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

被引：125

作者：

Claassen, Shantelle ^{[1
]}

du Toit, Elloise ^{[1
]}

Kaba, Mamadou ^{[1
,4
]}

Moodley, Clinton ^{[2
]}

Zar, Heather J. ^{[3
]}

Nicol, Mark P. ^{[1
,4
]}

机构：

[1] Univ Cape Town, Dept Clin Lab Sci, Div Med Microbiol, ZA-7925 Cape Town, South Africa

[2] Groote Schuur Hosp, Natl Inst Communicable Dis, Natl Hlth Lab Serv, ZA-7925 Cape Town, South Africa

[3] Univ Cape Town, Red Cross War Mem Childrens Hosp, Dept Paediat & Child Hlth, ZA-7925 Cape Town, South Africa

[4] Univ Cape Town, Inst Infect Dis & Mol Med, ZA-7925 Cape Town, South Africa

来源：

JOURNAL OF MICROBIOLOGICAL METHODS | 2013年 / 94卷 / 02期

关键词：

Bacteria; DGGE; DNA extraction; Faeces; Real-time PCR; T-RFLP; GRADIENT GEL-ELECTROPHORESIS; 16S RIBOSOMAL-RNA; INTESTINAL MICROBIOTA; PCR; POPULATIONS; DIVERSITY; COMMUNITIES; GENES;

D O I：

10.1016/j.mimet.2013.05.008

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

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 (R) Virus/Bacteria Midi Kit (kit QS), ZR Fecal DNA MiniPrep (TM) (kit Z), QIAamp (R) DNA Stool Mini Kit (kit QA), Ultradean (R) Fecal DNA Isolation Kit (kit U) and PowerSoil (R) 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. (C) 2013 Elsevier R.V. All rights reserved.

引用

页码：103 / 110

页数：8

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