Evaluation of Lysis Methods for the Extraction of Bacterial DNA for Analysis of the Vaginal Microbiota.
Evaluation of Lysis Methods for the Extraction of Bacterial DNA for Analysis of the Vaginal Microbiota.
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DOI：
10.1371/journal.pone.0163148
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Darby AC
中科院分区：
文献类型：
--
作者：
Gill C;van de Wijgert JH;Blow F;Darby AC
Recent studies on the vaginal microbiota have employed molecular techniques such as 16S rRNA gene sequencing to describe the bacterial community as a whole. These techniques require the lysis of bacterial cells to release DNA before purification and PCR amplification of the 16S rRNA gene. Currently, methods for the lysis of bacterial cells are not standardised and there is potential for introducing bias into the results if some bacterial species are lysed less efficiently than others. This study aimed to compare the results of vaginal microbiota profiling using four different pretreatment methods for the lysis of bacterial samples (30 min of lysis with lysozyme, 16 hours of lysis with lysozyme, 60 min of lysis with a mixture of lysozyme, mutanolysin and lysostaphin and 30 min of lysis with lysozyme followed by bead beating) prior to chemical and enzyme-based DNA extraction with a commercial kit. After extraction, DNA yield did not significantly differ between methods with the exception of lysis with lysozyme combined with bead beating which produced significantly lower yields when compared to lysis with the enzyme cocktail or 30 min lysis with lysozyme only. However, this did not result in a statistically significant difference in the observed alpha diversity of samples. The beta diversity (Bray-Curtis dissimilarity) between different lysis methods was statistically significantly different, but this difference was small compared to differences between samples, and did not affect the grouping of samples with similar vaginal bacterial community structure by hierarchical clustering. An understanding of how laboratory methods affect the results of microbiota studies is vital in order to accurately interpret the results and make valid comparisons between studies. Our results indicate that the choice of lysis method does not prevent the detection of effects relating to the type of vaginal bacterial community one of the main outcome measures of epidemiological studies. However, we recommend that the same method is used on all samples within a particular study.
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影响因子：
4.2
作者：
Brooks JP;Edwards DJ;Harwich MD Jr;Rivera MC;Fettweis JM;Serrano MG;Reris RA;Sheth NU;Huang B;Girerd P;Vaginal Microbiome Consortium;Strauss JF 3rd;Jefferson KK;Buck GA
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Buck GA
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3.7
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影响因子：
4.5
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通讯作者：
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影响因子：
17.1
作者：
Gajer P;Brotman RM;Bai G;Sakamoto J;Schütte UM;Zhong X;Koenig SS;Fu L;Ma ZS;Zhou X;Abdo Z;Forney LJ;Ravel J
通讯作者：
Ravel J
影响因子：
15.5
作者：
Fadrosh DW;Ma B;Gajer P;Sengamalay N;Ott S;Brotman RM;Ravel J
通讯作者：
Ravel J