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

D. A. Koch, G. A. Duncan, T. J. Parsons, K. P. Pruess, T. O. Powers

Research output: Contribution to journalArticle

16 Scopus citations

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 amplications. 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.

Original languageEnglish (US)
Pages (from-to)314-318
Number of pages5
JournalJournal of medical entomology
Volume35
Issue number3
DOIs
StatePublished - May 1998

All Science Journal Classification (ASJC) codes

  • Parasitology
  • veterinary(all)
  • Insect Science
  • Infectious Diseases

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