Enhancement of M. tuberculosis Line Probe Assay Sensitivity through Whole Genome Amplification of Low-Quantity DNA Released from Sputum and Archived on Chemically-Coated Cellulose Matrix Using an Isothermal Enzymatic Strand-Displacement Process

In this study, thirty-nine sputum samples from tuberculosis (TB)-positive patients undergoing first-line therapy were collected and archived on a chemical-coated cellulose matrix. DNA was extracted from these matrices and tested for Mycobacterium tuberculosis using the Xpert MTB/RIF Ultra assay. Seven samples tested positive for M. tuberculosis, with low levels of detection. End-point PCR yielded faint signals in four samples, but no signal in the remaining three. A Line Probe Assay (LPA) detected pathogen DNA in only one of the three PCR-negative samples. Remarkably, the LPA-negative samples were successfully detected by LPA and end-point PCR following isothermal, strand displacement-based whole genome amplification (WGA) of the stock DNA. The drug sensitivity profile of these samples was consistent with the Xpert MTB/RIF Ultra results obtained from the original stock DNA. Additionally, sputum DNA from healthy individuals spiked with 125 ng, but not with 1.25 ng, of M. tuberculosis culture DNA was detected by LPA. Following WGA, the 1.25 ng sample was also detected successfully by LPA, and its drug sensitivity profile matched that of the 125-ng sample. These findings indicate that WGA of sputum DNA from a cellulose matrix, even with low pathogen load, can enhance the detection capabilities of LPA by enriching the genome target through an isothermal enzymatic strand displacement method. This method holds promise for improving diagnostic sensitivity in TB detection.