›› 2019, Vol. 46 ›› Issue (8): 2211-2219.doi: 10.16431/j.cnki.1671-7236.2019.08.003

• Biotechnology • Previous Articles     Next Articles

Establishment of Gene Chip Detection Method for 5 Common Canine Diarrhea Viruses

SU Xia, ZHOU Hongzhuan, CHANG Yanyan, QI Qi, LIN Lulu, ZHANG Jin, XU Fuzhou, YANG Bing   

  1. Beijing Key Laboratory for Prevention and Control of Infectious Diseases in Livestock and Poultry, Institute of Animal Science and Veterinary Medicine, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China
  • Received:2019-03-08 Online:2019-08-20 Published:2019-08-17

Abstract:

This study was aimed to establish a simple, rapid and efficient method for the diagnosis of canine adenovirus type 1 (CAV-1), canine adenovirus type 2 (CAV-2), canine coronavirus (CCV), canine distemper virus (CDV) and canine parvovirus (CPV).5 canine diarrhea viruses CAV-1, CAV-2, CCV, CDV and CPV were used as target viruses.The primers were designed in the conserved region according to the viral gene sequences included in NCBI.Then 2 to 3 probes for each virus were designed according to the variation region.After optimizing the reaction conditions of the detection system and detecting the specificity and sensitivity of the detection method, the gene chip detection method which could simultaneously distinguish 5 viruses was established.The results showed that the chip was able to simultaneously detect the above five kinds of canine diarrhea viruses.The optimized reaction system in this study was that the annealing temperature of PCR was 55℃, the extension time was 1 min 15 s, the hybridization temperature was 40℃, and the hybridization time was 2.5 h.The detection limit of 5 virus standard were as follows:CAV-1 0.2 fg/μL, CAV-2 2 fg/μL, CCV 2 fg/μL, CDV 20 pg/μL and CPV 0.02 fg/μL.The specificity results showed that no positive signals were found for the CPIV.The detection of 14 clinical diarrhea samples showed that the positive detection rates of CAV-1, CAV-2, CCV, CDV and CPV by the gene chip method were 28.57%, 50.00%, 64.28%, 14.28% and 85.71%, respectively, and the sensitivity of the microarray assay was 10 to 100 times higher than that of PCR.These results indicated that the developed method in this study was specific and sensitive, and had diagnostic meaning for the clinical mixed infection of canine viruses.

Key words: canine diarrhea virus; gene chip; diagnosis

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