Y Peng1, J Jin, C Wu, J Yang, X Li. 1. School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China.
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 (Mg(2+), 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), Mg(2+) (F = 181.000, P = 0.001) and dNTP (F = 27.000, P = 0.011). The optimal ERIC-PCR condition was containing 200 micromol l(-1) dNTP, 2.5 mmol l(-1) Mg(2+), 0.4 micromol l(-1) primer, 1 U HotstarTaq DNA polymerase namely 25 microl 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 Mg(2+), 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.
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 (Mg(2+), 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), Mg(2+) (F = 181.000, P = 0.001) and dNTP (F = 27.000, P = 0.011). The optimal ERIC-PCR condition was containing 200 micromol l(-1) dNTP, 2.5 mmol l(-1) Mg(2+), 0.4 micromol l(-1) primer, 1 U HotstarTaq DNA polymerase namely 25 microl 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 Mg(2+), 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.
Authors: M Chatterjee; X Ge; Y Kostov; P Luu; L Tolosa; H Woo; R Viscardi; S Falk; R Potts; G Rao Journal: Physiol Meas Date: 2015-04-02 Impact factor: 2.833
Authors: Xudong Ge; Prosper Adangwa; Ja Young Lim; Yordan Kostov; Leah Tolosa; Richard Pierson; Daniel Herr; Govind Rao Journal: Med Eng Phys Date: 2018-04-05 Impact factor: 2.242