Method for Reduction of Inhibition in a Mycobacterium tuberculosis -Specific Ligase Chain Reaction DNA Amplification Assay

Abstract

The present study describes the identification of inhibitors of aMycobacterium tuberculosis-specific gap ligase chain reaction (LCR) DNA amplification assay as well as a method for their removal. A major contributor to inhibition was deduced to be a calcium phosphate precipitate, CaHPO4. The precipitate forms duringN-acetyl-l-cysteine–sodium hydroxide (NALC-NaOH) decontamination, digestion, and concentration of respiratory specimens. The solubility product of CaHPO4 precipitate at pH 7.8, the pH at which gap LCR is optimized, indicates that the precipitate releases an amount of phosphate ions sufficient to inhibit amplification. A method for removal of the precipitate was identified. The precipitate is dissociated by exposing it to a mildly acidic (pH 4.1) buffer during the first of two centrifugation steps; the inhibitory phosphate ions are removed by the centrifugation steps. When 100 NALC-NaOH respiratory sediments were tested by gap LCR, none of the sediments were inhibitory when the acidic buffer was used while 24 samples were inhibitory when TE buffer, pH 7.8, was used. In another study, when the acidic buffer wash was applied to 1,440 NALC-NaOH respiratory sediments, only 10 sediments were found to be inhibitory. None of the inhibited sediments were culture positive for M. tuberculosis. This work demonstrates that when inhibition mechanisms are identified, relatively simple protocols can be used to obtain low inhibition rates and to allow the use of larger volume equivalents in amplification reactions.

Multicenter Evaluation of the Abbott LCx Mycobacterium tuberculosis Ligase Chain Reaction Assay

“…The LCx assay integrates well into the routine diagnostic laboratory. Because the amplification and detection steps are automated, once specimen preparation is completed, the assay requires minimal involvement from laboratory staff, with the time to test completion being approximately 5 h. Specimen preparation involves two washing steps and appears to reduce the potential for specimen inhibitors to interfere with the assay (23). An internal amplification control is not included in the assay, so a second tube should be spiked with whole MTBC cells to check for the presence of inhibitors.…”