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Published on: June 11, 2013
Comparison of DNA concentration and bacterial pathogen PCR detection when using two DNA extraction kits for
Dam Khan1, Shola-Able Thomas1, Peggy-Estelle Tientcheu1
1Medical Research Council Unit The Gambia at the London School of Hygiene & Tropical Medicine, Banjul, The Gambia.
Introduction:
Several important human pathogens that cause life-threatening infections are asymptomatically carried in the Nasopharynx/Oropharynx (NP/OP). DNA extraction is a prerequisite for most culture-independent techniques used to identify pathogens in the NP/OP. However, components of DNA extraction kits differ thereby giving rise to differences in performance. We compared the DNA concentration and the detection of three pathogens in the NP/OP using the discontinued DNeasy PowerSoil Kit (Kit DP) and the DNeasy PowerLyzer PowerSoil Kit (Kit DPP).
Methods:
DNA was extracted from the same set of 103 NP/OP samples using the two kits. DNA concentration was measured using the Qubit 2.0 Fluorometer. Real-time Polymerase Chain reaction (RT-PCR) was done using the QuantStudio 7-flex system to detect three pathogens: S. pneumoniae, H. influenzae, and N. meningitidis. Bland-Altman statistics and plots were used to determine the threshold cycle (Ct) value agreement for the two kits.
Results:
The average DNA concentration from kit DPP was higher than Kit DP; 1235.6 ng/ml (SD = 1368.3) vs 884.9 ng/ml (SD = 1095.3), p = 0.002. Using a Ct value cutoff of 40 for positivity, the concordance for the presence of S. pneumoniae was 82% (84/102); 94%(96/103) for N. meningitidis and 92%(95/103) for H. influenzae. Kit DP proportionately resulted in higher Ct values than Kit DPP for all pathogens. The Ct value bias of measurement for S. pneumoniae was +2.4 (95% CI, 1.9-3.0), +1.4 (95% CI, 0.9-1.9) for N. meningitidis and +1.4 (95% CI, 0.2-2.5) for H. influenzae.
Conclusion:
The higher DNA concentration obtained using kit DPP could increase the chances of recovering low abundant bacteria. The PCR results were reproducible for more than 90% of the samples for the gram-negative H. influenzae and N. meningitidis. Ct value variations of the kits must be taken into consideration when comparing studies that have used the two kits.
Insights
The DNeasy PowerLyzer PowerSoil Kit (Kit DPP) yielded higher DNA concentrations and comparable pathogen detection to the DNeasy PowerSoil Kit (Kit DP). This suggests Kit DPP may improve recovery of low-abundance bacteria in Nasopharynx/Oropharynx samples.
Area of Science:
- Microbiology
- Molecular Biology
- Clinical Diagnostics
Background:
- Nasopharynx/Oropharynx (NP/OP) asymptomatic carriage of human pathogens necessitates accurate identification.
- DNA extraction is crucial for culture-independent pathogen detection methods.
- Variations in DNA extraction kit components can impact assay performance.
Purpose of the Study:
- To compare the DNA yield and pathogen detection performance of two DNA extraction kits: DNeasy PowerSoil Kit (Kit DP) and DNeasy PowerLyzer PowerSoil Kit (Kit DPP).
- To evaluate the impact of kit differences on the detection of Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis in NP/OP samples.
Main Methods:
- DNA was extracted from 103 NP/OP samples using both Kit DP and Kit DPP.
- DNA concentration was quantified using Qubit 2.0 Fluorometer.
- Real-time Polymerase Chain Reaction (RT-PCR) was employed to detect S. pneumoniae, H. influenzae, and N. meningitidis, with Bland-Altman analysis for Ct value agreement.
Main Results:
- Kit DPP resulted in significantly higher average DNA concentration (1235.6 ng/ml) compared to Kit DP (884.9 ng/ml) (p=0.002).
- High concordance (>82%) was observed for pathogen detection between the kits, with Kit DP yielding higher Ct values (indicating lower DNA concentration or detection efficiency).
- Reproducible PCR results were achieved for >90% of samples for H. influenzae and N. meningitidis.
Conclusions:
- The higher DNA yield from Kit DPP may enhance the detection of low-abundance bacteria in NP/OP samples.
- Both kits demonstrated good concordance for detecting common NP/OP pathogens, particularly gram-negative bacteria.
- Variations in Ct values between kits should be considered when comparing results across studies using different DNA extraction methods.
