Orthogonal array design in optimizing ERIC-PCR system for fingerprinting rat’s intestinal microflora

Abstract

Aims: The aim of the present study was to rapidly optimize enterobacterial repetitive intergenic consensus (ERIC)‐PCR amplification systems for fingerprinting rat’s intestinal microflora. Methods and Results: Orthogonal array design and statistic analysis methods were attempted to rapidly optimize ERIC‐PCR reaction system for fingerprinting intestinal microflora. The results showed that variations of the four factors (Mg2+, dNTP, primer and HotstarTaq polymerase concentrations) changed the fingerprinting patterns significantly. The order of effects of those factors on fingerprinting patterns was primers (F = 274·000, P = 0·000), Hotstar Taq polymerase (F = 197·000, P = 0·001), Mg2+ (F = 181·000, P = 0·001) and dNTP (F = 27·000, P = 0·011). The optimal ERIC‐PCR condition was containing 200 μmol l−1 dNTP, 2·5 mmol l−1 Mg2+, 0·4 μmol l−1 primer, 1 U HotstarTaq DNA polymerase namely 25 μl reaction system, which is proved to be a simple, fast and reliable method suitable for fingerprinting rat’s intestinal microflora. Conclusions: The results suggest that Mg2+, dNTP, primer and HotstarTaq polymerase concentrations play important roles on ERIC‐PCR fingerprinting patterns. Orthogonal array design is a considerable method to optimize ERIC‐PCR reaction system for its rapidness, simplicity, potential to investigate mutual effects of parameters. Significance and Impact of the Study: It is the first report on optimization of ERIC‐PCR amplification systems for fingerprinting intestinal microflora using orthogonal array design or statistic analysis methods and systematically observing the effects of variables of reaction conditions.

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