|
|
|
| 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.
|
|
Received: 04 July 2019
|
|
|
|
Corresponding Authors:
*rsghb110@126.com
|
|
|
|
[1] 曹三杰, 黄小波, 文心田, 等. 2009.猪传染性胃肠炎病毒检测基因芯片的构建[J]. 中国兽医学报, 29(2): 121-124.
(Cao S J, Huang X B, Wen X T, et al.Construction of detecting genechip for transmissible gastroenteritis virus[J]. Chinese Journal of Veterinary Science, 29(2): 121-124.)
[2] 陈静, 杨盼盼, 雷喜梅, 等. 2015. PEDV中国流行株和疫苗株S蛋白免疫反应性差异分析[J]. 中国兽医学报, 35(10): 1573-1577.
(Chen J, Yang P P, Lei X M, et al.2015.Differences in immunoreactivity of spike protein between Porcine epidemic diarrhea virus variant and CV777 vaccine strain[J]. Journal of Veterinary Science, 35(10): 1573-1577.)
[3] 滑翔, 胡中凯, 黄小波, 等. 2015. 检测猪流行性腹泻病毒、猪传染性胃肠炎病毒和猪轮状病毒的cDNA芯片的构建[J]. 畜牧兽医学报, 46(12): 2235-2242.
(Hua X, Hu Z K, Huang X B, et al.2015. Construction of cDNA Microarray for detecting Porcine epidemic diarrhea virus, Porcine transmissible gastroenteritis virus and Porcine rotavirus[J]. Chinese Journal of Animal and Veterinary Sciences, 46(12): 2235-2242.)
[4] 黄小波, 徐璐, 曹三杰, 等. 2009.猪轮状病毒双抗体夹心ELISA检测方法的建立[J]. 中国兽医科学, 2009, 39(08): 723-727.
(Huang X B, Xu L, Cao S J, et al.2009.Development of a double antibody sandwich ELISA for detection of Porcine rotavirus[J]. Chinese Veterinary Science, 39(08): 723-727.)
[5] 罗尚星, 范京惠, 刘宝京, 等. 2018.猪丁型冠状病毒与猪流行性腹泻病毒双重实时荧光定量RT-PCR方法的建立和初步应用[J]. 畜牧兽医学报, 49(04): 852-858.
(Luo S X, Fan J H, Liu B J, et al.2018. Establishment and application of the real-time reverse transcription quantitative PCR assay for Porcine epidemic diarrhea virus and Porcine deltacoronavirus[J]. Chinese Journal of Animal and Veterinary Sciences, 49(04): 852-858.)
[6] 欧阳达, 姜来生, 黄小波, 等. 2015.猪繁殖与呼吸综合征病毒和日本脑炎病毒二联基因芯片的构建与应用[J]. 中国兽医科学, 45(05): 509-516.
(Ouyang D, Jiang L S, Huang X B, et al.2018. Development and application of a microarray assay for simultaneous detection of Porcine reproductive and respiratory syndrome virus and Japanese encephalitis virus[J]. Chinese Veterinary Science, 45(05): 509-516.)
[7] 施开创, 龙飞翔, 粟艳琼, 等. 2017.猪流行性腹泻病毒强毒株和疫苗株多重RT-PCR鉴别检测方法的建立及应用[J]. 中国兽医科学, 47(01): 1-8.
(Shi K C, Long F X, Su Y Q, et al.2017. Establishment and application of a multiplex RT-PCR assay for differential detection of wild-type and vaccine strains of PEDV[J]. Chinese Veterinary Science, 47(01): 1-8.)
[8] 施开创, 王睿敏, 黎宗强, 等. 2019. PEDV、TGEV、PDCoV、PRoV多重TaqMan荧光定量RT-PCR检测方法的建立及应用[J]. 中国预防兽医学报, 41(06): 595-600.
(Shi K C,Wang R M, Li Z Q, et al.2019. Establishment and application of multiplex TaqMan real-time RT-PCR for differential detection of PEDV, TGEV, PDCoV and PRoV[J]. Chinese Journal of Preventive Veterinary Medicine, 41(06): 595-600.)
[9] 王黎, 李碧, 周远成, 等. 2015.猪传染性胃肠炎病毒RT-PCR检测方法的建立及临床应用[J]. 中国兽医学报, 35(02): 190-194.
(Wang L, Li B, Zhou Y C, et al.2015. Establishment of a RT-PCR method for detection of transmissible gastroenteritis of swine virus and its clinical application[J]. Chinese Journal of Veterinary Science, 35(02): 190-194.)
[10] 肖驰, 曹三杰, 文心田, 等. 2006. PRRS-CSF-PCV2诊断DNA芯片构建研究[J]. 畜牧兽医学报, 37(8): 799-803.
(Xiao C, Cao S J, Wen X T, et al.2006. Development of PRRS-CSF-PCV2 diagnostic DNA microarray[J]. Acta Veterinaria et Zootechnica Sinica, 37(8): 799-803.)
[11] 阳酉萍, 黄小波, 曹三杰, 等. 2015.四川部分地区猪流行性腹泻的分子流行病学调查[J]. 中国兽医学报, 35(05): 704-710.
(Yang Y P, Huang X B, Cao S J, et al.2015.Molecular epidemiological investigation porcine epidemic diarrhea in some areas of Sichuan[J]. Chinese Journal of Veterinary Science, 35(05): 704-710.)
[12] 张坤, 何启盖. 2010.猪流行性腹泻病毒、猪传染性胃肠炎病毒和猪A群轮状病毒多重RT-PCR检测方法的建立及临床应用[J]. 畜牧兽医学报, 41(8): 1001-1005.
(Zhang K, He Q G.2010. Establishment and clinical application of a multiplex reverse transcription-PCR for Porcine epidemic diarrhea virus, Porcine transmissible gastroenteritis virus and Porcine group A rotavirus[J]. Acta Veterinaria et Zootechnica Sinica, 41(8): 1001-1005.)
[13] 张雪, 杨倩, 于红欣, 等. 2015.猪传染性胃肠炎病毒实时荧光定量PCR检测方法的建立与应用[J]. 中国兽医科学, 45(06): 644-648.
(Zhang X, Yang Q, Yu H X, et al.2015. Establishment and application of a fluorescent quantitative PCR for detection of transmissible gastroenteritis virus of swine[J]. Chinese Veterinary Science, 45(06): 644-648.)
[14] Badiee A, Eiken H G, Steen V M, et al.2003. Evaluation of five different cDNA labeling methods for microarrays using spike controls[J]. BMC Biotechnology, 3(1): 23.
[15] He Z, Zhou J.2008. Empirical evaluation of a new method for calculating signal-to-noise ratio for microarray data analysis[J]. Applied & Environmental Microbiology, 74(10): 2957-2966.
[16] Hengpraprohm S, Chongstitvatana P.2014. Feature selection by weighted-SNR for cancer microarray data classification[J]. International Journal of Innovative Computing Information & Control Ijicic, 5(12(A)): 4627-4635.
[17] Jung K, Hu H, Saif L J.2016. Porcine deltacoronavirus infection: Etiology, cell culture for virus isolation and propagation, molecular epidemiology and pathogenesis[J]. Virus Research, 226: 50-59.
[18] Kandeil A, Gomaa M, Shehata M, et al.2019. Middle East respiratory syndrome coronavirus infection in non-camelid domestic mammals[J]. Emerging Microbes Infections, 8(1): 103-108.
[19] Pan Y.2012. Isolation and characterization of a variant Porcine epidemic diarrhea virus in China[J]. Virology Journal, 9(1): 195.
[20] Rennie C, Noyes HA, Kemp S J, et al.2008. Strong position-dependent effects of sequence mismatches on signal ratios measured using long oligonucleotide microarrays[J]. BMC Genomics, 9(1): 317.
[21] Varghese V, Das S, Singh N B, et al.2003. Molecular characterization of a human rotavirus reveals porcine characteristics in most of the genes including VP6 and NSP4[J]. Archives of Virology, 149(1): 155-172.
[22] Wang L, Zhang Y, Beverly B.2014. Development and evaluation of a duplex real-time RT-PCR for detection and differentiation of virulent and variant strains of porcine epidemic diarrhea viruses from the United States[J]. Journal of Virological Methods, 207(10): 154-157.
[23] Yauk C L, Berndt M L.2007. Review of the literature examining the correlation among DNA microarray technologies[J]. Environmental and Molecular Mutagenesis, 48(5): 380-394.
[24] Zhang H L, Liang Q Q, Li B X, et al.2019. Prevalence, phylogenetic and evolutionary analysis of Porcine deltacoronavirus in Henan province, China[J]. Preventive Veterinary Medicine, 166: 8-15.
[25] Zhang J, Guo L, Xu Y, et al.2017. Characterization of Porcine epidemic diarrhea virus infectivity in human embryonic kidney cells[J]. Archives of Virology, 162(8): 1-5.
[26] Zhang Q, Hu R, Tang X, et al.2013. Occurrence and investigation of enteric viral infections in pigs with diarrhea in China[J]. Archives of Virology, 158(8): 1631-1636.
[27] Zhao J, Shi B J, Huang X G, et al.2013. A multiplex RT-PCR assay for rapid and differential diagnosis of four porcine diarrhea associated viruses in field samples from pig farms in East China from 2010 to 2012[J]. Journal of Virological Methods, 194(1-2): 107-112. |
|
|
|
