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.
The transient thermal fracture problem of a crack (perpendicular to the gradient direction) in a graded orthotropic strip is investigated. Most of the materials properties are assumed to vary as an exponential function of thickness direction. The transient two-dimensional temperature problem is analyzed by the methods of Laplace and Fourier transformations. A system of singular integral equations are obtained and solved numerically. Numerical results are figured out to show the variation of the temperature on the crack faces and extended line and stress intensity factors for different material parameters with dimensionless time.
Approximately 94% of the total 16S rDNA of Pseudomonas andropogonis strain ACH 01053A was sequenced and compared with that of strain ATCC 23061 obtained from the GenBank database. The two sequences were highly homologous with 1.3% difference. Alignment of sequences with those from closely related bacteria revealed two possible regions for design of a specific primer suitable for detection of Ps. andropogonis. Using primers designed to these variable regions in a PCR test, an amplification product of approximately 410 bp was specifically produced by 40 strains of Ps. andropogonis. No other bacterial species showed an amplification product under optimized PCR conditions. As few as 1000 cells per reaction were detected.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.