Evaluación de Cinco Métodos de Extracción de ADN e Identificación de Biotipos de Spodoptera frugiperda (J.E. Smith) Mediante PCR-RFLP

The extraction of DNA in insects is a limiting factor in genetic studies due to the presence of inhibitors that interfere on the polymerase chain reaction (PCR) and molecular markers such as RFLP-PCR. Thus, it is important to use methods that allow sufficient amounts of DNA with high purity, quality, and concentration. Five DNA extraction methods based on CTAB, H. Guanidine, STE, Lysis Buffer, and Qiagen Kit, were evaluated in fourth-and fifth-instar larvae of Spodoptera frugiperda (J.E. Smith). The DNA obtained from the five protocols, was evaluated for quality, purity, cost, and concentration. This allowed selection of the best methodology for biotype identification of the insect pest by PCR-RFLP. The average concentrations of DNA extraction were 1158.5, 404, 610, 188.7, and 25.9 ng/μl, for the methods CTAB, H. Guanidine, STE buffer, Lysis Buffer, and Qiagen kit, respectively. The DNA obtained with the STE method presented a greater amount of protein impurities and cell debris (A260/A280 = 1.37), followed by lysis buffer, H. Guanidine, Qiagen kit, and CTAB (2.13, 1.89, and 1.85). Results showed the best method in terms of DNA purity and quality was by the Qiagen kit; however, it is the protocol with the lowest concentration. The CTAB method represented the best extraction protocol with the highest concentration and reliable purity. With the Qiagen kit, CTAB, and Lysis Buffer methods, 100% was obtained in amplification of the 569 pb fragment of the S. frugiperda COI gene, compared to the rest of the methods evaluated. The best results were obtained using CTAB and Qiagen kit methods, but the CTAB method is recommended from this study because its highest concentration, required quality, and low cost of DNA extraction, in addition to detecting and identifying the S. frugiperda biotype in maize of Sinaloa, Mexico. All the DNA extraction methods evaluated were efficient in identifying the biotype of the fall armyworm by PCR-RFLP analysis.