Abstract：Foot-and-mouth disease (FMD) is an acute, hot, and highly contagious animal disease caused by Foot-and-mouth disease virus (FMDV). This study is to establish a gene chip that distinguishes FMDV serotypes O, A and AsiaⅠ. Three specific primers were designed according to the VP1 gene sequences of FMDV, and three target fragments of 248, 206 and 239 bp were obtained by RT-PCR. Three kinds of probes were designed and labeled with primer markers. A genotyping microarray encoding O, A and AsiaⅠ was established and its sensitivity, specificity, reproducibility and shelf life were evaluated. The optimum reaction conditions were as follows: hydration temperature was 37 ℃, confluent concentration was 0.25% BH4Na, 25% ethanol, and hybridization temperature was 42 ℃. Sensitivity test showed that FMDV-O, FMDV-A and FMDV-AsiaⅠ were 10-5, 10-7, 10-5 after the band is not visible. The PCR detection limits were: FMDV-O 118 pg/mL, 4.3×106 copies/μL; FMDV-A 18.4 pg/mL, 8.1×104 copies/μL; FMDV-AsiaⅠ 129 pg/mL, 4.9×105 copies/μL. The FMDV-O dilution reaches 10-6; the FMDV-A dilution reaches 10-8; the FMDV-AsiaⅠ dilution reaches 10-6, the sensitivity of the method was 10~100 times higher than that of a conventional PCR. There was no cross-hybrid among the detecting surfaces O, A and A1; the chip was reproducible and the prepared chip could be stored for at least three months. In this study, the experimental conditions were optimized, and a fast and simple FMDV classification method was established to provide a new technique for the classification of FMDV.
[1] Hayama Y, Muroga N, Nishida T, et al. Risk factors for local spread of foot-and-mouth disease, 2010 epidemic in Japan [J]. Research in Veterinary Science, 2012, 93(2): 631-635.[2] 刘在新. 全球FMD防控技术及病原特性研究概观 [J]. 中国农业科学, 2015, 48(17): 3547-3564.[3] Reid S M, Grierson S S, Ferris N P, et al. Evaluation of automated RT-PCR to accelerate the laboratory diagnosis of foot-and-mouth disease virus [J]. Journal of virological methods, 2003, 107(2): 129.[4] Bachanek-Bankowska K, Mero H R, Wadsworth J, et al. Development and evaluation of tailored specific real-time RT-PCR assays for detection of foot-and-mouth disease virus serotypes circulating in East Africa [J]. Journal of virological methods, 2016, 237(114-120.[5] Yamazaki W, Mioulet V, Murray L, et al. Development and evaluation of multiplex RT-LAMP assays for rapid and sensitive detection of foot-and-mouth disease virus [J]. Journal of virological methods, 2013, 192(1-2): 18-24.[6] Dong Y, Xu Y, Liu Z, et al. Rapid screening swine foot-and-mouth disease virus using micro-ELISA system [J]. Lab on A Chip, 2011, 11(13): 2153.[7] Lei W, Tao J, Lu Z J, et al. Development and validation of a prokaryotically expressed foot-and-mouth disease virus non-structural protein 2C'3AB-based immunochromatographic strip to differentiate between infected and vaccinated animals [J]. Virology Journal, 2011, 8(1): 1-9.[8] 张骞, 盛军. 基因芯片技术的发展和应用 [J]. 中国医学科学院学报, 2008, 30(3): 344-347.[9] Wang J D, Guo J H, Yang C S, et al. Progress on Gene Chip Technolodgy and Its Application in Foot-and-mouth Disease [J]. Progress in Veterinary Medicine, 2007, [10] 李铁锋, 李坚, 王照, 等. 膜基因芯片技术鉴定布鲁杆菌的初步研究 [J]. 中华地方病学杂志, 2015, 34(8): 582-585.[11] 吴回君, 罗丽琳, 张玉勤. 透明导电薄膜载体材料在无标记电化学基因芯片中的应用 [J]. 中国组织工程研究, 2010, 14(29): 5479-5482.[12] Li Y, Xiong T, Wu H, et al. Visual DNA microarray coupled with multiplex-PCR for the rapid detection of twelve genetically modified maize [J]. BioChip Journal, 2016, 10(1): 42-47.[13] Jung W S, Kim S, Hong S I, et al. DNA probe chip system for multiple detection of food poisoning microorganisms [J]. Materials Science & Engineering C, 2004, 24(1): 47-51.[14] Sterrenburg E, Turk R, Boer J M, et al. A common reference for cDNA microarray hybridizations [J]. Nucleic Acids Research, 2002, 30(21): e116.[15] Vora G J, Meador C E, Stenger D A, et al. Nucleic acid amplification strategies for DNA microarray-based pathogen detection [J]. Applied & Environmental Microbiology, 2004, 70(5): 3047-3054.[16] Qin L X, Kerr K F. Empirical evaluation of data transformations and ranking statistics for microarray analysis [J]. Nucleic Acids Research, 2004, 32(18): 5471-5479.
