Development of Nested Polymerase Chain Reaction with Novel Specific Primers for Detection of Tritrichomonas muris...

Area of Science:

• Veterinary Parasitology
• Molecular Diagnostics
• Laboratory Animal Science

Background:

• Rodent cecal parasitism by Tritrichomonas muris (T. muris), a flagellated protozoan, is common.
• Current detection methods for T. muris in laboratory mice lack ideal accuracy and specificity.
• Development of novel molecular diagnostic tools is crucial for effective T. muris detection.

Purpose of the Study:

• To develop and validate a highly sensitive and specific molecular method for detecting T. muris in laboratory mice.
• To compare the diagnostic performance of the new method against traditional smear microscopy.

Main Methods:

• Morphological characterization of T. muris using staining and Scanning Electron Microscopy (SEM).
• Design and optimization of a nested PCR assay targeting conserved regions of the SSU rRNA gene of T. muris.
• Evaluation of nested PCR sensitivity and specificity using known parasite loads and fecal DNA concentrations.
• Comparative analysis of nested PCR and smear microscopy for T. muris detection in fecal samples from five mouse strains.

Main Results:

• The nested PCR system demonstrated high sensitivity, detecting as few as 100 T. muris trophozoites/mL or 0.1 ng/μL fecal DNA.
• The assay could detect 176 T. muris trophozoites per gram of mouse feces.
• Nested PCR identified T. muris in 18.96% of samples, significantly higher than smear microscopy (14.05%).
• The nested PCR assay achieved 100% sensitivity and specificity, showing a 26% increase in diagnostic sensitivity over smear microscopy.

Conclusions:

• A novel nested PCR assay based on T. muris SSU rRNA gene primers is accurate, specific, and sensitive.
• This molecular method significantly improves the detection rate and diagnostic sensitivity for T. muris infections in laboratory mice compared to smear microscopy.
• The developed nested PCR provides a valuable tool for precise diagnosis and monitoring of T. muris in research settings.