Partial inhibition of amplifications by primers of EHEC genes.
Diagnostic value of multiplex polymerase chain reaction (PCR) was examined by using three primer pairs, specific for the common conserved region of stx1 and stx2, eae and an enterohaemolysin A gene (ehxA). The sensitivity in respect of each amplicon decreased with three exponents comparing to the individual PCR reactions. These PCR reactions were partially inhibited by the presence of certain additional primers. This inhibitory effect was template-concentration dependent, and was partially balanced by usage of increased amount of dNTP. Taq DNA polymerase in a range of 0.3-1.25 U/reaction did not influence the inhibition. The same inhibition was detected if the annealing temperature was changed from 48 degrees C to 57 degrees C. Pairs of EHEC primers inhibited a Salmonella enteritidis virulence-plasmid specific gene amplification, as well. Theoretical inhibiting effects were predicted by Primer Premier software but our observations can be sufficiently explained neither by the competitions between the specific and aspecific amplifications nor by the inhibition caused by dimerization of primers.
National Center for Epidemiology B. Johan, Budapest, Hungary.
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Gadó
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Ruzsics
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Tóth
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Király
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Milch
D000818:Animals; D003092:Colitis / Q000382:microbiology; D017931:DNA Primers; D003967:Diarrhea / Q000382:microbiology; D004927:Escherichia coli Infections / Q000175:diagnosis / Q000382:microbiology; D019453:Escherichia coli O157 / Q000235:genetics* / Q000302:isolation & purification*; D005069:Evaluation Studies as Topic; D005243:Feces / Q000382:microbiology; D005784:Gene Amplification; D006801:Humans; D008892:Milk / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012480:Salmonella Infections / Q000175:diagnosis / Q000382:microbiology; D012481:Salmonella Infections, Animal / Q000175:diagnosis / Q000382:microbiology; D012477:Salmonella enteritidis / Q000235:genetics* / Q000302:isolation & purification*; D012680:Sensitivity and Specificity
D017931:DNA Primers