Construction and Application of Oligonucleotide Microarray for the Simultaneous Detection of Four Porcine (Sus scrofa) Diarrhea Viruses
HU Jing-Fei1, HUA Xiang1, HUANG Xiao-Bo1,2,3*, ZHAO Yu-Jia1, CAO San-Jie1,2,3, WEN Xin-Tian1,2,3, WEN Yi-Ping1,2,3, WU Rui1,2,3, ZHAO Qin1,2,3
1 Research Center of Swine Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China;
2 Sichuan Science-observation station Veterinary Drugs and Veterinary Diagnostic Technology, Ministry of Agriculture, Chengdu 611130, China;
3 National Animal Experiment Teaching Demonstration Center, Sichuan Agricultural University, Chengdu 611130, China
Abstract：Diarrhea is common in piglets and causes serious economic losses. In this study, an oligonucleotide microarray was developed for simultaneous the detection of Porcine epidemic diarrhea virus (PEDV), Transmissible gastroenteritis virus (TGEV), Porcine rotavirus group A (PRoV A) and Porcine delta-coronavirus (PDCoV). Specific primers were designed according to the genes of PEDV spike gene (S), membrane gene(M), TGEV spike gene (S) nucleocapsid gene (N), PoRV A outer capsid protein 7 gene (VP7), non-structural protein 4 gene (NSP4) and PDCoV membrane gene (M), nucleocapsid gene (N), and the corresponding oligonucleotide probes were designed according to the conservative region of each target gene fragment. The 5' end of downstream primers was labelled Cy3 fluorophores directly for multiple PCR. The microarray reaction parameters of each reaction step were optimized, which were as follows: The dilution ratio of 100 μmol/L original probe and sample buffer was 1∶2, the optimal hybridization condition was 46 ℃ for 120 min. Signal-to-noise ratio (SNR)≥2 or signal value >1 300 was defined as positive. The sensitivity of chip was 105 copies/μL. No cross reaction was detected with other non-target pathogens. The shelf life test showed that the microarray can be stored at 4 ℃ for at least 120 d. The detection of the 209 clinical samples by oligonucleotide microarray showed the positive rates of PEDV, TGEV, PDCoV and PoRV A were 68.42%, 4.30%, 0.95% and 2.87%, respectively, which was consistent with the results by Reverse transcription PCR. In conclusion, the constructed oligonucleotide microarray provides a new alternative method for high-through screening of the 4 porcine enteric diarrheic viruses.
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