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ORIGINAL RESEARCH article

Front. Vet. Sci.
Sec. Veterinary Experimental and Diagnostic Pathology
Volume 11 - 2024 | doi: 10.3389/fvets.2024.1421427

Performance of three multiplex real-time PCR assays for simultaneous detection of 12 infectious pathogens in mice affected with respiratory and digestive diseases

Provisionally accepted
Hye-young Wang Hye-young Wang 1*Jaeil Ahn Jaeil Ahn 1Jonghoon Lee Jonghoon Lee 2Sang C. Kang Sang C. Kang 2Hyunil Kim Hyunil Kim 1
  • 1 Optipharm, Inc., Cheongwon, Republic of Korea
  • 2 Optipharm Animal Disease Diagnostic Center, Cheongju, North Chungcheong, Republic of Korea

The final, formatted version of the article will be published soon.

    Research quality can be improved with reliable and reproducible experimental results when animal experiments are conducted using laboratory animals with guaranteed microbiological and genetic quality through health monitoring. Therefore, health monitoring requires the rapid and accurate diagnosis of infectious diseases in laboratory animals. This study presents a performance evaluation of a commercially available multiplex real-time PCR (mRT-PCR) assay for the rapid detection of 12 infectious pathogens (Set 1: Sendai virus [SeV, formally murine respirovirus], Mycoplasma spp., Rodentibacter. pneumotropicus, and Rodentibacter heylii; Set 2: Helicobacter spp., Murine norovirus [MNV], Murine hepatitis virus [MHV], and Salmonella spp.; Set 3: Staphylococcus aureus,Streptobacillus moniliformis, Corynebacterium kutscheri, and Pseudomonas aeruginosa). The assay's detection limit ranged from 1 to 100 copies per reaction. Specificity testing involving various viruses and bacteria indicated no cross-reactivity between strains. Additionally, the assay exhibited good reproducibility, with mean coefficients of variation for inter-and intra assay variation below 3%. To evaluate the efficacy of the mRT-PCR assay, 102 clinical samples encompassing fecal and cecal specimens were analyzed. The resulting data were then compared with the findings from sequence analysis for validation. The overall positive rate was 52.9% (n = 54), with the mRT-PCR assay findings matching sequence analysis results (κ = 1). MHV (n = 29, 28.4%) was the most prevalent pathogen, followed by Helicobacter spp. (n = 28, 27.5%), R. heylii (n = 18, 17.6%), Mycoplasma spp.(n = 14, 13.7%), MNV (n = 12, 11.8%), S. aureus (n = 9, 8.8%), P. aeruginosa (n = 4, 3.9%), and R. pneumotropicus (n = 1, 0.9%). This assay offers a rapid turnaround time of 100 min, including 30 min for DNA preparation and 70 min for target DNA/RNA amplification. It ensures accuracy, minimizing false positives or negatives, making it a convenient tool for the simultaneous detection of infectious diseases in many samples. Overall, the proposed assay holds promise for the effective detection of the most important pathogens in laboratory animal health monitoring.

    Keywords: Mouse, health monitoring, Multiplex real-time PCR, diagnosis, Sequence Analysis

    Received: 23 Apr 2024; Accepted: 27 Jun 2024.

    Copyright: © 2024 Wang, Ahn, Lee, Kang and Kim. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

    * Correspondence: Hye-young Wang, Optipharm, Inc., Cheongwon, Republic of Korea

    Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.