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


Autoři: Yu-Sheng Wang aff001;  Tian-Mei Dai aff001;  Hu Tian aff001;  Fang-Hao Wan aff001;  Gui-Fen Zhang aff001
Působiště autorů: State Key Laboratory for Biology of Plant Diseases and Insect Pests / Key Laboratory of Integrated Pest Management of Crop, Ministry of Agriculture and Rural Affairs of the People’s Republic of China, Institute of Plant Protection, Chinese Academy of Agri aff001;  Hunan Provincial Key Laboratory for Control of Forest Diseases and Pests, College of Forestry, Central South University of Forestry and Technology, Changsha, China aff002;  Caofeidian Sub-Center of Hebei Entry-Exit Inspection and Quarantine Technical Center, Tangshan, China aff003;  Center for Management of Invasive Alien Species, Ministry of Agriculture and Rural Affairs of the People’s Republic of China, Beijing, China aff004
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article
prolekare.web.journal.doi_sk: 10.1371/journal.pone.0226818

Souhrn

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.

Klíčová slova:

DNA – DNA electrophoresis – DNA extraction – Gene amplification – Nymphs – Polymerase chain reaction – Ribonucleases – RNA extraction


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2019 Číslo 12