DIRECT PCR USING Spodoptera frugiperda EGGS AS TEMPLATE

LOTO FLAVIA; BAIGORI, MARIO; PERA, LICIA

Tafi del Valle, Tucumán

Congreso; XXXV Jornadas Científicas de la Asociación de Biología de Tucumán; 2008

Asociación de Biología de Tucumán

Introduction: PCR is widely employed in a variety of experimen-tal applications such as studies of Spodoptera frugiperda (Sf), animportant pest in America. Objective: To optimize direct PCR tech-nique using Sf samples. Material and methods: PCR reactionswere performed in 25 μL containing: 21.4 μl sterile water, 0.1 μl ofeach oligonucleotide primers (JM76 and JM77), 5 μl 5X Taq bufferand 0.2 μl Taq DNA polymerase. One, two or three eggs were addedto the reaction mixture. Negative and positive controls were per-formed with distilled water and purified DNA, respectively. Am-plification was performed with the following program: An initialdenaturation step at 97ºC for 5 min and then 35 cycles of the fol-lowing: 1 min at 94ºC, 1 min at 58ºC, 2 min at 72ºC, and a finalextension at 72ºC 2 min. Results and Discussion: Samples with 2eggs were amplified with the same quality as samples amplifiedwith purified DNA. Only one of three amplifications assays waspositive in samples with one egg. Samples with 3 eggs were notamplified, probably due to excessive DNA or PCR inhibitors. Themethod is simple, fast and cost saving.This work was supported by grants PICTO-UNT 761 and PIP 6062