Development of Nested Polymerase Chain Reaction with Novel Specific Primers for Detection of Tritrichomonas muris Infection in Laboratory Mice

SIMPLE SUMMARY: Tritrichomonas muris (T. muris) mainly parasitizes the ceca of different rodent species worldwide. To date, there are no ideal methods for the detection of T. muris infections in laboratory mice. This study developed a nested PCR reaction using a novel specific primer based on the conserved regions of the SSU rRNA gene of T. muris for a molecular epidemiological survey (investigation) of T. muris infections in laboratory mice. The results showed that the nested PCR system has higher sensitivity, reliability, and specificity. The nested PCR system showed an infection rate of T. muris of 18.96% (58/306), which was higher than the infection rate of 14.05% (43/306) that was detected via smear microscopy in fecal samples from five mouse strains. The sensitivity and specificity of nested PCR in detecting T. muris was found to be 100%, and it demonstrated a 26% increase in diagnostic sensitivity compared to the smear microscopy method. The present study provides a new method for the molecular epidemiological investigation of T. muris infections in laboratory mice. ABSTRACT: A variety of rodent ceca are parasitized by Tritrichomonas muris (T. muris), a flagellated protozoan. To date, there are no ideal methods for the detection of T. muris infections in laboratory mice; thus, new molecular methodologies for its specific detection need to be developed. In this study, using staining and SEM, it was observed that T. muris has a pear-shaped body and contains three anterior flagella. A nested PCR system with novel specific primers was designed based on the conserved regions of the SSU rRNA gene of T. muris. The nested PCR system for T. muris showed good specificity and high sensitivity for at least 100 T. muris trophozoites/mL and 0.1 ng/μL of fecal genomic DNA, which means that 176 trophozoites per gram of mouse feces could be detected. When using this nested PCR system, the detection rate was 18.96% (58/306), which was higher than the detection rate of 14.05% (43/306) detected via smear microscopy in fecal samples from five mouse strains. The sensitivity and specificity of nested PCR in detecting T. muris was found to be 100%, and it demonstrated a 26% increase in diagnostic sensitivity compared to the smear microscopy method in the present study. In conclusion, the nested PCR developed with novel primers based on the SSU rRNA gene of T. muris has good accuracy, specificity, and sensitivity for the detection of T. muris infections in laboratory mice.