Optimization of Random Amplified Polymorphic DNA-PCR for Genotyping Salmonella enterica subspecies enterica serovar Typhi Using a Mathematical Approach

摘要

Aim: A mathematical approach was employed to optimize and observe the interactive effects of Random Amplified Polymorphic DNA-PCR (RAPD-PCR) master mix with a view to resolving its limited reproducibility for reliable diagnostic and biomarker discovery for Salmonella Typhi. Methodology: A gradient PCR for RAPD primer annealing temperature was performed, and a rotatable centred central composite design (RCCCD) using Design Expert? software was used to generate 82 experiments with six replicated centre points. Master mix components optimized include concentrations of DNA template, PCR buffer, MgCl₂, RAPD primer, dNTPs and Taq DNA polymerase. Results: The result of this study showed that significant interactions that yielded higher numbers of amplified DNA bands existed between PCR buffer and MgCl₂; dNTPs and MgCl₂; and RAPD primer and MgCl₂. Although not statistically significant, good interactive relationships that recorded higher numbers of bands were recorded between Taq with MgCl₂, and between RAPD primers and dNTPs. Reproducible RAPD-PCR results were obtained following the optimization with a co-efficient of variation (CV) value of 2.19%. Conclusion: It was concluded that stringent and interactive master mix optimization is necessary if the simplicity and cost effectiveness of RAPD-PCR is to be utilized, and RSM offers a rapid and cost effective solution to this potentially tedious task.