I am working on ERIC-PCR for clinical isolates of Pseudomonas aeruginosa. I am using the following primers:

ERIC1 (5’-ATGTAAGCTCCTGGGGATTCAC-3’) and

ERIC2 (5’-AAGTAAGTACTGGGGTGAGCG-3’)

at the following conditions: denaturation at 94°C for 7 min followed by 35 cycles of: denaturation at 94°C for 1 min, annealing at 52°C for 1 min, extension at 72°C for 2min, and a final extension step at 72°C for 5 min. I tried using a primer concentration of 10μM as cited in (Khosravi et al., 2016 ), and the maximum number of bands I got was 7 with some isolates showing only 1 band and some not showing any bands at all, and I always got a huge primer dimer. So I went to change the primer concentration to 2.5 pM as cited by (Savari et al., 2016) and I did not get any bands at all. If anyone had worked on ERIC-PCR for Pseudomonas aeruginosa what is the optimal primer concentration that should be used? and what kind of errors could be leading to these results? Also how can I compare my results to other people’s results? For example: I am using P. aeruginosa ATCC 27853 strain as a control but I can’t find any recourse mentioning the pattern of bands that it should give so that I can compare my results to it. Is there a problem in the reproducibility of this method of genotyping?

References:

Khosravi, A. D., Hoveizavi, H., Mohammadian, A., Farahani, A., & Jenabi, A. (2016). Genotyping of multidrug-resistant strains of Pseudomonas aeruginosa isolated from burn and wound infections by ERIC-PCR. Acta Cirurgica Brasileira, 31(3), 206-211. doi:10.1590/s0102-865020160030000009

Savari, M., Rostami, S., Ekrami, A., & Bahador, A. (2016). Characterization of Toxin-Antitoxin (TA) Systems in Pseudomonas aeruginosa Clinical Isolates in Iran. Jundishapur Journal of Microbiology, 9(1). doi:10.5812/jjm.26627