Comparison of phenol-chloroform and a commercial deoxyribonucleic acid extraction kit for identification of bloodmeal sources from triatomines (Hemiptera: Reduviidae).

Andressa Noronha Barbosa Da Silva,Rita De Cássia Moreira De Souza,Egler Chiari,Nathan Ravi Medeiros Honorato,Rand Randall Martins,Antônia Claudia Jácome Da Câmara,Lúcia Maria Da Cunha Galvão

doi:10.1590/0037-8682-0189-2020

Abstract

INTRODUCTION: Knowledge of triatomine bloodmeal sources is essential for understanding vector-host interactions in Trypanosoma cruzi transmission cycles. Expensive commercial deoxyribonucleic acid (DNA) extraction kits are widely used for bloodmeal identification. This study assessed the performance of an inexpensive phenol-chloroform DNA extraction protocol for identification of triatomine bloodmeal sources, comparing it with a commercially available kit. METHODS: Both methods were used to obtain DNA from the intestinal contents of Triatoma brasiliensis blood-fed on either Columba sp., Mus musculus, or Gallus gallus. Subsequently, the mitochondrial 12S ribosomal ribonucleic acid (rRNA) gene was amplified by polymerase chain reaction, sequenced, and compared with GenBank data. RESULTS: Twelve (80%) samples extracted with the commercial kit and four (26.7%) with phenol-chloroform were pure (according to the A260/A280 ratio). Samples extracted with phenol-chloroform, except for Columba sp. samples, had higher DNA concentration than those extracted with the commercial kit. Samples extracted using phenol-chloroform and blood-fed on G. gallus had significantly higher DNA concentration than those blood-fed on Columba sp. (p-value <0.001) and M. musculus (p-value <0.001). The 215-base-pair 12S rRNA fragment was amplified from all samples and produced reliable sequences, enabling the identification of the bloodmeal source, most of which showed identity and coverage above 95%. The phenol-chloroform method was much less expensive than the commercial kit but took considerably more time to perform. CONCLUSIONS: Our data showed that both DNA extraction methods produced reliable sequences enabling identification of triatomine bloodmeal sources but differed greatly in cost and time required.

Highlights

Knowledge of triatomine bloodmeal sources is essential for understanding vector-host interactions in Trypanosoma cruzi transmission cycles
There was no significant difference in the average A260/280 ratios of deoxyribonucleic acid (DNA) samples collected from triatomines fed on the same bloodmeal source but extracted using different methods: (1) for Columba sp., the mean and standard deviation for the commercial kit were 1.84 ± 0.02, and for phenol-chloroform were 1.78 ± 0.08 (p-value = 0.916); (2) for M. musculus, the average for the commercial kit was 1.76 ± 0.12, and for phenol-chloroform was 1.67 ± 0.16 (p-value = 0.892); and (3) for G. gallus, the average for the commercial kit was 1.83 ± 0.01, and for phenol-chloroform was 1.75 ± 0.04 (p-value = 0.995)
With regard to the A260/A230 ratio, for both methods the presence of some contamination was apparent, the samples extracted with the commercial kit showed values closer to the appropriate range when compared to those extracted using phenol-chloroform

Summary

Introduction

Knowledge of triatomine bloodmeal sources is essential for understanding vector-host interactions in Trypanosoma cruzi transmission cycles. This study assessed the performance of an inexpensive phenol-chloroform DNA extraction protocol for identification of triatomine bloodmeal sources, comparing it with a commercially available kit. Conclusions: Our data showed that both DNA extraction methods produced reliable sequences enabling identification of triatomine bloodmeal sources but differed greatly in cost and time required. Triatomines (Hemiptera, Reduviidae, and Triatominae) are obligatorily hematophagous insects in their nymphal and adult life cycle stages[1] They are potential transmitters of Trypanosoma cruzi, the etiological agent of Chagas disease or American trypanosomiasis[2]. The vertebrate hosts are numerous species of mammals from several orders, including Didelphimorphia, Rodentia, Carnivora, and Primates[4] Despite their intrinsic refractoriness to T. cruzi infection[5,6], birds and www.scielo.br/rsbmt I www.rsbmt.org.br

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