A colorimetric assay for Neospora caninum utilizing the loop-mediated isothermal amplification technique

Area of Science:

• Veterinary Parasitology
• Molecular Diagnostics
• Livestock Health

Background:

• Neospora caninum (N. caninum) is a protozoan parasite posing significant risks to livestock through infection in various animals.
• Traditional diagnostic methods for N. caninum can be time-consuming and less sensitive.
• Loop-Mediated Isothermal Amplification (LAMP) presents a promising alternative for rapid, sensitive, and cost-effective parasite detection.

Purpose of the Study:

• To develop and optimize a precise, rapid, and visually assessable colorimetric LAMP assay for N. caninum detection.
• To compare the diagnostic performance of the developed LAMP method against conventional Polymerase Chain Reaction (PCR) techniques.
• To evaluate the potential of the LAMP assay for on-site and field applications in livestock diagnostics.

Main Methods:

• Optimal primer sets for N. caninum detection were identified through rigorous screening.
• The LAMP reaction was optimized at 65°C for 40 minutes using 270 μmol/L neutral red.
• Specificity was confirmed experimentally, and the method was applied to sheep and bovine serum samples.
• Performance was compared to nested PCR using the intragroup correlation coefficient (ICC).

Main Results:

• The optimized colorimetric LAMP method demonstrated high specificity for N. caninum.
• The LAMP assay exhibited a lower detection limit compared to traditional PCR.
• Prevalence detection in sheep serum was 5.61% (LAMP) vs. 3.37% (PCR), and in bovine serum was 3.38% (LAMP) vs. 1.69% (PCR).
• A high ICC value (0.999) indicated excellent agreement between LAMP and PCR methods.

Conclusions:

• The developed colorimetric LAMP assay is a sensitive, specific, and rapid diagnostic tool for N. caninum.
• This method offers significant advantages in convenience and cost-effectiveness over conventional PCR.
• The optimized LAMP technique holds strong potential for on-site and field-based detection of N. caninum in livestock.