Validation of digital PCR assay for the quantification of Mycobacterium avium subsp. paratuberculosis in bovine faeces according to the ISO 20395:2019

Paratuberculosis is an enteric disease caused by Mycobacterium avium subs. Paratuberculosis (MAP). Quantifying the load of MAP in faeces samples offers the advantage of determining the stage of infection and planning control measures. Currently, detection of MAP in faecal specimens relies on cultural assays and quantitative PCR (qPCR), but both methods have limitations such as prolonged isolation times for cultural assay and the absence of nucleic acid standards for qPCR. Digital PCR (dPCR) represents an advancement over qPCR as it allows direct quantification of nucleic acid in a sample without the need for a standard curve.The present paper reports about the validation process, following ISO 20395:2019 guidelines, of a F57 digital PCR assay for quantifying MAP cells in faecal samples.Based on our validation, the Limit Of Detection (LOD) corresponds to 7.85 104 MAP cells/g, and the Limit Of Quantification (LOQ) to 7.85 105 MAP cells, with an efficiency of recovery at LOQ estimated about 4.5%. To assess precision, we evaluated the same faecal sample extracted by two different operators at different times. The standard deviation under repeatability conditions (S Repeatability) and intersession variability conditions (S Intermediate) were calculated, resulting in values of 0.43 and 0.26, respectively. Trueness was determined at LOQ and a value ten times higher, yielding percentages of 3.35% and 5.16%, respectively. Linearity showed a R2 value of 0.998, indicating strong linear correlation. Measurement uncertainty was 26% in absolute value and 3% on a logarithmic base 10 scale. Overall, the assay exhibits good specificity and robustness. Our validation underlines the good performance of the quantification method and allow the laboratory to provide quantitative results of MAP/cells on faecal samples.