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International Journal of Biotech Trends and Technology (IJBTT) |  |
| © 2025 by IJBTT Journal | ||
| Volume - 15 Issue - 1 | ||
| Year of Publication : 2025 | ||
| Authors : Ikechukwu Jude Onyia, Uzoamaka Ogechi George-Okafor, Ujunwa Felicia Nwachukwu | ||
| DOI : 10.14445/22490183/IJBTT-V15I1P102 |
How to Cite?
"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 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.
References
[1] Soumitesh Chakravorty et al., “The New Xpert MTB/RIF Ultra: Improving Detection of Mycobacterium Tuberculosis and Resistance to Rifampin in an Assay Suitable for Point-of-Care Testing,” MBio, vol. 8, no. 4, 2017.
[CrossRef] [Google Scholar] [Publisher Link]
[2] Frank B. Dean et al., “Comprehensive Human Genome Amplification Using Multiple Displacement Amplification,” Proceedings of the National Academy of Sciences of the United States of America, vol. 99, no. 8, pp. 5261–5266, 2002.
[CrossRef] [Google Scholar] [Publisher Link]
[3] J. Dinnes et al., “A Systematic Review of Rapid Diagnostic Tests for the Detection of Uberculosis Infection,” Health Technology Assessment, vol. 11, no. 3, pp. 1–196, 2007.
[CrossRef] [Google Scholar] [Publisher Link]
[4] Krishna H. Goyani, and Pratap N. Mukhopadhyaya, “Estimating Limit of Detection of M. Tuberculosis DNA as an Analyte Released from a Chemically Coated Solid Matrix and Using a WHO-Approved CB-NAAT Platform,” BioRxiv, pp. 1-6, 2023.
[CrossRef] [Google Scholar] [Publisher Link]
[5] Krishna H. Goyani, and Pratap N. Mukhopadhyaya, “Sputum-spotted Solid Matrix Designed to Release Diagnostic-Grade Mycobacterium Tuberculosis DNA Demonstrate Optimal Biocontainment Property,” BioRxiv, 2023.
[CrossRef] [Google Scholar] [Publisher Link]
[6] A.J. Hance et al., “Detection and Identification of Mycobacteria by Amplification of Mycobacterial DNA,” Molecular Microbiology, vol. 3, no. 7, pp. 843–849, 1989.
[CrossRef] [Google Scholar] [Publisher Link]
[7] L.B. Heifets, and G.A. Cangelosi, “Drug Susceptibility Testing of Mycobacterium Tuberculosis: A Neglected Problem at the Turn of the Century,” The International Journal of Tuberculosis and Lung Disease, vol. 3, no. 7, pp. 564–581, 1999.
[Google Scholar] [Publisher Link]
[8] Patricia T. Kent, Public Health Mycobacteriology: A Guide for the Level III Laboratory, U.S. Department of Health and Human Services, Centers for Disease Control, pp. 1-207, 1985.
[Google Scholar] [Publisher Link]
[9] Roger S. Lasken, and Michael Egholm, “Whole Genome Amplification: Abundant Supplies of DNA from Precious Samples or Clinical Specimens,” Trends in Biotechnology, vol. 21, no. 2, pp. 153–158, 2003.
[Google Scholar] [Publisher Link]
[10] Stephen D. Lawn, and Mark P. Nicol, “Xpert® MTB/RIF Assay: Development, Evaluation, and Implementation of a New Rapid Molecular Diagnostic for Tuberculosis and Rifampicin Resistance,” Future Microbiology, vol. 6, no. 9, pp. 1067–1082, 2011.
[CrossRef] [Google Scholar] [Publisher Link]
[11] Stephen D. Lawn et al., “Advances in Tuberculosis Diagnostics: The Xpert MTB/RIF Assay and Future Prospects for a Point-of-Care Test,” The Lancet Infectious Diseases, vol. 13, no. 4, pp. 349–361, 2013.
[Google Scholar] [Publisher Link]
[12] J.J. Leary, D.J. Brigati, and D.C. Ward, “Rapid and Sensitive Colorimetric Method for Visualizing Biotin-labeled DNA Probes Hybridized to DNA or RNA Immobilized on Nitrocellulose: Bio-Blots,” Proceedings of the National Academy of Sciences of the United States of America, vol. 80, no. 13, pp. 4045–4049, 1983.
[CrossRef] [Google Scholar] [Publisher Link]
[13] Gardner Middlebrook, and Maurice L. Cohn, “Bacteriology of Tuberculosis: Laboratory Methods,” American Journal of Public Health and the Nation's Health, vol. 48, no. 7, pp. 844–853, 1958.
[Google Scholar] [Publisher Link]
[14] Carl Nathan, “Resisting Antimicrobial Resistance,” Nature Reviews Microbiology, vol. 18, pp. 259–260, 2020.
[CrossRef] [Google Scholar] [Publisher Link]
[15] Marilyn M. Ninan et al., “The Diagnostic Utility of Line Probe Assays for Multidrug-Resistant Tuberculosis,” Pathogens and Global Health, vol. 110, no. 4–5, pp. 194–199, 2016.
[CrossRef] [Google Scholar] [Publisher Link]
[16] G. Roscigno, “Drug Resistant TB and New Diagnostics for People Living with HIV: Emerging Results from FIND,” IAS Conference: Catalysing HIV/TB Research, 2009.
[Google Scholar]
[17] Kuldeep Singh Sachdeva et al., “Use of Xpert MTB/RIF in Decentralized Public Health Settings and Its Effect on Pulmonary TB and DR-TB Case Finding in India,” Plos One, vol. 10, no. 5, 2015.
[CrossRef] [Google Scholar] [Publisher Link]
[18] Peter M. Small, and Madhukar Pai, “Tuberculosis Diagnosis—Time for a Game Change,” The New England Journal of Medicine, vol. 363, pp. 1070–1071, 2010.
[CrossRef] [Google Scholar] [Publisher Link]
[19] Ha Kan Telenius et al., “Degenerate Oligonucleotide-Primed PCR: General Amplification of Target DNA by a Single Degenerate Primer,” Genomics, vol. 13, no. 3, pp. 718–725, 1992.
[CrossRef] [Google Scholar] [Publisher Link]
[20] Grant Theron et al., “Evaluation of the Xpert MTB/RIF Assay for the Diagnosis of Pulmonary Tuberculosis in a High HIV Prevalence Setting,” American Journal of Respiratory and Critical Care Medicine, vol. 184, no. 1, 2011.
[CrossRef] [Google Scholar] [Publisher Link]
[21] World Health Organization, “Policy Statement: Molecular Line Probe Assays for Rapid Screening of Patients at Risk of Multidrug resistant Tuberculosis (MDR-TB),” pp. 1-9, 2008.
[Publisher Link]
[22] World Health Organization, “Multidrug and Extensively Drug-resistant TB (M/XDR-TB): 2010 Global Report on Surveillance and Response,” pp. 1-71, 2010.
[Publisher Link]
[23] World Health Organization, “Guidelines for the Programmatic Management of Drug-resistant Tuberculosis: 2011 Update,” 2011.
[Publisher Link]
[24] World Health Organization, “Global Programme on Tuberculosis & Lung Health,” 2023.
[Publisher Link]
[25] Konstantin A. Blagodatskikh et al., “Improved DOP-PCR (iDOP-PCR): A Robust and Simple WGA Method for Efficient Amplification of Low Copy Number Genomic DNA,” Plos One, vol. 12, no. 9, 2017.
[CrossRef] [Google Scholar] [Publisher Link]
[26] Yohei Nishikawa et al., “Monodisperse Picoliter Droplets for Low-Bias and Contamination-Free Reactions in Single-Cell Whole Genome Amplification,” Plos One, vol. 10, no. 9, 2015.
[CrossRef] [Google Scholar] [Publisher Link]