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

PLoS One. 2019 Dec 31;14(12):e0226818. doi: 10.1371/journal.pone.0226818. eCollection 2019.

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • DNA / isolation & purification*
  • Female
  • Hemiptera / genetics*
  • Nymph / genetics
  • Polymerase Chain Reaction / methods
  • Preservation, Biological / methods*

Substances

  • DNA

Grants and funding

This work was supported by the National Key Research and Development Program of China (2017YFC1200600, 2016YFC1201200), and the Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences (no. caascx-2013-2018-IAS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.