Evaluation of a cellulose-based dipstick method for DNA extraction from Leptotrombidium mites: A comparative study

Abstract

The Leptotrombidium mites are vectors of Orientia tsutsugamushi and a significant public health threat worldwide: scrub typhus is endemic in Sri Lanka. In this respect, the need to extract DNA from the Leptotrombidium mites in the field is critical for rapid diagnosis and surveillance; nevertheless, this presents some difficulties, including sample degradation and lack of specialist equipment. This study aims to validate a cellulose-based dipstick method against a commercial kit for efficient DNA extraction from Leptotrombidium mites. The DNA extraction from samples was thus performed with three methods: the cellulose-based dipstick method, the simple heat-lysis method, and the DNeasy Blood and Tissue Kit as a commercial benchmark. Assessments of DNA quality and quantity were done spectrophotometrically. Real-time PCR was done with primers of 16S rRNA targeting O. tsutsugamushi DNA. Agarose gel electrophoresis was conducted for the determination of the amplification. Descriptive statistics, paired Wilcoxon signed rank tests for DNA concentration and purity ratios, and Kruskal-Wallis tests for cycle threshold values were computed from the data. The DNA concentration and the total yield were highest in the DNeasy method. The dipstick method had a DNA concentration of 0.933 ± 0.138 μg/mL, while the mean values of the DNeasy and the heat-lysis methods were 3.240 ± 0.276 μg/mL and 0.840 ± 0.035 μg/mL, respectively. The dipstick method had the most variable A260/A280 ratios at 1.773 ± 0.215, while that of the DNeasy and the heat-lysis methods were 1.496 ± 0.087 and 1.418 ± 0.000, respectively. The comparison of Ct values did not show any statistically significant difference between methods, P = 0.2697. In conclusion, the cellulose-based dipstick method performed comparably to the heat-lysis method. Despite DNA yield and purity differences, all three methods produced similar PCR results. This method encompasses simplicity in conduct, cost-effectiveness, and field applicability, showing promising further developments in on-site DNA extraction.