Multiplex Detection of Five Canine Viral Pathogens for Dogs as Laboratory Animals by the Luminex xTAG Assay.

Miaoli Wu,Meili Chen,Ren Huang,Feng Cong,Pengju Guo,Yuexiao Lian,Yujun Zhu

doi:10.3389/fmicb.2018.01783

Miaoli Wu, Meili Chen + Show 5 more

Open Access

https://doi.org/10.3389/fmicb.2018.01783

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Abstract

More and more dogs have been used as a disease model for medical research and drug safety evaluation. Therefore, it is important to make sure that the dogs and their living houses are special pathogen free. In this study, the development and evaluation of a Luminex xTAG assay for simultaneous detection of five canine viruses was carried out, including canine distemper virus, canine parvovirus, canine parainfluenza virus, canine adenovirus, and rabies virus. Assay specificity was accomplished by targeting conserved genomic regions for each virus. Hybridization between multiplexed PCR products and the labeled fluorescence microspheres was detected in a high throughput format using a Luminex fluorescence reader. The Luminex xTAG assay showed high sensitivity with limits of detection for the five viruses was 100 copies/μL. Specificity of the xTAG assay showed no amplification of canine coronavirus, pseudorabies virus and canine influenza virus indicating that the xTAG assay was specific. Seventy-five clinical samples were tested to evaluate the xTAG assay. The results showed 100% coincidence with the conventional PCR method. This is the first report of a specific and sensitive multiplex Luminex xTAG assay for simultaneous detection of five major canine viral pathogens. This assay will be a useful tool for quality control and environmental monitoring for dogs used as laboratory animals, may even be applied in laboratory epidemiological investigations.

Highlights

Dogs has been extensively used as animal models for medical research and drug evaluation, such as studying human breast cancer carcinogenesis (Abdelmegeed and Mohammed, 2018), Duchenne muscular dystrophy (DMD) (Wells, 2018), management of furcation defect (Afifi et al, 2018), chronic myocardial infarction (MI) (Xiong et al, 2018), comparative pathogenesis study of H3N2 canine influenza virus (CIV) challenged (Luo et al, 2018), long term safety and efficacy of AAV gene therapy (Lee et al, 2018)
Nucleic acid of pseudorabies virus (PRV), CIV, and canine coronavirus (CCV) were used as templates in this study to evaluate the specificity of the multiplex-PCR primers
Sensitivity was tested using 10-fold serial dilutions of standards and the limit of detection was defined as the highest dilution that resulted in an mean fluorescence intensity (MFI) value above the cutoff value

Summary

Introduction

Dogs has been extensively used as animal models for medical research and drug evaluation, such as studying human breast cancer carcinogenesis (Abdelmegeed and Mohammed, 2018), Duchenne muscular dystrophy (DMD) (Wells, 2018), management of furcation defect (Afifi et al, 2018), chronic myocardial infarction (MI) (Xiong et al, 2018), comparative pathogenesis study of H3N2 canine influenza virus (CIV) challenged (Luo et al, 2018), long term safety and efficacy of AAV gene therapy (Lee et al, 2018). It is critical to make sure that the dogs used as laboratory animals are free of special pathogens. In China, according to the national standard of laboratory dogs, the rabies virus (RV), canine parvovirus (CPV), canine adenovirus (CAV), and Canine Pathogens Detected by xTAG.

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