Revista da Sociedade Brasileira de Medicina Tropical · 2020
Comparison of phenol-chloroform and a commercial deoxyribonucleic acid extraction kit for identification of bloodmeal sources from triatomines (Hemiptera: Reduviidae).
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Silva Andressa Noronha Barbosa da, Souza Rita de Cássia Moreira de, Honorato Nathan Ravi Medeiros, Martins Rand Randall, Câmara Antônia Claudia Jácome da, Galvão Lúcia Maria da Cunha, Chiari Egler
Аннотация
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.
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.