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.

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.

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

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