Orthogonal array design in optimizing ERIC-PCR system for fingerprinting rat's intestinal microflora
2007
Peng, Y. | Jin, J. | Wu, C. | Yang, J. | Li, Xunjing
The aim of the present study was to rapidly optimize enterobacterial repetitive intergenic consensus (ERIC)-PCR amplification systems for fingerprinting rat's intestinal microflora. 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. 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. 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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