Maintenance of host DNA integrity in field-preserved mosquito (Diptera: Culicidae) blood meals for identification by DNA barcoding
| Autor: | Phillip E. Kaufman, Akito Y. Kawahara, Jennifer L. Gillett-Kaufman, Lawrence E. Reeves, Chris J. Holderman |
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| Jazyk: | angličtina |
| Předmět: |
Degraded DNA
0301 basic medicine Blood meal analysis Polymerase Chain Reaction DNA barcoding law.invention chemistry.chemical_compound Aedes aegypti 0302 clinical medicine Aedes law Food science Polymerase chain reaction Cold Temperature Blood Infectious Diseases Host preference Vector ecology Blood meal identification Paper DNA preservation Preservation Biological 030231 tropical medicine Cold storage Mosquito Vectors Biology Specimen Handling Blood meal preservation 03 medical and health sciences COI barcoding Animals DNA Barcoding Taxonomic Desiccation Ethanol business.industry Host (biology) Research DNA Feeding Behavior Blood meal Biotechnology 030104 developmental biology Parasitology chemistry Vector business Filtration |
| Zdroj: | Parasites & Vectors |
| ISSN: | 1756-3305 |
| DOI: | 10.1186/s13071-016-1791-z |
| Popis: | Background Determination of the interactions between hematophagous arthropods and their hosts is a necessary component to understanding the transmission dynamics of arthropod-vectored pathogens. Current molecular methods to identify hosts of blood-fed arthropods require the preservation of host DNA to serve as an amplification template. During transportation to the laboratory and storage prior to molecular analysis, genetic samples need to be protected from nucleases, and the degradation effects of hydrolysis, oxidation and radiation. Preservation of host DNA contained in field-collected blood-fed specimens has an additional caveat: suspension of the degradative effects of arthropod digestion on host DNA. Unless effective preservation methods are implemented promptly after blood-fed specimens are collected, host DNA will continue to degrade. Preservation methods vary in their efficacy, and need to be selected based on the logistical constraints of the research program. Methods We compared four preservation methods (cold storage at -20 °C, desiccation, ethanol storage of intact mosquito specimens and crushed specimens on filter paper) for field storage of host DNA from blood-fed mosquitoes across a range of storage and post-feeding time periods. The efficacy of these techniques in maintaining host DNA integrity was evaluated using a polymerase chain reaction (PCR) to detect the presence of a sufficient concentration of intact host DNA templates for blood meal analysis. We applied a logistic regression model to assess the effects of preservation method, storage time and post-feeding time on the binomial response variable, amplification success. Results Preservation method, storage time and post-feeding time all significantly impacted PCR amplification success. Filter papers and, to a lesser extent, 95 % ethanol, were the most effective methods for the maintenance of host DNA templates. Amplification success of host DNA preserved in cold storage at -20 °C and desiccation was poor. Conclusions Our data suggest that, of the methods tested, host DNA template integrity was most stable when blood meals were preserved using filter papers. Filter paper preservation is effective over short- and long-term storage, while ethanol preservation is only suitable for short-term storage. Cold storage at -20 °C, and desiccation of blood meal specimens, even for short time periods, should be avoided. |
| Databáze: | OpenAIRE |
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