Validation and application of qPCR test kit for detection of Mycoplasma dispar DNA

Introduction. Currently, Mycoplasma dispar is widely spread and circulating in livestock farms around the world, including in the Russian Federation. The implementation of a real-time polymerase chain reaction test kit for detecting Mycoplasma dispar DNA in veterinary practice is highly relevant, as this pathogen can cause respiratory diseases in cattle and contribute to calf mortality, leading to significant economic losses in livestock production.

Objective. To introduce a newly developed real-time polymerase chain reaction test kit Mycoplasma dispar DNA detection kit into veterinary practice and determine its major validation parameters.

Materials and methods. Mycoplasma dispar reference strain (ATCC No. 27140) was cultured in 1699 Revised Mycoplasma Medium recommended by the American Type Culture Collection. DNA was extracted using a commercial kit, real-time polymerase chain reaction was performed using pre-selected parameters. The major validation parameters of the test kit were determined: analytical sensitivity, analytical specificity, amplification efficiency, repeatability and reproducibility. Applicability of real-time polymerase chain reaction test kit for detection of Mycoplasma dispar DNA was demonstrated.

Results. The Mycoplasma dispar DNA detection test kit demonstrated an analytical sensitivity (detection limit) of 10 copies/μL (100 copies/reaction), 100% specificity (exclusive to Mycoplasma dispar DNA), 99.01% amplification efficiency, and an average repeatability coefficient of variation of 0.91%. Reproducibility coefficient of variation ranged from 0.66% to 1.26% across 5 replicates and was 0.91% across 15 replicates. The test kit was validated using 228 biological samples from cattle from 13 regions of the Russian Federation, while Mycoplasma dispar DNA was detected in 39.47% of the samples tested.

Conclusion. The developed Mycoplasma dispar DNA test kit has demonstrated high validation performance and is suitable for diagnosing bovine mycoplasmosis.

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