All experiments were conducted in duplicate, with a positive internal amplification control (IAC) present in each sample and a separate negative
control lacking template DNA included with PCR amplifications. The specific PCR product amplified with primers ASP_GEN_MTSSU_F1 and ASP_GEN_MTSSU_R1 was firstly digested using the restriction enzyme SnaBI (New England BioLabs, Ipswich, MA, USA), and a 154 bp fragment containing the annealing site for primer ASP_GEN_MTSSU_F1 then cloned into the vector pGEMTeasy (Promega, Madison, WI, USA) according selleckchem to standard protocols. Following cloning, a recombinant strain was stored as a glycerine culture at −80°C. Plasmid DNA was isolated and 10 pg used as an IAC template in all PCR reactions, together with the pGEM®-T Easy-targeting reverse primer M13, annealing to position 176 on the pGEM®-T Easy plasmid vector DNA sequence. CP-868596 datasheet mtDNA SSU rDNA PCR RFLP analysis The potential of the mtDNA SSU rRNA gene amplicon for inter-specific differentiation was investigated based upon polymorphism
in restriction sites. The target mtDNA sequence was analysed in each of the Selleckchem GSI-IX Aspergillus species available at Genbank, which included six Aspergillus species with complete mitochondrial genome sequences [54]. Restriction maps were generated for each species using the program Sequence Manipulation Suíte (http://www.bioinformatics.org/sms2/rest_map.html). Following identification of suitable restriction sites for differentiation, RFLP digestion of the specific mtDNA amplicons was then tested across the section Flavi species and additional Aspergillus species isolated from Brazil nut. Each digest reaction volume of 30 μL contained 1 mg of PCR product, 1 × restriction enzyme buffer React 1 (Invitrogen, Carlsbad, CA, USA), and 1 U of the selected restriction enzyme DraI (Invitrogen, Carlsbad, CA, USA). Following a two hour incubation period
at 37°C, digest fragments were electrophoresed in 1% agarose gels at 5 V cm−1 in the presence of ethidium bromide (1 μg mL−1), and visualized under UV BCKDHA at 254 nm. The marker Low DNA Mass ladder® (Invitrogen, Carlsbad, CA, USA) was included on gels for digest fragment size estimation. Acknowledgements This work was funded by EMBRAPA (Project “Inovações tecnológicas para o controle da contaminação da castanha-do-Brasil por aflatoxinas”) and CNPq (Project “Aspectos epidemiológicos e moleculares no diagnóstico e controle da contaminação da castanha-do-Brasil por aflatoxinas”). GEOM was supported by a fellowship from CAPES at Universidade de Brasília. RNGM was supported by a fellowship from CNPq. We thank anonymous reviewers for their useful comments on the manuscript. Electronic supplementary material Additional file 1: MtDNA SSU rRNA gene Dra I restriction mapping data for Aspergillus species. (DOCX 17 KB) Additional file 2: Ribosomal DNA ITS, beta-tubulin and calmodulin gene sequences deposited at Genbank for Aspergillus ex-type strains.