Confirmation of the presence of Mycobacterium tuberculosis and other mycobacteria in mycobacterial growth indicator tubes (MGIT) by multiplex strand displacement amplification

Abstract

Multiplex strand displacement amplification (mSDA) is capable of amplifying three distinct DNA sequences simultaneously. These include sequences present in most genera of mycobacteria, a sequence specific for Mycobacterium tuberculosis, and an internal control. mSDA was used to detect the presence of these target sequences in 154 (72 positive, 76 negative, and 6 failed) clinical specimens cultured in the mycobacterial growth indicator tube (MGIT) system. A wide variety of specimen types were processed and cultured. Once these cultures were deemed positive by MGIT fluorescence or were deemed negative after 8 weeks of incubation, MGIT culture aliquots were processed for mSDA analyses. A chemiluminescent microwell assay was used to detect the amplified products. The procedure was relatively simple and took less than 6 h to complete. The sensitivity of mSDA for detecting acid-fast bacilli was 96.4% compared to that of MGIT culture. Sensitivity and specificity were 97.2 and 96.1%, respectively, when all clinical criteria were considered. mSDA was shown to be a rapid and effective method for confirming the presence of M. tuberculosis and other mycobacteria in positive MGIT cultures.

Impact of drug resistance on fitness of Mycobacterium tuberculosis strains of the W-Beijing genotype

“…A volume of 0.5 ml of the dilution (1.5×10 5 bacterial cells) was added to the BBL MGIT Mycobacteria Growth Indicator Tube (BACTEC, Becton Dickinson Diagnostic Systems, Sparks, MD, USA). The tubes were entered into the BACTEC MGIT 960 System (BACTEC, Becton Dickinson Diagnostic Systems, Sparks, MD, USA) [28], incubated at 37 °C, and monitored for increasing fluorescence. The BACTEC MGIT 960 System performs monitoring for fluorescence (in units) every hour.…”

Strand Displacement Amplification for Multiplex Detection of Nucleic Acids

“…After amplification of the target strands by the primers, the adapter sequences start to bind to the amplified target sequences and begin extension and displacement, which results in a cascade of exponential amplification of the target sequences using the adapters rather than the primers. This method was also used for multiplexed SDA of three distinct DNA sequences of Mycobacterium tuberculosis and other mycobacteria [56]. Furthermore, multiplex SDA was used to amplify multiple SNPs simultaneously with molecular beacon probe-assisted fluorescent signal readout [57].…”