Abstract
Mouse reovirus type 3 (Reo-3) infection is a viral disease that is harmful for laboratory mice. No rapid and accurate detection methods are currently available for this infection. In this study, we describe a rapid, simple, closed-tube, one step, reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for Reo-3 and compare our assay with indirect enzyme-linked immunosorbent assay (ELISA). Three sets of RT-LAMP primers were designed by sequence analysis of a specific conserved sequence of the Reo-3 S1 gene. Using RS2 primer set, the RT-LAMP assay required 60 min at 65 °C to amplify the S1 gene in one step by using Reo-3 RNA template and had no cross-reactivity with the other related pathogens, such as Sendai virus (SV), pneumonia virus of mice (PVM), mouse hepatitis virus (MHV), Ectromelia virus (Ect), minute virus of mice (MVM), P. pneumotropica, B. bronchiseptica, K. pneumonia and P. aeruginosa. in our LAMP reaction system. The limit of detection (LOD) of our RT-LAMP assay is 4 fg/μL. The established RT-LAMP assay enabled visual detection when fluorescence detection reagents were added, and was demonstrated to be effective and efficient. We tested 30 clinical blood samples and five artificial positive samples from SPF mice, the concordance between the two methods for blood samples was 100% compared with indirect ELISA and RT-PCR. Considering its performance, specificity, sensitivity, and repeatability, the developed RT-LAMP could be a valuable tool to supply a more effective Reo-3 detection method in laboratory animal quality monitoring.
Highlights
Mouse reovirus type 3 (Reo-3) infection is a viral disease that is harmful for laboratory mice
Reo-3, which was assigned to the genus Orthoreovirus, the family Reoviridae, was first isolated from the human body in the 1950s, and thereafter has been found in a variety of mammals[5]
Reo-3 is mainly transmitted through the digestive tract, the respiratory tract, the air and the fecal–oral route, and it can survive stably in ordinary environments[4,6]
Summary
Mouse reovirus type 3 (Reo-3) infection is a viral disease that is harmful for laboratory mice. Reo-3 plays an immunostimulatory role in the host response to environmental c arcinogens[6,7,8] It severely interferes with animal testing and is one of the most serious viral diseases in laboratory mice. Indirect enzyme-linked immunosorbent assay (ELISA) was recommended as the anti-Reo-3 antibodies detection method with certain limitations[9,10,11]. This method is not suitable for the detection of serum antibodies in cases in which: 1) antibody has not been produced after infection, or the level of serum antibody in mice with latent infections is too low to be serologically tested; 2) nude mice have immunodeficiency and Scientific Reports | (2021) 11:3508. This study aimed to establish an efficient, convenient and stable RT-LAMP detection method for Reo-3 nucleic acid detection
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