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GeXP analyzer-based multiplex reverse-transcription PCR assay for the simultaneous detection and differentiation of eleven duck viruses.

ABSTRACT: Duck viral pathogens primarily include the avian influenza virus (AIV) subtypes H5, H7, and H9; duck hepatitis virus (DHV); duck tembusu virus (DTMUV); egg drop syndrome virus (EDSV); duck enteritis virus (DEV); Newcastle disease virus (NDV); duck circovirus (DuCV); muscovy duck reovirus (MDRV); and muscovy duck parvovirus (MDPV). These pathogens cause great economic losses to China's duck breeding industry.A rapid, specific, sensitive and high-throughput GeXP-based multiplex PCR assay consisting of chimeric primer-based PCR amplification with fluorescent labeling and capillary electrophoresis separation was developed and optimized to simultaneously detect these eleven viral pathogens. Single and mixed pathogen cDNA/DNA templates were used to evaluate the specificity of the GeXP-multiplex assay. Corresponding specific DNA products were amplified from each pathogen. Other pathogens, including duck Escherichia coli, duck Salmonella, duck Staphylococcus aureus, Pasteurella multocida, infectious bronchitis virus, and Mycoplasma gallisepticum, did not result in amplification products. The detection limit of GeXP was 10(3)copies when all twelve pre-mixed plasmids containing the target genes of eleven types of duck viruses were present. To further evaluate the reliability of GeXP, 150 clinical field samples were evaluated. Comparison with the results of conventional PCR methods for the field samples, the GeXP-multiplex PCR method was more sensitive and accurate.This GeXP-based multiplex PCR method can be utilized for the rapid differential diagnosis of clinical samples as an effective tool to prevent and control duck viruses with similar clinical symptoms.

SUBMITTER: Zhang YF

PROVIDER: S-EPMC4628294 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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GeXP analyzer-based multiplex reverse-transcription PCR assay for the simultaneous detection and differentiation of eleven duck viruses.

Zhang Yan-Fang YF Xie Zhi-Xun ZX Xie Li-Ji LJ Deng Xian-Wen XW Xie Zhi-Qin ZQ Luo Si-Si SS Huang Li L Huang Jiao-Ling JL Zeng Ting-Ting TT

BMC microbiology 20151030

<h4>Background</h4>Duck viral pathogens primarily include the avian influenza virus (AIV) subtypes H5, H7, and H9; duck hepatitis virus (DHV); duck tembusu virus (DTMUV); egg drop syndrome virus (EDSV); duck enteritis virus (DEV); Newcastle disease virus (NDV); duck circovirus (DuCV); muscovy duck reovirus (MDRV); and muscovy duck parvovirus (MDPV). These pathogens cause great economic losses to China's duck breeding industry.<h4>Result</h4>A rapid, specific, sensitive and high-throughput GeXP-b ...[more]

PMID: 26518004

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Project description:Foot-and-mouth disease virus (FMDV), Bluetongue virus (BTV), Vesicular stomatitis Virus (VSV), Bovine viral diarrheal (BVDV), Bovine rotavirus (BRV), and Bovine herpesvirus 1 (IBRV) are common cattle infectious viruses that cause a great economic loss every year in many parts of the world. A rapid and high-throughput GenomeLab Gene Expression Profiler (GeXP) analyzer-based multiplex PCR assay was developed for the simultaneous detection and differentiation of these six cattle viruses. Six pairs of chimeric primers consisting of both the gene-specific primer and a universal primer were designed and used for amplification. Then capillary electrophoresis was used to separate the fluorescent labeled PCR products according to the amplicons size. The specificity of GeXP-multiplex PCR assay was examined with samples of the single template and mixed template of six viruses. The sensitivity was evaluated using the GeXP-multiplex PCR assay on serial 10-fold dilutions of ssRNAs obtained via in vitro transcription. To further evaluate the reliability, 305 clinical samples were tested by the GeXP-multiplex PCR assay. The results showed that the corresponding virus specific fragments of genes were amplified. The detection limit of the GeXP-multiplex PCR assay was 100 copies/μL in a mixed sample of ssRNAs containing target genes of six different cattle viruses, whereas the detection limit for the Gexp-mono PCR assay for a single target gene was 10 copies/μL. In detection of viruses in 305 clinical samples, the results of GeXP were consistent with simplex real-time PCR. Analysis of positive samples by sequencing demonstrated that the GeXP-multiplex PCR assay had no false positive samples of nonspecific amplification. In conclusion, this GeXP-multiplex PCR assay is a high throughput, specific, sensitive, rapid and simple method for the detection and differentiation of six cattle viruses. It is an effective tool that can be applied for the rapid differential diagnosis of clinical samples and for epidemiological investigation.

Dataset Information

GeXP analyzer-based multiplex reverse-transcription PCR assay for the simultaneous detection and differentiation of eleven duck viruses.

Publications

GeXP analyzer-based multiplex reverse-transcription PCR assay for the simultaneous detection and differentiation of eleven duck viruses.

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