[Optimization and primary application of one molecular typing method of Salmonella, enterobacteia repetitive intergenic consensus sequences-based PCR in Salmonella].
OBJECTIVE
To optimize the reaction conditions of enterobacteia repetitive intergenic consensus sequences-based PCR (ERIC-PCR), a molecular typing method of Salmonella, study the genomic DNA ERIC-PCR fingerprint maps of Salmonella standard strains and epidemic isolates, and supply data for a system of Salmonella epidemiology investigation and homology tracing.


METHODS
Genomic DNA of S. typhimurium was abstracted and used as the template for PCR. Enterobacteia repetitive intergenic consensus sequences were used as primers to amplify the target sequences in S. typhimurium genomic DNA: Amplification products were separated by agarose gel electrophoresis, and electrophoresis maps were analyzed by gel image analysis system. To optimize template concentration, Mg2+ concentration, primers concentration and annealing temperature of PCR, the factor to be optimized was designed in different concentration grads and other factors were fixed. Analyzed 16 Salmonella strains and one E. coli strain by PCR conditions optimized.


RESULTS
The electrophoresis bands of amplified products were entire and most clear when template concentration was 100 ng/25 microl, Mg2+ concentration was 2.0 mmol/L, primers concentrations were 0.4 micromol/L respectively in the total volume of 25 microl of the reaction system, and annealing temperature was 52 degrees C. The ERIC-PCR fingerprint maps of different Salmonella strains with different sources were different. From 250 to 5000 bp, there were 3 to 13 bands, and in those there was a specific 250 bp band.


CONCLUSIONS
Important reaction conditions of ERIC-PCR had been optimized in this study. ERIC-PCR technique could discriminate Salmonella strains isolated from different regions. It could be used in Salmonella epidemiology investigation and homology tracing, so as to make up for the flaws of the traditional classification methods for salmonella.
Wang
Chuan
C
Department of Medical Technology, West China School of Public Health, Sichuan University, Chengdu 610041, China. wangchuan1974@sina.com
Liu
Hengchuan
H
Jiang
Xian
X
Yu
Qian
Q
D015373:Bacterial Typing Techniques / Q000379:methods*; D016172:DNA Fingerprinting / Q000379:methods; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D012475:Salmonella / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*
D004269:DNA, Bacterial