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, Thomas O Powers

Research output: Contribution to journalArticle

16 Citations (Scopus)

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 - Jan 1 1998

Fingerprint

Simuliidae
Diptera
Parasites
digestive system
polymerase chain reaction
parasites
Polymerase Chain Reaction
DNA
Larva
Ethanol
ethanol
Solar System
methodology
larvae
animal parasitic nematodes
solar drying
DNA Primers
Mitochondrial Genes
DNA primers
Acetic Acid

Keywords

  • Black flies
  • DNA extraction
  • Insect preservation methods
  • Polymerase chain reaction
  • Randomly amplified polymorphic DNA
  • Simulium vittatum

ASJC Scopus subject areas

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

Cite this

Effects of Preservation Methods, Parasites, and Gut Contents of Black Flies (Diptera : Simuliidae) on Polymerase Chain Reaction Products. / Koch, D. A.; Duncan, G. A.; Parsons, T. J.; Pruess, K. P.; Powers, Thomas O.

In: Journal of medical entomology, Vol. 35, No. 3, 01.01.1998, p. 314-318.

Research output: Contribution to journalArticle

@article{77d54b83ed2c4ae7989c7e2bc7ee105e,
title = "Effects of Preservation Methods, Parasites, and Gut Contents of Black Flies (Diptera: Simuliidae) on Polymerase Chain Reaction Products",
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.",
keywords = "Black flies, DNA extraction, Insect preservation methods, Polymerase chain reaction, Randomly amplified polymorphic DNA, Simulium vittatum",
author = "Koch, {D. A.} and Duncan, {G. A.} and Parsons, {T. J.} and Pruess, {K. P.} and Powers, {Thomas O}",
year = "1998",
month = "1",
day = "1",
doi = "10.1093/jmedent/35.3.314",
language = "English (US)",
volume = "35",
pages = "314--318",
journal = "Journal of Medical Entomology",
issn = "0022-2585",
publisher = "Entomological Society of America",
number = "3",

}

TY - JOUR

T1 - Effects of Preservation Methods, Parasites, and Gut Contents of Black Flies (Diptera

T2 - Simuliidae) on Polymerase Chain Reaction Products

AU - Koch, D. A.

AU - Duncan, G. A.

AU - Parsons, T. J.

AU - Pruess, K. P.

AU - Powers, Thomas O

PY - 1998/1/1

Y1 - 1998/1/1

N2 - 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.

AB - 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.

KW - Black flies

KW - DNA extraction

KW - Insect preservation methods

KW - Polymerase chain reaction

KW - Randomly amplified polymorphic DNA

KW - Simulium vittatum

UR - http://www.scopus.com/inward/record.url?scp=0032060479&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0032060479&partnerID=8YFLogxK

U2 - 10.1093/jmedent/35.3.314

DO - 10.1093/jmedent/35.3.314

M3 - Article

VL - 35

SP - 314

EP - 318

JO - Journal of Medical Entomology

JF - Journal of Medical Entomology

SN - 0022-2585

IS - 3

ER -