# Multiplex One-Step qPCR/RT-qPCR Assays for Detection of Ectromelia Virus, Murine Hepatitis Virus, Reovirus Type 3, and Parvoviruses

## TL;DR

A new multiplex PCR test detects four dangerous viruses in lab mice more accurately and efficiently than previous methods, helping ensure reliable research results.

## Contribution

A novel one-step multiplex real-time PCR assay for simultaneous detection of four key mouse pathogens with optimized sensitivity and specificity.

## Key findings

- The assay detected 21.88% ECTV positives in tissue samples compared to 10.16% with conventional PCR.
- Detection limits ranged from 1.08 × 10¹ to 2.38 × 10¹ copies/μL for the four viruses.
- The assay showed high reproducibility with coefficients of variation below 2%.

## Abstract

Laboratory mice are fundamental to biomedical research; however, their health status has a critical influence on experimental outcomes. Ectromelia virus (ECTV), murine hepatitis virus (MHV), reovirus type 3 (Reo-3), and murine parvoviruses (MUV) are four key pathogens requiring exclusion from specific pathogen-free (SPF) mouse colonies to ensure data reliability. This study developed a novel one-step multiplex real-time PCR (mrt-PCR) assay for simultaneous detection of these viruses, optimizing primer and probe concentrations via response surface methodology to achieve balanced amplification efficiency. The assay demonstrated high specificity (no cross-reactivity with other murine pathogens), exceptional sensitivity (detection limits of 1.08 × 101–2.38 × 101 copies/μL), and excellent reproducibility (coefficients of variation < 2%). When applied to 128 clinical mouse tissue samples, it significantly outperformed conventional PCR (e.g., detecting 21.88% vs. 10.16% ECTV positives), proving superior for identifying low-level infections. This mrt-PCR method provides a robust and efficient tool for microbial quality control and early infection prevention in laboratory mouse facilities, thereby safeguarding the validity of animal model-based research.

The use of experimental animals with unified quality standards is an important condition for ensuring the effectiveness of scientific research. Ectromelia virus (ECTV), murine hepatitis virus (MHV), reovirus type 3 (Reo-3), and murine parvoviruses (MUV) are the four pathogens that need to be eliminated from SPF (Specific Pathogen-Free) level mice. These four pathogens present fast transmission and high pathogenicity, making it difficult to control. The previously described detection methods present substantial limitations in efficiency and accuracy. Thus, there is an urgent need for rapid and precise diagnostic methods to improve prevention and diagnosis efforts. In this study, we developed a one-step multiplex real-time PCR (mrt-PCR) detection method that can simultaneously detect four key viral pathogens causing diseases in laboratory mice without cross-reactivity with other mouse susceptible pathogens. We tested 128 suspected diseased mouse tissue samples collected from Beijing, and the results showed that this new method has higher sensitivity and specificity than ordinary PCR. The detection limit for ECTV, MHV, and MUV was determined to be 1.08 × 101 copies/μL, 1.14 × 101 copies/μL, 2.38 ×101 copies/μL, and 1.08 × 101 copies/μL, respectively. In addition, the assay showed excellent reproducibility, with a coefficient of variation below 1.5%, strong linear correlation (R2 > 0.996), and amplification efficiency between 90% and 100%. In summary, the mrt-PCR serves not only as a rapid and accurate detection and early prevention method for laboratory mice but also constitutes a robust tool for microbial quality control in laboratory mice.