Development of quantitative triplex real time PCR for the simultaneous detection of Mycobacterium avium subsp. avium and M. a. subsp. hominissuis

Abstract
Mycobacterium avium subsp. avium (MAA) and M. a. subsp. hominissuis (MAH) belong to the Mycobacterium avium complex and are frequently associated with diseases in animals and humans. In animals it causes tuberculous lesions in parenchymatous organs. Infections with MAA and MAH in humans are rather scarce, but when present they cause severe complications that lead to the chronic stage or sometimes even to the death of individual. The aim of this study was to develop a system for rapid and accurate quantitative real time PCR (qPCR) identification and quantification of MAA and MAH. The developed triplex qPCR reaction was based on the simultaneous detection of specific insertion sequences, IS901 and IS1245 and an internal amplification control. The specificity and sensitivity of the qPCR were determined as well as the limit of detection for both pathogens isolated from tissue samples. In order to quantify both pathogens as precise as possible, the recovery of MAA and MAH cells after DNA isolation was established. To test the triplex qPCR assay coupled with the DNA isolation, tissue samples from 22 per os artificially infected pigs, of which ten were infected with MAA, ten with MAH and two were present as a negative control group were tested. From each animal, 21 different tissue samples as well as blood were tested by microscopy, culture and qPCR. In both groups of experimentally infected animals, the newly developed triplex qPCR assay proved to be more specific and sensitive in comparison with the other methods used. Contrary to culture examination, triplex qPCR confirmed the infection in all animals infected with MAA, and in eight animals infected with MAH. In conclusion, we developed a quick and sufficiently sensitive triplex qPCR for MAA and MAH detection in tissue samples that represents a suitable alternative to the culture.
This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic “AdmireVet” (CZ 1.05/2.1.00/01.0006 and No. ED 0006/01/01), the Ministry of Agriculture of the Czech Republic (Grant No. MZe0002716202) and the EU grant PathogenCombat.