猪病毒性腹泻检测基因芯片的两种样品标记技术比较

doi:10.11843/j.issn.0366-6964.2016.04.015

摘要/Abstract

摘要：

对猪病毒性腹泻检测芯片的两种样品标记方法进行比较。分别选取猪流行性腹泻病毒（PEDV）的S和M基因，猪传染性胃肠炎病毒（TGEV）的N和S基因，A型猪轮状病毒（GAR）的VP7和NSP4基因设计引物，用PCR扩增制备靶基因，纯化后制备猪病毒性腹泻联合检测基因芯片。提取样品核酸，采用Cy3直接标记法和间接标记法进行PCR扩增标记，标记的样品再与芯片杂交、扫描和数据分析，同时对两种标记方法的特异性、敏感性等进行了比较。结果表明，两种方法标记的样品均能与基因芯片特异性杂交，但直接法的中位信号值（median）均高于间接法的中位信号值，直接法的芯片杂交信噪比是用间接法的5倍以上。两种标记方法制备样品特异性好，猪蓝耳病毒、猪瘟病毒、猪乙型脑炎病毒、猪伪狂犬病毒验证检测为阴性；直接法和间接法的最低检测浓度分别为10⁵和10⁷ copies•μL^-1，前者的灵敏性是后者的100倍。应用优化的直接标记法处理56份临床样品，进行芯片的检测应用，结果与RT-PCR一致。本研究结果表明直接法荧光标记样品可明显提高病毒性腹泻检测芯片的检测效果。

Abstract:

Two samples labeling methods of microarray detection for diarrheal viruses are compared respectively in this study.Primers were designed based on the sequences of S and M gene of Porcine Epidemic Diarrhea Virus(PEDV)，S and N gene of Transmissible Gastroenteritis Virus(TGEV)，VP7 and NSP4 gene of Group A Porcine Rotavirus(GAR)，and then were used to amplify the target genes by PCR.The target genes were purified using ethanol precipitation to manufacture simultaneous detection microarray.Fluorescence molecules were imported to PCR products by direct-labeling method and indirect-labeling method respectively after extraction of virus RNA from samples.The specificity and sensitivity of two labeling methods were compared by scanning and analyzing the results after PCR products hybridized with microarray.The results showed that microarray based on two different labeling methods could both hybridize with target genes with high specificity.However，the median signal volumes of direct-labeling were higher than those of indirect-labeling and Signal to Noise Ratio(SNR) of direct-labeling was more than five times higher compared with that of indirect-labeling.Specific test results showed that two labeling methods were both specific and CSFV，PRRSV，PCV-2，JEV detection showed negative result.Sensitivity test results suggested that the sensitivity of direct-labeling were 100 times higher than that of indirect-labeling.The minimum concentration of direct-labeling method and indirect-labeling method can be detected reliably were 10⁵copies•μL^-1 and 10⁷ copies•μL^-1，respectively.A total of 56 clinical samples were treated with direct-labeling and hybridized with the microarray，the results were consistent with those of RT-PCR.This study indicate that direct-labeling technology could effectively improve the detection effect of diarrhea microarray.

中图分类号:

S854.43

马锐，尹人杰，黄小波，文心田，杨国淋，常晓霞，张仙，滑翔，赵玉佳，曹三杰，文翼平，伍锐. 猪病毒性腹泻检测基因芯片的两种样品标记技术比较[J]. 畜牧兽医学报, 2016, 47(4): 752-761.

MA Rui，YIN Ren-jie，HUANG Xiao-bo，WEN Xin-tian，YANG Guo-lin，CHANG Xiao-xia，ZHANG Xian，HUA Xiang，ZHAO Yu-jia，CAO San-jie，WEN Yi-ping，WU Rui. Comparision of Two Fluorescence-labeling Methods of Porcine Diarrheal Viruses Detection Microarrays[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(4): 752-761.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.xmsyxb.com/CN/10.11843/j.issn.0366-6964.2016.04.015

https://www.xmsyxb.com/CN/Y2016/V47/I4/752

参考文献

［1］BOWMAN A S，KROGWOLD R A，PRICE T，et al.Investigating the introduction of porcine epidemic diarrhea virus into an Ohio swine operation［J］.BMC Vet Res，2015，11：38.
［2］WANG L，ZHANG Y，BYRUM B.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］.J Virol Methods，2014，207：154-157.
［3］KIM S Y，SONG D S，PARK B K.Differential detection of transmissible gastroenteritis virus and porcine epidemic diarrhea virus by duplex RT-PCR［J］.J Vet Diagn Invest，2001，13(6)：516-520.
［4］PENSAERT M B，DE BOUCK P.A new coronavirus-like particle associated with diarrhea in swine［J］.Arch Virol，1978，58(3)：243-247.
［5］VAN REETH K，PENSAERT M.Prevalence of infections with enzootic respiratory and enteric viruses in feeder pigs entering fattening herds［J］.Vet Rec，1994，135(25)：594-597.
［6］TAKAHASHI K，OKADA K，OHSHIMA K.An outbreak of swine diarrhea of a new-type associated with coronavirus-like particles in Japan［J］.Nihon Juigaku Zasshi，1983，45(6)：829-832.
［7］PARK S J，KIM H K，SONG D S，et al.Molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (PEDV) field isolates in Korea［J］.Arch Virol，2011，156(4)：577-585.
［8］LEE S，LEE C.Outbreak-related porcine epidemic diarrhea virus strains similar to US strains，South Korea，2013［J］.Emerg Infect Dis，2014，20(7)：1223-1226.
［9］PURANAVEJA S，POOLPERM P，LERTWATCHARASARAKUL P，et al.Chinese-like strain of porcine epidemic diarrhea virus，Thailand［J］.Emerg Infect Dis，2009，15(7)：1112-1115.
［10］CHEN J，WANG C，SHI H，et al.Molecular epidemiology of porcine epidemic diarrhea virus in China［J］.Arch Virol，2010，155(9)：1471-1476.
［11］ZHANG Q，HU R，TANG X，et al.Occurrence and investigation of enteric viral infections in pigs with diarrhea in China［J］.Arch Virol，2013，158(8)：1631-1636.
［12］CHEN Q，LI G，STASKO J，et al.Isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the United States［J］.J Clin Microbiol，2014，52(1)：234-243.
［13］JUNG K，SAIF L J.Porcine epidemic diarrhea virus infection：Etiology，epidemiology，pathogenesis and immunoprophylaxis［J］.Vet J，2015，204(2)：134-143.
［14］GERBER P F，GONG Q，HUANG Y W，et al.Detection of antibodies against porcine epidemic diarrhea virus in serum and colostrum by indirect ELISA［J］.Vet J，2014，202(1)：33-36.
［15］SONG D，PARK B.Porcine epidemic diarrhoea virus：a comprehensive review of molecular epidemiology，diagnosis，and vaccines［J］.Virus Genes，2012，44(2)：167-175.
［16］OPRIESSNIG T，XIAO C T，GERBER P F，et al.Porcine epidemic diarrhea virus RNA present in commercial spray-dried porcine plasma is not infectious to nave pigs［J］.PLoS One，2014，9(8)：e104766.
［17］ROSELL R，CABEZÓN O，PUJOLS J，et al.Identification of a porcine pestivirus as a border disease virus from naturally infected pigs in Spain［J］.Vet Rec，2014，174(1)：18.
［18］ENGEL E A，ESCOBAR P F，ROJAS L A，et al.A diagnostic oligonucleotide microarray for simultaneous detection of grapevine viruses［J］.J Virol Methods，2010，163(2)：445-451.
［19］TOWNSEND M B，DAWSON E D，MEHLMANN M，et al.Experimental evaluation of the FluChip diagnostic microarray for influenza virus surveillance［J］.J Clin Microbiol，2006，44(8)：2863-2871.
［20］LUO Y，DU J，ZHAN Z，et al.A diagnostic gene chip for hereditary spastic paraplegias［J］.Brain Res Bull，2013，97：112-118.
［21］LIU Y C，HUANG G S，WU M C，et al.Detection of foot and mouth disease and porcine reproductive and respiratory syndrome viral genes using microarray chip［J］.Vet Res Commun，2006，30(2)：191-204.
［22］曹三杰，黄小波，文心田，等.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，2009，29(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，2009，29(2)：121-124.(in Chinese)
［23］曹三杰，文心田，肖驰，等.应用基因芯片技术检测禽 4 种主要疫病的研究检测基因芯片的构建及制备［J］.中国兽医学报，2007，27(3)：311-314，318.
CAO S J，WEN X T，XIAO C，et al.Detection of four avian infectious diseases using microarray technology Ⅱ.Construction and preparation of detection microarray［J］.Chinese Journal of Veterinary Science，2007，27(3):311-314，318.(in Chinese)
［24］SCHENA M，SHALON D，DAVIS R W，et al.Quantitative monitoring of gene expression patterns with a complementary DNA microarray［J］.Science，1995，270(5235)：467-470.
［25］KNAUER M，IVLEVA N P，NIESSNER R，et al.A flow-through microarray cell for the online SERS detection of antibody-captured E.coli bacteria［J］.Anal Bioanal Chem，2012，402(8)：2663-2667.
［26］KIM Y K，LIM S I，CHO Y Y，et al.The CSFV DNAChip：a novel diagnostic assay for classical swine fever virus［J］.J Virol Methods，2014，204：44-48.
［27］BANÉR J，GYARMATI P，YACOUB A，et al.Microarray-based molecular detection of foot-and-mouth disease，vesicular stomatitis and swine vesicular disease viruses，using padlock probes［J］. J Virol Methods，2007，143(2)：200-206.
［28］LUNG O，FISHER M，BEESTON A，et al.Multiplex RT-PCR detection and microarray typing of vesicular disease viruses［J］.J Virol Methods，2011，175(2)：236-245.
［29］LI X，QI X，MIAO L，et al.Detection and subtyping of influenza A virus based on a short oligonucleotide microarray［J］.Diagn Microbiol Infect Dis，2009，65(3)：261-270.
［30］CAO X，WANG Y F，ZHANG C F，et al.Visual DNA microarrays for simultaneous detection of Ureaplasma urealyticum and Chlamydia trachomatis coupled with multiplex asymmetrical PCR［J］.Biosens Bioelectron，2006，22(3)：393-398.
［31］HE Y，ZHANG X，ZHANG S，et al.Visual detection of single-base mismatches in DNA using hairpin oligonucleotide with double-target DNA binding sequences and gold nanoparticles［J］.Biosens Bioelectron，2012，34(1)：37-43.
［32］HEGDE P，QI R，ABERNATHY K，et al.A concise guide to cDNA microarray analysis［J］.Biotechniques，2000，29(3):548-550.
［33］MOREIRA B G，YOU Y，OWCZARZY R.Cy3 and Cy5 dyes attached to oligonucleotide terminus stabilize DNA duplexes：Predictive thermodynamic model［J］.Biophys Chem，2015，198：36-44.
［34］ZHANG Q，HU R，TANG X，et al.Occurrence and investigation of enteric viral infections in pigs with diarrhea in China［J］.Arch Virol，2013，158(8)：1631-1636.
［35］SUN R Q，CAI R J，CHEN Y Q，et al.Outbreak of porcine epidemic diarrhea in suckling piglets，China［J］.Emerg Infect Dis，2012，18(1)：161-163.
［36］RODÁK L，VALÍEK L，SMÍD B，et al.An ELISA optimized for porcine epidemic diarrhoea virus detection in faeces［J］.Vet Microbiol，2005，105(1):9-17.
［37］CHAE C，KIM O，CHOI C，et al.Prevalence of porcine epidemic diarrhoea virus and transmissible gastroenteritis virus infection in Korean pigs［J］.Vet Rec，2000，147(21)：606-608.

[1]	李昕, 孙燕燕, 林密, 陈夏辉, 李峰松, 包艳芳, 杨光, 蒋韬. O、A、Asia 1型口蹄疫病毒胶体金定量检测免疫层析方法的建立[J]. 畜牧兽医学报, 2021, 52(4): 1042-1052.
[2]	张雪婷, 陈峥嵘, 赵雯雯, 韩丽, 陈建国. 区分犬瘟热病毒Asia-Ⅰ型野毒株及疫苗株的AS-PCR方法[J]. 畜牧兽医学报, 2020, 51(10): 2490-2499.
[3]	徐璐, 夏应菊, 张乾义, 张雯雯, 王震, 李翠, 邹兴启, 朱元源, 徐嫄, 王兆, 赵启祖, 王琴. 猪瘟病毒抗体ELISA检测国家级能力验证结果与分析[J]. 畜牧兽医学报, 2020, 51(9): 2250-2256.
[4]	王俊伟, 陈芳洲, 库旭钢, 李畅, 何启盖. 基于PCV3-Cap蛋白间接ELISA检测方法的建立及临床应用[J]. 畜牧兽医学报, 2019, 50(2): 454-460.
[5]	朱建波, 杨振兴, 肖雷, 高林, 苗海生, 何于雯, 孟锦昕, 杨恒, 李华春. 流行性出血病多克隆抗体C-ELISA检测方法的建立[J]. 畜牧兽医学报, 2018, 49(7): 1440-1450.
[6]	罗尚星, 范京惠, 刘宝京, 师乾凯, 侯林杉, 左玉柱. 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, 2018, 49(4): 852-858.
[7]	魏后军, 范志宇, 王芳, 宋艳华, 胡波, 仇汝龙, 陈萌萌, 徐为中, 薛家宾. 检测兔出血症病毒抗体胶体金试纸条的研制及初步应用[J]. 畜牧兽医学报, 2018, 49(3): 614-619.
[8]	王佳, 熊祺琰, 华利忠, 刘蓓蓓, 甘源, 冯志新, 刘茂军, 武昱孜, 邵国青. 猪鼻支原体血清抗体间接ELISA检测方法的建立[J]. 畜牧兽医学报, 2017, 48(10): 1932-1938.
[9]	滑翔，胡中凯，黄小波，文心田，曹三杰，文翼平，伍锐，邓静，赵松，尹人杰，常晓霞，欧阳达，张仙. 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, 2015, 46(12): 2235-2242.
[10]	张改梅，贾红，侯绍华，鑫婷，郭晓宇，袁维峰，刘淑清，吴竞，高新桃，李明，董瑞凯，朱鸿飞. 检测牛IFN-γ双抗体夹心ELISA的建立及在牛结核病诊断上的初步应用[J]. 畜牧兽医学报, 2014, 45(10): 1693-1698.
[11]	彭武丽，季新成，于学辉，史茜，王科珂，李娜. 多重PCR检测奶牛布鲁菌、鹦鹉热衣原体和贝纳氏柯克斯氏体[J]. 畜牧兽医学报, 2014, 45(1): 123-128.
[12]	王鑫，崔治中，龚建森，高明燕，范建华，俞燕，朱静，徐步. ELISA与IFA用于检测J亚型禽白血病病毒的比较分析[J]. 畜牧兽医学报, 2013, 44(9): 1499-1503.
[13]	黄娇玲，谢芝勋，罗思思，谢志勤，谢丽基，刘加波，庞耀珊，范晴. 基于纳米材料的电化学免疫传感器检测H5N1亚型禽流感病毒的研究[J]. 畜牧兽医学报, 2013, 44(6): 911-918.
[14]	智晓莹, 任维维, 祁光宇, 吕建亮, 梁仲, 蒋韬. O型口蹄疫抗体金标检测试纸条判定标准的确立[J]. 畜牧兽医学报, 2012, 43(9): 1415-1421.
[15]	杨春华, 周延, 孙思扬, 钟毅, 王川庆. 猪细环病毒PCR-DHPLC检测技术的建立及应用[J]. 畜牧兽医学报, 2012, 43(9): 1422-1428.