Effects of Preservation Methods, Parasites, and Gut Contents of Black Flies (Diptera: Simuliidae) on Polymerase Chain Reaction Products

Authors

• D. A. Koch, Nebraska Wesleyan University
• G. A. Duncan, Nebraska Wesleyan UniversityFollow
• T. J. Parsons, University of Nebraska-Lincoln
• K. P. Pruess, University of Nebraska-LincolnFollow
• T. O. Powers, University of Nebraska-LincolnFollow

Document Type

Article

Date of this Version

5-1998

Citation

Journal of Medical Entomology 35:3 (May 1998), pp. 314–318.

doi 10.1093/jmedent/35.3.314

Comments

Copyright © 1998 Entomological Society of America; published by Oxford University Press. Used by permission.

Abstract

Molecular analysis of biological specimens usually requires extraction of high-molecular-weight DNA free of foreign DNA contaminants. DNA was extracted from black flies at different life stages that had been preserved by 4 methods: larvae and adults in ethanol, larvae in Carnoy’s solution, adults on card-points, and adults hand-swatted and sun-dried. Using specific primers for the mitochondrial ND4 gene, a 257-bp amplicon was obtained from specimens preserved by ethanol, card-point mounting, and sun-drying. Successful amplification often required DNA dilutions ≥ 1:20 (<1–10 ng). DNA from specimens preserved in Carnoy’s solution (ethanol: acetic acid, 3:1) yielded degraded DNA, resulting in fewer successful amplifications. Parasitic nematodes and, to a lesser extent, gut contents resulted in extra products when amplified with randomly amplified polymorphic DNA (RAPD) primers. Sufficient DNA was extracted from the head of a larva for a successful polymerase chain reaction (PCR), eliminating the need to remove the contaminating gut and parasites.