A comparison of the efficiency of five different commercial DNA extraction kits for extraction of DNA from faecal samples

Abstract

Differences in the composition of the gut microbiota have been associated with a range of diseases using culture-independent methods. Reliable extraction of nucleic acid is a key step in identifying the composition of the faecal microbiota. Five widely used commercial deoxyribonucleic acid (DNA) extraction kits (QIAsymphony® Virus/Bacteria Midi Kit (kit QS), ZR Fecal DNA MiniPrep™ (kit Z), QIAamp® DNA Stool Mini Kit (kit QA), Ultraclean® Fecal DNA Isolation Kit (kit U) and PowerSoil® DNA Isolation Kit (kit P)) were evaluated, using human faecal samples. Yield, purity and integrity of total genomic DNA were compared spectrophotometrically and using gel electrophoresis. Three bacteria, commonly found in human faeces were quantified using real time polymerase chain reaction (qPCR) and total bacterial diversity was studied using denaturing gradient gel electrophoresis (DGGE) as well as terminal restriction fragment length polymorphism (T-RFLP). The measurements of DNA yield and purity exhibited variations between the five kits tested in this study. Automated kit QS exhibited the best quality and highest quantity of DNA. All kits were shown to be reproducible with CV values≤0.46 for DNA extraction. qPCR results showed that all kits were uniformly efficient for extracting DNA from the selected target bacteria. DGGE and T-RFLP produced the highest diversity scores for DNA extracted using kit Z (H'=2.30 and 1.27) and kit QS (H'=2.16 and 0.94), which also extracted the highest DNA yields compared to the other kits assessed.

Keywords: Bacteria; DGGE; DNA extraction; Faeces; Real-time PCR; T-RFLP.

Fig. 1

DNA yield and purity extracted from faecal specimens using five commercial kits. DNA was extracted in triplicate from two starting weights (100 mg and 200 mg) of faeces per sample (n = 8 samples per kit). Median values are indicated by the line within the box plot. The box extends from the 25th to 75th percentiles and whiskers indicate the minimum and maximum values. * p < 0.05; ** p ≤ 0.01. A) Boxplot showing DNA yields obtained for all five kits evaluated. Kit QA values after RNA degradation and clean-up. B) Figure showing absorbance ratios at 260/280 for all five kits assessed. Kit QA values after RNA degradation. QS: QIAsymphony® Virus/Bacteria Mini Kit (Qiagen); Z: ZR Fecal DNA Isolation Kit™ (Zymo Research); QA: QIAamp® DNA Stool Mini Kit (Qiagen); U: Ultraclean® Fecal DNA Isolation Kit (Mobio); P: PowerSoil® DNA Isolation Kit (Mobio).

Fig. 2

Absolute DNA concentration for three targeted bacterial strains/groups using template DNA extracted with five commercial kits. DNA was extracted in triplicate from 200 mg of faeces per sample (n = 8 samples per kit). Median values are indicated by the line within the box plot. The box extends from the 25th to 75th percentiles and whiskers indicate the minimum and maximum values. A) Boxplot showing absolute DNA concentration extracted for Bacteroides/Prevotella group; B) Bifidobacterium genus; and C) Escherichia coli. QS: QIAsymphony® Virus/Bacteria Mini Kit (Qiagen); Z: ZR Fecal DNA Isolation Kit™ (Zymo Research); QA: QIAamp® DNA Stool Mini Kit (Qiagen); U: Ultraclean® Fecal DNA Isolation Kit (Mobio); P: PowerSoil® DNA Isolation Kit (Mobio).

Fig. 3

DGGE profiles of the 16S rDNA genes from DNA extracted using five commercial kits. DNA was extracted in triplicate from 200 mg of faeces per sample (n = 8 samples per kit) and run on a 45–65% DGGE gel gradient. A); Infant 1 (I1) and Adult 1 (A1); B); Infant 2 (I2) and Adult 2 (A2); C); Infant 3 (I3) and Adult 3 (A3); D); Infant 4 (I4) and Adult 4 (A4). QS: QIAsymphony® Virus/Bacteria Mini Kit (Qiagen); Z: ZR Fecal DNA Isolation Kit™ (Zymo Research); QA: QIAamp® DNA Stool Mini Kit (Qiagen); U: Ultraclean® Fecal DNA Isolation Kit (Mobio); P: PowerSoil® DNA Isolation Kit (Mobio).

Fig. 4

Bacterial diversity obtained from DNA extracted from faecal specimens using five commercial kits. Diversity (H′ indices) was determined from DNA extracted in triplicate from 200 mg of faeces per sample (n = 8 samples per kit), using two fingerprinting techniques. Mean H′ indices are indicated with a “+” within each box-and-whisker plot. Median values are indicated by the line within the box plot. The box extends from the 25th to 75th percentiles and the whiskers indicated the minimum and maximum values obtained. **p ≤ 0.01; ***p ≤ 0.001; **** p ≤ 0.0001 QS: QIAsymphony® Virus/Bacteria Mini Kit (Qiagen); Z: ZR Fecal DNA Isolation Kit™ (Zymo Research); QA: QIAamp® DNA Stool Mini Kit (Qiagen); U: Ultraclean® Fecal DNA Isolation Kit (Mobio); P: PowerSoil® DNA Isolation Kit (Mobio).