DNA was extracted from sediments using the GenoLyse kit (Hain Lifescience, Germany)8 . DNA was amplified using two ramp rates: the manufacturer-recommended ramp rate (2.2 °C/s), and 4.0 °C/s, the most frequently used incorrect ramp rate in the survey, using a CFX96 (Bio-Rad, United States), which was the only machine available with a customisable ramp rate. This instrument undergoes annual servicing and calibration by the manufacturer. Hybridisation was done with the GT-Blot 48 (Hain Lifescience, Germany)26 . An experienced reader interpreted bands in a blinded manner.
Gt blot 48
Manufactured by Hain Lifescience
Sourced in Germany
The GT-Blot 48 is a laboratory equipment designed for Western blotting analysis. It features a 48-well format and is capable of performing multiple blotting experiments simultaneously.
Automatically generated - may contain errors
4 protocols using gt blot 48
1
DNA Extraction from Sediments
2
Mycobacterial DNA Extraction and PCR Detection
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Mycobacterial DNA was extracted using a GenoLyse kit (Hain Lifescience, Nehren, Germany)-based manual method; however, instead of the 0.5 mL recommended by the manufacturer, we used 1 mL of decontaminated sputum sample. Polymerase chain reaction (PCR) was performed using pre-made amplification mixes (amplification mix A and amplification mix B) that contained all the necessary components. Hybridization was performed using an automated GT Blot 48 device (Hain Lifescience, Nehren, Germany), and the results were interpreted based on the operating manual provided by the manufacturer [5 , 6 (link)].
3
GenoType Mycobacterium CM Assay Protocol
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GenoType Mycobacterium CM assay (Hain Life Science, Nehren, Germany) was performed to identify mycobacteria species, according to the manufacturer’s instructions. The assay was run on a GT-Blot 48 (Hain Life Science, Nehren, Germany). The volume of the PCR mixture was 50 μL, comprised of 35 μL PNM, 5 μL 10x PCR buffer for HotStarTaq DNA Polymerase (Qiagen, Germany), 2 μL 25 mM MgCl2 solution, 1 U HotStarTaq DNA Polymerase, 3 μL deionized water, and 5 μL DNA solution. PCR was performed by denaturation steps at 95℃ for 15 min, 10 cycles of 95℃ for 30s and 58℃ for 2 min, 20 cycles of 95℃ for 25s, 53℃ for 40s, and 70℃ for 40s, followed by an extension at 70℃ for 8 min. After PCR completion, the results were automatically analyzed by the integrated software.
4
Mycobacterium Species Identification
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DNA was extracted from positive cultures using a GenoLyse® DNA Extraction kit from Hain Lifescience according to instructions.
Per the manufacturer’s instructions, M. tuberculosis was amplified and genotyped using a GenoType Mycobacterium CM kit. Amplicon hybridization was performed using a GenoType Mycobacterium CM VER 2.0 kit (Hain Lifescience) in an automated GT–Blot 48 (Hain Lifescience) hybridization apparatus. Visible hybridization bands on the DNA strips were compared to a reference key to differentiate and speciate between the M. tuberculosis complex and 27 clinically relevant Nontuberculous Mycobacteria (NTM).
Per the manufacturer’s instructions, M. tuberculosis was amplified and genotyped using a GenoType Mycobacterium CM kit. Amplicon hybridization was performed using a GenoType Mycobacterium CM VER 2.0 kit (Hain Lifescience) in an automated GT–Blot 48 (Hain Lifescience) hybridization apparatus. Visible hybridization bands on the DNA strips were compared to a reference key to differentiate and speciate between the M. tuberculosis complex and 27 clinically relevant Nontuberculous Mycobacteria (NTM).
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