Evaluation of a Chelex 100-based DNA extraction procedure for morphologically identified adult cyathostomins

Abstract

Cyathostomins (Nematoda: Strongylida: Cyathostominae) are common gastrointestinal nematodes of horses and comprise a taxonomically diverse group. The aim of the study was to evaluate an adapted Chelex 100-based procedure for DNA extraction from individual morphologically identified adult cyathostomins and to assess the yield and spectrophotometric characteristics of the obtained DNA. Eleven adult cyathostomins were morphologically identified and processed individually. DNA concentration and absorbance ratios A260/A280 and A260/A230 were determined by microvolume spectrophotometry. Measurable nucleic acid was obtained from all 11 specimens. DNA concentrations ranged from 1.9 to 106.6 ng/µL, with a median of 12.5 ng/µL and a mean of 29.9 ng/µL. The A260/A280 ratio ranged from 1.80 to 6.33; the median value was 1.97, and six of the eleven extracts had values between 1.80 and 2.00. The extracts were additionally tested by PCR. Amplification was obtained from the prepared DNA samples, confirming the presence of amplifiable template. The Chelex 100/proteinase K procedure provided DNA from individually processed, morphologically identified adult cyathostomins without column purification or organic solvents and with a limited number of sample-transfer steps. The method can be used as a simple preparatory approach in studies combining morphological identification of individual cyathostomins with molecular genetic analysis.