同时监测8种牛常见病原体的GeXP高通量快速检测技术

doi:10.11843/j.issn.0366-6964.2017.10.015

Abstract

Abstract:

The aim of this study was to develop a multiple PCR assay on a basis of Genome Lab Genetic Expression Analysis System (GeXP) for simultaneous detection of 8 pathogens of bovine infectious diseases, including food-and-mouth disease virus (FMDV), bluetongue virus (BTV) ,vesicular stomatitis virus (VSV), bovine viral diarrheal virus (BVDV), bovine rotavirus (BRV), enterotoxigenic E. coli (ETEC), bovine herpesvirus 1 (IBRV), Peste des petits ruminants virus (PPRV). The GeXP profilerutilisesgene-specific primers containing 5'-universal adaptor sequences. Eight gene-specific primers consist of a universal sequence fused to the 5'-end and a gene-specific sequence were designed according to the conserved sequence of each pathogen and used for amplification.The reaction system and condition were optimized. The specificity of GeXP were examined with samples of the single and mixture of 8 viruses' co-infection. The sensitivity was evaluated by performing the assay on serial 10-fold dilutions of cloned plasmids. And 305 clinical samples were detected by GeXP to further evaluate the reliability. The results showed that the corresponding specific fragments of genes were amplified respectively. The detection limit of GeXP was 100 copies·μL^-1 when all of 8 pre-mixed plasmids containing target genes of 8 bovine pathogens. In detection of 305 clinical samples, the results of GeXP were consistent with real-time PCR completely. Analysis of positive samples by sequencing demonstrated that the GeXP assay had no false positive samples of nonspecific amplification. In conclusion, this GeXP assay is a high throughput, specific, sensitive, rapid and simple method for the detection of 8 pathogens of bovine infectious diseases. It is an effective tool applied to differential diagnosis rapidly for clinical samples and epidemiological investigation.

CLC Number:

S858.235

FAN Qing, XIE Zhi-xun, XIE Zhi-qin, XIE Li-ji, HUANG Li, HUANG Jiao-ling, ZHANG Yan-fang, ZENG Ting-ting, WANG Sheng, LUO Si-si, DENG Xian-wen, LIU Jia-bo, PANG Yao-shan. Development of a GeXP Assay for Simultaneous Differentiation of 8 Pathogens of Bovine Infectious Diseases[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(10): 1920-1931.

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Development of a GeXP Assay for Simultaneous Differentiation of 8 Pathogens of Bovine Infectious Diseases

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