Detection of Mycobacterium chelonae, Mycobacterium abscessus Group, and Mycobacterium fortuitum Complex by a Multiplex Real-Time PCR Directly from Clinical Samples Using the BD MAX System

Abstract

A new multiplex PCR test was designed to detect Mycobacterium chelonae, Mycobacterium abscessus group, and Mycobacterium fortuitum complex on the BD MAX System. A total of 197 clinical samples previously submitted for mycobacterial culture were tested using the new protocol. Samples were first treated with proteinase K, and then each sample was inoculated into the BD MAX Sample Buffer Tube. Extraction and multiplex PCR were performed by the BD MAX System, using the BD MAX ExK TNA-3 extraction kit and BD TNA Master Mix, along with specific in-house designed primers and probes for each target. The limit of detection of each target, as well as specificity, was evaluated. Of 197 clinical samples included in this study, 133 were positive and 60 were negative for mycobacteria by culture, and another 4 negative samples were spiked with M. chelonae ATCC 35752. The new multiplex PCR on the BD MAX had 97% concordant results with culture for M. abscessus group detection, 99% for M. chelonae, and 100% for M. fortuitum complex. The new multiplex PCR test performed on the BD MAX System proved to be a sensitive and specific test to detect M. chelonae, M. abscessus group, and M. fortuitum complex by real-time PCR on an automated sample-in results-out platform.

Host and pathogen response to bacteriophage engineered against Mycobacterium abscessus lung infection

“…At transplant the participant's lungs were surveyed for evidence of M. abscessus. Bronchoalveolar lavage (BAL), swabs of unprocessed endobronchial secretions, gross pathology and homogenized tissue from each lobe, and the mainstem bronchovascular margins were stained for acid fact bacilli (AFB), cultured for NTM and bacteria, and analyzed by real-time quantitative PCR (qPCR) (Rocchetti et al, 2017;Figure 1G). All sample types were negative for AFB by stain in all lobes.…”

“…Culture-independent markers demonstrate evidence of M. abscessus lysis Molecular quantification of M. abscessus DNA from total DNA of 17 sputum samples, ranging from À136 days pre-phage to 361 days post-phage, was analyzed by real-time qPCR (Rocchetti et al, 2017) as a complement to traditional cultures (Figure 4A). Compared with a standard curve with known concentrations of M. abscessus, sputum samples from days À136 to 82 demonstrated consistent detection of M. abscessus DNA corresponding with positive cultures during that time period (Figures 4B and S2).…”

Closed-type pre-treatment device for point-of-care testing of sputum

“…fortuitum . The forward and reverse primers used for qPCR were synthesized as 5′-TCACCTGATCTGCACATAATGT-3′ and 5′-AGCACCTCATGCGACTT-3′, respectively (Bioneer, Korea) 23 . To sterilized PCR tubes was added 10 μL of genomic DNA extracted from M .…”

Development, Optimization, and Validation of a Multiplex Real-Time PCR Assay on the BD MAX Platform for Routine Diagnosis of Acanthamoeba Keratitis

“…The BD MAX platform can be used for adapting previously developed in-house PCR methods into a fully integrated and automated on-demand molecular diagnostic system, and it has already been used and validated for many other microbiological targets. 11,12,23,24 The linear dynamic range revealed a theoretical lower limit of detection of 0.2 genomes per PCR, but the automated extraction protocol of the BD MAX is performed on 950/ 1250 mL from the BD MAX sample tube, so, if there is only one cyst in the extraction tube, there is a small probability (1 in 4) of the cyst not being extracted. Thus, although AK-BD MAX can detect one or two cysts in the sample tube, at least three or four cysts would likely be a more comfortable input for ensuring maximal sensitivity of the test.…”