International Journal of Biotech
Trends and Technology

Research Article | Open Access | Download PDF
Volume 15 | Issue 1 | Year 2025 | Article Id. IJBTT-V15I1P102 | DOI : https://doi.org/10.14445/22490183/IJBTT-V15I1P102

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

Krishna H. Goyani, Chirajyoti Deb, Daisy Patel, Shalin Vaniawala, Pratap N. Mukhopadhyaya

Received Revised Accepted Published
18 Nov 2024 05 Mar 2025 26 Mar 2025 11 Apr 2025

Citation :

Krishna H. Goyani, Chirajyoti Deb, Daisy Patel, Shalin Vaniawala, Pratap N. Mukhopadhyaya, "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," International Journal of Biotech Trends and Technology (IJBTT), vol. 15, no. 1, pp. 8-18, 2025. Crossref, https://doi.org/10.14445/22490183/IJBTT-V15I1P102

Abstract

In this study, thirty-nine sputum samples from tuberculosis (TB)-positive patients undergoing first-line therapy were collected and archived on a chemically 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 pathogenic DNA in only one of the three PCR-negative samples. Remarkably, 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 profiles of these samples were consistent with the Xpert MTB/RIF Ultra results obtained using the original stock DNA. Additionally, sputum DNA from healthy individuals spiked with 125 ng, but not 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 a low pathogen load, can enhance the detection capabilities of LPA by enriching the genome target through an isothermal enzymatic strand displacement method. This method is promising for improving the diagnostic sensitivity of TB detection.

Keywords

Cellulose matrix, Whole Genome Amplification, Line Probe Assay, Xpert, End point PCR, Tuberculosis.

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