Method for absolute quantification of microbial communities by using both microarrays and competitive PCR

Abstract

Methods for the robust quantification of bacterial communities are still under development. In this context, the present study aimed to evaluate a method combining competitive PCR (cPCR) and microarray assays for the determination of absolute content of total bacteria and individual bacterial species in samples. For this, a competitor DNA for cPCR and microarrays containing three types of DNA probes was prepared. A calibration curve was generated with genomic DNA samples as standards, which was then utilized for cPCR-based determination of the total number (in moles) of 16S rRNA genes in other bacterial samples. Moreover, scatter plots of species-specific probes versus total bacteria probe for each genomic DNA of known concentration was fit to the regression model, and the obtained slope value was defined as the hybridization affinity ratio.The cPCR assay was performed for both a commercially available mixed genomic DNA sample and human oral bacterial DNA samples, and the total number of moles of 16S rRNA genes was determined. These values were distributed among each species on the basis of the signal intensities of species-specific probes and the hybridization affinity ratio. The total number of bacterial genomes and those of individual species were determined by dividing the copy number of 16S rRNA genes per genome. The obtained results were confirmed by quantitative real-time PCR (qPCR). For values of >1 × 102 copies determined by qPCR, the ratio of the values measured by DNA chips to by qPCR was 1.53-fold on average and <2.6-fold for all data. These results show that the combined method of cPCR and microarray is useful to quantify the absolute numbers of several types of bacteria in a sample at one time.