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

Abstract

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.

Key words: porcine diarrhea virus, microarray, direct-labeling, indirect-labeling, comparison

CLC Number:

S854.43

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.

References

［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]	LI Xiang, YAN Xinyi, LUO Hanpeng, LIU Lin, GUO Gang, WANG Xinyu, WANG Yachun. Genetic Parameters Estimation for Productive Life of Chinese Holsteins by Different Models [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(6): 1162-1170.
[2]	LI Xiang, YAN Xinyi, LUO Hanpeng, LIU Lin, GUO Gang, WANG Xinyu, WANG Yachun. Genetic Parameters Estimation for Productive Life of Chinese Holsteins by Different Models [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(6): 1162-1170.
[3]	JI Gai-ge, SHU Jing-ting, SHAN Yan-ju, ZHANG Ming, TU Yun-jie, LIU Yi-fan, JU Xiao-jun, ZOU Jian-min. Identification of Differentially Expressed Genes between Different Positions of Chicken Skin Based on Gene Expression Microarray [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(1): 36-45.
[4]	JI Gai-ge, SHU Jing-ting, SHAN Yan-ju, ZHANG Ming, TU Yun-jie, LIU Yi-fan, JU Xiao-jun, ZOU Jian-min. Identification of Differentially Expressed Genes between Different Positions of Chicken Skin Based on Gene Expression Microarray [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(1): 36-45.
[5]	LIU Hong-xiang，SONG Wei-tao，HU Yan，SONG Chi，SHU Jing-ting，JI Gai-ge，LI Hui-fang. The Comparison of Growth Patterns of Body Weight，Breast Muscle and Leg Muscle between Different Duck Breeds via Growth Models [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(6): 1112-1123.
[6]	LIU Hong-xiang，SONG Wei-tao，HU Yan，SONG Chi，SHU Jing-ting，JI Gai-ge，LI Hui-fang. The Comparison of Growth Patterns of Body Weight，Breast Muscle and Leg Muscle between Different Duck Breeds via Growth Models [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(6): 1112-1123.
[7]	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.
[8]	LIU Bin，YANG Jun，A Yun-ga，WU Ri-gexilatu，NA Shun-qimuge，HAO Ba-yasihuliang，ZHAO Cun-fa，LI Yu-rong. Exogenous Melatonin Promotes Expression Differences of Skin Hair Follicles Related Genes in Cashmere Goat [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(11): 2210-2217.
[9]	LIU Bin，YANG Jun，A Yun-ga，WU Ri-gexilatu，NA Shun-qimuge，HAO Ba-yasihuliang，ZHAO Cun-fa，LI Yu-rong. Exogenous Melatonin Promotes Expression Differences of Skin Hair Follicles Related Genes in Cashmere Goat [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(11): 2210-2217.
[10]	SHU Jing-ting，ZHANG Ming，SONG Chi，SHAN Yan-ju，XU Wen-juan，SONG Wei-tao，LI Hui-fang. Identification of Differentially Expressed Genes for Myofiber Characteristics in Soleus Muscles between Qingyuan Partridge Chickens and Cobb Broilers [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(1): 25-33.
[11]	SHU Jing-ting，ZHANG Ming，SONG Chi，SHAN Yan-ju，XU Wen-juan，SONG Wei-tao，LI Hui-fang. Identification of Differentially Expressed Genes for Myofiber Characteristics in Soleus Muscles between Qingyuan Partridge Chickens and Cobb Broilers [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(1): 25-33.
[12]	WANG Xiao-shuo，YU Ying，LIU Hui-jie，XIAO Li，ZHAI Li-wei，AO Hong，WANG Chu-duan. Mining Anti Classic Swine Fever Virus-related Genes Based on Microarray and Analyzing Their Transcriptional Expression under Different CSF-antibody Levels [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(1): 14-22.
[13]	WANG Xiao-shuo，YU Ying，LIU Hui-jie，XIAO Li，ZHAI Li-wei，AO Hong，WANG Chu-duan. Mining Anti Classic Swine Fever Virus-related Genes Based on Microarray and Analyzing Their Transcriptional Expression under Different CSF-antibody Levels [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(1): 14-22.
[14]	CHEN Xing-yong, XIE Shan-shan, ZHOU Li, JIANG Run-shen, GENG Zhao-yu. Identification of Differentially Expressed Genes in Skin of Wanxi-white Goose during Regeneration of Downy Feather [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2013, 44(7): 1030-1036.
[15]	CHEN Xing-yong, XIE Shan-shan, ZHOU Li, JIANG Run-shen, GENG Zhao-yu. Identification of Differentially Expressed Genes in Skin of Wanxi-white Goose during Regeneration of Downy Feather [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2013, 44(7): 1030-1036.

Comparision of Two Fluorescence-labeling Methods of Porcine Diarrheal Viruses Detection Microarrays

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments