Orthogonal array design in optimizing ERIC-PCR system for fingerprinting rat's intestinal microflora.
OBJECTIVE
The aim of the present study was to rapidly optimize enterobacterial repetitive intergenic consensus (ERIC)-PCR amplification systems for fingerprinting rat's intestinal microflora.


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.


CONCLUSIONS
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.
Peng
Y
Y
School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China.
Jin
J
J
Wu
C
C
Yang
J
J
Li
X
X
D000818:Animals; D015373:Bacterial Typing Techniques; D016384:Consensus Sequence; D016172:DNA Fingerprinting / Q000379:methods*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis*; D004755:Enterobacteriaceae / Q000235:genetics*; D020869:Gene Expression Profiling; D007422:Intestines / Q000382:microbiology*; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction / Q000379:methods; D051381:Rats; D017208:Rats, Wistar
D017931:DNA Primers; D004269:DNA, Bacterial