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Comparative analysis of eight DNA extraction methods for molecular research in mealybugs.

ABSTRACT: For molecular research, the quality and integrity of DNA obtained will affect the reliability of subsequent results. Extracting quality DNA from scale insects, including mealybugs, can be difficult due to their small body size and waxy coating. In this study, we evaluate eight commonly used DNA extraction methods to determine their efficacy in PCR analysis across life stages and preservation times. We find that fresh samples, immediately upon collection or after 2 wks, resulted in the most effective DNA extraction. Methods using the DNeasy Blood & Tissue kit, NaCl, SDS-RNase A, and SDS isolated DNA of sufficient quality DNA. The SDS method gave high DNA yield, while the NaCl and SDS-RNase A methods gave lower yield. NaCl, SDS-RNase A, SDS, chloroform-isopentyl alcohol, and the salting-out methods all resulted in sufficient DNA for PCR, and performed equal to or better than that of the DNeasy Blood & Tissue kit. When time and cost per extraction were considered, the SDS method was most efficient, especially for later life stages of mealybug, regardless of preservation duration. DNA extracted from a single fresh sample of a female adult mealybug was adequate for more than 10,000 PCR reactions. For earlier stages, including the egg and 1st instar nymph samples, DNA was most effectively extracted by the Rapid method. Our results provide guidelines for the choice of effective DNA extraction method for mealybug or other small insects across different life stages and preservation status.

SUBMITTER: Wang YS

PROVIDER: S-EPMC6938366 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Comparative analysis of eight DNA extraction methods for molecular research in mealybugs.

Wang Yu-Sheng YS Dai Tian-Mei TM Tian Hu H Wan Fang-Hao FH Zhang Gui-Fen GF

PloS one 20191231 12

For molecular research, the quality and integrity of DNA obtained will affect the reliability of subsequent results. Extracting quality DNA from scale insects, including mealybugs, can be difficult due to their small body size and waxy coating. In this study, we evaluate eight commonly used DNA extraction methods to determine their efficacy in PCR analysis across life stages and preservation times. We find that fresh samples, immediately upon collection or after 2 wks, resulted in the most effec ...[more]

PMID: 31891602

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Project description:BackgroundThe microbiota of the lower female genital tract plays an important role in women's health. Microbial profiling using the chaperonin60 (cpn60) universal target (UT) improves resolution of vaginal species associated with negative health outcomes compared to the more commonly used 16S ribosomal DNA target. However, the choice of DNA extraction and PCR product purification methods may bias sequencing-based microbial studies and should be optimized for the sample type and molecular target used. In this study, we compared two commercial DNA extraction kits and two commercial PCR product purification kits for the microbial profiling of cervicovaginal samples using the cpn60 UT.MethodsDNA from cervicovaginal secretions and vaginal lavage samples as well as mock community standards were extracted using either the specialized QIAamp DNA Microbiome Kit, or the standard DNeasy Blood & Tissue kit with enzymatic pre-treatment for enhanced lysis of gram-positive bacteria. Extracts were PCR amplified using well-established cpn60 primer sets and conditions. Products were then purified using a column-based method (QIAquick PCR Purification Kit) or a gel-based PCR clean-up method using the QIAEX II Gel Extraction Kit. Purified amplicons were sequenced with the MiSeq platform using standard procedures. The overall quality of each method was evaluated by measuring DNA yield, alpha diversity, and microbial composition.ResultsDNA extracted from cervicovaginal samples using the DNeasy Blood and Tissue kit, pre-treated with lysozyme and mutanolysin, resulted in increased DNA yield, bacterial diversity, and species representation compared to the QIAamp DNA Microbiome kit. The column-based PCR product purification approach also resulted in greater average DNA yield and wider species representation compared to a gel-based clean-up method. In conclusion, this study presents a fast, effective sample preparation method for high resolution cpn60 based microbial profiling of cervicovaginal samples.

Dataset Information

Comparative analysis of eight DNA extraction methods for molecular research in mealybugs.

Publications

Comparative analysis of eight DNA extraction methods for molecular research in mealybugs.

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