Efficient Differentiation ofMycobacterium aviumComplex Species and Subspecies by Use of Five-Target Multiplex PCR

Authors:
Publisher website
Google Scholar
Abstract
Infections caused by theMycobacterium aviumcomplex (MAC) are on the rise in both human and veterinary medicine. A means of effectively discriminating among closely related yet pathogenetically diverse members of the MAC would enable better diagnosis and treatment as well as further our understanding of the epidemiology of these pathogens. In this study, a five-target multiplex PCR designed to discriminate MAC organisms isolated from liquid culture media was developed. This MAC multiplex was designed to amplify a 16S rRNA gene target common to allMycobacteriumspecies, a chromosomal target called DT1 that is unique toM. aviumsubsp.aviumserotypes 2 and 3, toM. aviumsubsp.silvaticum, and toM. intracellulare, and three insertion sequences, IS900, IS901, and IS1311.The pattern of amplification results allowed determination of whether isolates were mycobacteria, whether they were members of the MAC, and whether they belonged to one of three major MAC subspecies,M. aviumsubsp.paratuberculosis,M. aviumsubsp.avium, andM. aviumsubsp.hominissuis. Analytical sensitivity was 10 fg ofM. aviumsubsp.paratuberculosisgenomic DNA, 5 to 10 fg ofM. aviumsubsp.aviumgenomic DNA, and 2 to 5 fg of DNA from other mycobacterial species. Identification accuracy of the MAC multiplex was evaluated by testing 53 bacterial reference strains consisting of 28 different mycobacterial species and 12 nonmycobacterial species. Identification accuracy in a clinical setting was evaluated for 223 clinical MAC isolates independently identified by other methods. Isolate identification agreement between the MAC multiplex and these comparison assays was 100%. The novel MAC multiplex is a rapid, reliable, and simple assay for discrimination of MAC species and subspecies in liquid culture media.

This publication has 42 references indexed in Scilit: