Development and application of a rapid screening method for Rodentibacter heylii and Rodentibacter pneumotropicus in laboratory animals

Abstract Background: Rodentibacter pneumotropicus and Rodentibacter heylii (formerly known as [Pasteurella pneumotropica]) are pathogens that must be excluded from specific pathogen-free (SPF)-level laboratory animals. Currently, the isolated culture is troublesome. The loop-mediated isothermal amplification (LAMP) with lateral flow dipstick (LFD) is simple, fast, specific, sensitive, and the closed device to prevent aerosol contamination. Methods: In this study, the deoxynucleotide triphosphate (dNTPs), magnesium (Mg2+), and temperature were set to various settings to determine the reaction system. The specificity and sensitivity studies were used to confirm the accuracy of the method. Multiple polymerase chain reaction, Real-time Quantitative polymerase chain reaction (qPCR), and LAMP-LFD were used to compare and analyze the trachea and lung tissues. 842 SPF-level laboratory animal samples were detected with LAMP-LFD. Results: The optimal concentrations of dNTPs, Mg2+, and reaction temperature for Rodentibacter pneumotropicus and Rodentibacter heylii were 1.6 mM, 6 mM, 63°C and 64°C, respectively. The lower limit of detection for LAMP-LFD can reach 1×10-5 ng/μL, which can detect Rodentibacter pneumotropicus and Rodentibacter heylii, and it has a sensitivity of 10 orders of magnitude higher than PCR. LAMP-LFD had no cross-reaction with 15 strains of common pathogenic bacteria in laboratory animals. Among the 28 infected samples analyzed, Multiple PCR, qPCR, and LAMP-LFD demonstrated the ability to detect Rodentibacter pneumotropicus and Rodentibacter heylii in 21, 23, and 19 positive samples, respectively. Furthermore, the isolated culture, Multiple PCR, qPCR, and LAMP-LFD identified 2, 23, 41, and 37 positive cases of Rodentibacter pneumotropicus and Rodentibacter heylii in a total of 842 clinical samples. Conclusion: In conclusion, LAMP-LFD was established together with an integrated detection device which can accurate identification of R. heylii and R. pneumotropicus. This simple and practical method greatly facilitated the detection efforts of grassroots organizations.