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Updated: Jan 13, 2026
Matrix-based DNA Extraction for Targeted Next-Generation Sequencing on Decontaminated Sputum Samples
Published on: June 6, 2025
Development and evaluation of 'Quick TB DNA Extraction' kit for the rapid and efficient detection of
Rakesh Kumar Gupta1,2, Keerti Chauhan1, Ritu Singhal3
1Department of Experimental Medicine and Biotechnology, Postgraduate Institute of Medical Education and Research, Chandigarh, India.
Purpose:
We recently demonstrated the utility of the 'TB Concentration & Transport' kit for bio-safe, ambient-temperature transport of dried sputum samples on Trans-Filter, along with the 'TB DNA Extraction' kit for efficient DNA extraction from Trans-Filter for use in the Line Probe Assay (LPA) for diagnosing drug-resistant tuberculosis (TB). The present study aimed to develop and evaluate a new 'Quick TB DNA Extraction' kit ('Quick DNA' kit) for rapid DNA isolation from Trans-Filter samples and assess its compatibility with LPA for the detection of multidrug-resistant TB (MDR-TB).
Methods:
Consecutive presumptive TB/MDR-TB/XDR-TB patients (n = 1823) were screened using LED-FM and/or TBDetect microscopy at 2 Designated Microscopy Centres associated with the National Institute of Tuberculosis and Respiratory Diseases (NITRD), New Delhi. Smear-positive samples (n = 235) were processed in duplicate using the 'TB Concentration and Transport' kit. Dried sputum on bio-safe Trans-Filters was transported at ambient temperature, along with sputum samples, in a 3-layer packing in cooling conditions to NITRD Hospital (a National Reference Laboratory). DNA was extracted from Trans-Filters using 'Quick DNA' kit and the 'TB DNA Extraction' kit, and from sputum using Hain's GenoLyse® DNA Extraction kit for first-line LPA for MDR-TB detection.
Results:
Quick Kit-LPA and Kit-LPA (LPA with DNA extracted from Trans-Filter using 'Quick DNA' kit and 'TB DNA Extraction' kit, respectively) showed similar sensitivity of 88.9% (95% CI: 65.3-98.6) and 88.5% (95% CI: 69.9-97.5) and specificity of 100% (95% CI: 98.2-100) and 99.5% (95% CI: 97.3-99.9) for rifampicin and isoniazid resistance detection, respectively against Direct-LPA (LPA with DNA extracted from sputum samples using GenoLyse kit). User feedback obtained from laboratory technicians corroborated that the one-step 'Quick DNA' kit procedure was rapid (5 minutes), easy to perform, seamlessly integrated with LPA testing, and was suitable as a replacement for Kit-LPA or Direct-LPA.
- , Conclusion:
The gap between drug-resistant TB detection and treatment initiation can be narrowed through Universal-Drug Susceptibility Testing by implementing (i) bio-safe and ambient temperature transport of sputum from primary healthcare centres to central laboratories, and (ii) by using Quick Kit-LPA over Direct-LPA in patients residing in remote areas.
