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.

Fig 1. Electrophoresis of total DNA extracted from specimens preserved for four durations by using eight commonly used methods.

(A-D) DNA extracted from specimens preserved for different durations (A: fresh, B: short period, C: intermediate period, D: long period; coded as I-IV in Table 1). Five lanes are shown for each method, from left to right: DNA samples extracted from an individual egg, and 1st, 2nd, and 3rd instar nymphs, as well as female adult mealybugs. M: λ-Hind III digest DNA marker.

Fig 2. PCR amplification pattern of COI (A) and 28S (B) genes for testing the DNA quality extracted specimens preserved for four durations by using eight commonly used DNA extraction methods.

Preservation methods I-IV are fresh, short period, intermediate period, and long period. Five lanes are shown for each method, from left to right: DNA samples extracted from an adult mealybug, 3rd, 2nd and 1st instar nymphs, as well as individual eggs. M: Trans2K DNA Marker; NTC: negative control (ultrapure water).

Fig 3. Average absorbance ratio A260/280 (mean±SE) of DNA extracted from fresh and short period conserved samples.

Average DNA absorbance ratio A260/280 was calculated based on samples of the 3rd instar female nymph and female adult. Means with different lowercase letters above the bars are significantly different at P < 0.05 (one-way ANOVA, Tukey’s HSD test).

Fig 4. PCR amplification of the COI (A) and 28S (B) genes using a DNA template dilution series of fresh samples extracted using the SDS method.

Five individuals of female adults are a-e. Lanes 1–15: 166.20×10³, 83.10×10³, 41.55×10³, 20.78×10³, 10.39×10³, 5.19×10³, 2.60×10³, 1.30×10³, 649.22, 324.61, 162.30, 81.15, 40.58, 20.29, 10.14 pg·μL^-1, i.e., 1-, 2-, 4-, 8-, 16-, 32-, 64-, 128-, 256-, 512-, 1,024-, 2,048-, 4,096-, 8,192-, and 16,384-fold dilutions, respectively. M: Trans2K DNA Marker; NTC: negative control (ultrapure water).