) were screened by biological software,and three pairs of primers matched each other.After verification of three pair suitable primers with singleplex PCR,the reaction conditions of the multiplex PCR were optimized,including annealing temperature,concentrations of primers and Taq DNA polymerase.The simple multiplex PCR assay was successfully established after specificity,sensitivity and simplified test.The specific bands with lengths of 228 bp(MDV),400 bp (ILTV),499 bp(FPV) were amplified by the multiplex PCR,which were consistent with those expected.The results demonstrated that the method could simultaneously amplify these three viruses at a sensitivity of 100 fg when all three viruses were present.According to the principle of PCR technology,the classical PCR(a three step thermal cycle assay) was improved to be a two step thermal cycle assay,namely annealing and extension step were merged into one step (62 ℃),and the reaction time was shortened.This multiplex PCR for three DNA viruses was both highly specific and sensitive,and could be used as a rapid diagnostic assay for clinical samples.
Keywords：Marek's disease virusinfectious laryngotracheitis virusfowlpox virusDNA virusesmultiplex PCR
Publication Date：2017-01-01
Online Publishing Date：2026-09-12(First online date of this platform, not the publication date of the document)
Pages：5( 101-105 )