Evaluation of DNA Extraction Methods for Reliable Quantification of Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa

Abstract

Detection and quantification of DNA biomarkers relies heavily on the yield and quality of DNA obtained by extraction from different matrices. Although a large number of studies have compared the yields of different extraction methods, the repeatability and intermediate precision of these methods have been largely overlooked. In the present study, five extraction methods were evaluated, using digital PCR, to determine their efficiency in extracting DNA from three different Gram-negative bacteria in sputum samples. The performance of two automated methods (GXT NA and QuickPick genomic DNA extraction kit, using Arrow and KingFisher Duo automated systems, respectively), two manual kit-based methods (QIAamp DNA mini kit; DNeasy UltraClean microbial kit), and one manual non-kit method (CTAB), was assessed. While GXT NA extraction kit and the CTAB method have the highest DNA yield, they did not meet the strict criteria for repeatability, intermediate precision, and measurement uncertainty for all three studied bacteria. However, due to limited clinical samples, a compromise is necessary, and the GXT NA extraction kit was found to be the method of choice. The study also showed that dPCR allowed for accurate determination of extraction method repeatability, which can help standardize molecular diagnostic approaches. Additionally, the determination of absolute copy numbers facilitated the calculation of measurement uncertainty, which was found to be influenced by the DNA extraction method used.

Characterization and in-vitro plant-based control of hindgut bacteria isolated from Odontotermes obesus Rambur (Termitidae) and Heterotermes indicola Wasmann (Rhinotermitidae)

“…Bacterial DNA was extracted by using the CTAB method with some modifications. 12 Polymerase chain reaction (PCR) amplification of the 16SrRNA gene was performed by using the following set of primers UNR-5 ′ -GGACTACCAGGGTATCTAAT-3 ′ and UI-5 ′ -CCAGCAGCCGCGGTAAT ACG-3 ′ . PCR reaction was conducted by using a Thermo cycler (96 universal Gradient Peq Star Lab Peq UK) having a reaction mixture of 50 μL, comprising PCR water (33.3 μL),Taq buffer (5 μL), MgCl 2 (4 μL), dNTPs (1 μL), forward and reverse primer (2 μL), and Taq DNA polymerase (0.7 μL).…”

Whole-genome sequencing of Klebsiella pneumoniae isolated from clinical specimens in Chattogram, Bangladesh

“…The colony morphology was observed the next day, followed by biochemical tests [e.g., IMViC tests and Triple sugar iron (TSI) test] for initial detection of Kpn ( 7 , 8 ). Genomic DNA was extracted from pure colonies by following the manufacturer’s instruction of the DNA extraction kit (QIAmp DNA extraction kit, Qiagen, Germany) and extracted DNA quality was screened with a Nanodrop spectrophotometer (Thermo Fisher Scientific, USA) ( 9 ). The pair-end library was prepared by using a NEBNext Ultra II FS DNA library prep kit (Illumina, San Diego, CA) following manufacturer guideline, and library size was selected with AMPure XP beads (BECKMAN COULTER) ( 10 , 11 ).…”

Automated Protocol for Monitoring Droplets and Fomites on Surfaces

“…A Nextrator-48S system (Genolution, Seoul, Republic of Korea) was used according to the manufacturer's recommendations after testing the already available kits for different purposes (e.g., viral RNA, yeast DNA, nucleic acids from stools and other matrices), in particular VN141R, MD141 or SD151 (Genolution, Seoul, Republic of Korea), optimizing the conditions in preliminary experiments. Briefly, preliminary tests included DNA extraction using the highest starting volumes (100-200 µL) and different lysis volumes (50-100 µL), finally suggesting loading 200 µL of the sample into an extract lysis volume of 50 µL (Supplementary Materials, Tables S1 and S2) in accordance with Best Practice Recommendations for Internal Validation of DNA Extraction Methods [54,55]. Experiments were finally performed using kit CVN291, showing the highest level of accuracy and repeatability for processing environmental swab samples diluted in collection and storage media (Table S1) [50,[52][53][54][55][56].…”

“…Briefly, preliminary tests included DNA extraction using the highest starting volumes (100-200 µL) and different lysis volumes (50-100 µL), finally suggesting loading 200 µL of the sample into an extract lysis volume of 50 µL (Supplementary Materials, Tables S1 and S2) in accordance with Best Practice Recommendations for Internal Validation of DNA Extraction Methods [54,55]. Experiments were finally performed using kit CVN291, showing the highest level of accuracy and repeatability for processing environmental swab samples diluted in collection and storage media (Table S1) [50,[52][53][54][55][56]. Briefly, an aliquot of 200 µL was loaded into the well of the plate and extraction was performed using a program named VN in Nextractor-48S software system, following the manufacturer's instructions.…”