Restricted AP-PCR (resAP-PCR)

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This technique has been selected for oral presentation in the 8th International Meeting on Microbial Epidemiological Markers (IMMEM-8), to be held in Zakopane, Poland, May 14-17, 2008: Plenary session #1: New Typing Strategies and Technologies: Oral presentation #6 Application of resAP-PCR fingerprinting to strains from sequenced bacterial species I. Martinez-Ballesteros, A. Bernal, R. San Millán, A. Rementeria, J. Garaizar, J. Bikandi

This technique was primarely developed by in silico simulation, and latter it was applied to Salmonella strains. The performance was excelent. The methods used are described bellow: Organisms. A total of 27 Salmonella strains (check table bellow), grown on trypticasein soy agar at 37 ºC overnight, were examined.  The cultures were obtained from our collection of Salmonella spp. strains at the Department of Immunology, Microbiology and Parasitology, University of the Basque Country. They belong to 13 serotypes of Salmonella enterica. Serotype Strain number Serotype Strain number Typhimurium LT2 Anatum 331 Typhimurium 75 Anatum 342 Typhimurium 4,5,12:i:- 2B- California 327 Enteritidis 26 California 333 Enteritidis 28 Derby 343 Miami 261 Derby 345 Miami 8 Lexington 367 Arizonae 83 Lexington 378 Arizonae 20 Llandoff 334 Hadar 272 Llandoff 335 Hadar 275 Kentucky 531 Montevideo 328 Kentucky 534 Montevideo 454 Agona 263     Agona 337 DNA extraction. Bacterial colonies were resuspended in saline solution until a 2 McFarland concentration and 1ml of that solution was used for the DNA extraction. The DNA extraction was performed according to the protocol supplied in the DNeasy Blood and Tissue kit (Qiagen). The bacterial DNA was recovered in 200 µl of elution buffer supplied with the DNeasy Blood and Tissue kit. To this protocol it was added a DNA concentration phase with Sodium Acetate 3M. To determine the amount of DNA, 1 µl of each sample was measure with the NanoDrop ND-1000 Spectrophotometer. Digestion.1 µg of genomic DNA was digested with 10U of Hae III in 10X M Buffer (final volume, 10 µl) for 2h at 37ºC. Selection of Oligonucleotides for PCR amplification. The procedure is described bellow. PCR Amplification. Each 25 µl PCR mixture included 1 µl of template DNA (digested genomic DNA), 1U of Taq DNA polymerase (BIOLINE), deoxynucleoside triphosphates (200 µM each) (BIOLINE), 2.5 mM MgCl2 and the three oligonucleotide primers (1 µM) in 1X PCR buffer. Amplification was performed in a RoboCycler 40 (Stratagene) with an amplification profile that consisted of an initial denaturation step at 95ºC for 2 min and then 30 cycles with denaturation at 95ºC for 1 min, primer annealing at 32ºC for 30 s, and extension at 72ºC for 1 min. To ensure complete strand extension, the reaction mixture was kept at 72ºC for 4 min after the final cycle. All the experiments included negative controls which were processed with the samples. The amplified products were resolved on 2% agarose gels and the bands stained by ethidium bromide staining, visualized in a transilluminator. The banding patterns were compared visually. Reproducibility of the technique. DNA extractions were performed from each strain in two separated days. Each DNA sample was used for restriction with HaeIII and subsequence PCR amplification and separation in agarose gel in two different days. In total, four gel images were obtained to search for reproducibility.  The images are shown bellow:  Selection of primers Software. The programs used for selection of oligonucleotides were generated by our group. PHP scripting language was used to develop them, and they were run in an Apache/PHP environment Selection of oligonucleotides. The procedure for selection of 9 nucleotides long primers was performed in 5 steps: Selection of 9 bases long primers with Tm over 30 C and no hairpin or other unwished structure formation. A total of 37,913 oligonucleotides were selected. Selection of primers matching the genome of Salmonella Typhimurium LT2 at least 200 times after its theoretical restriction digest with HaeIII (GG'CC). Search for trios of primers from the selected ones in the previous step. Only trios yielding at least 8 theoretical bands by PCR for Salmonella Typhimurium LT2 cleaved with HaeIII were selected. The number of trios fulfilling the minimal bands requirement was 315, from which 81 had an A+T content equal to 2, and in 234 was equal to 3.  Search for PCR amplification with trios selected in previous step: Salmonella enterica subsp. enterica serovar Typhi (NC_003198), Salmonella typhi Ty2 (NC_004631), and Salmonella enterica subsp. enterica serovar Paratyphi A str. ATCC 9150 (NC_006511). Selection of trios of primers for wet experiments. The selection was visual, and no specific selection procedure was applied. PCR amplification with the proposed technique was applied to 27 strains of Salmonella. As wet lab results were excelent, the procedure was applied to all sequenced prokaryiotes. For some species the technique is not applicable (there are no oligonucleotide trios available), but this genotyping technique may be applied to most pathogens.

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