Assay for estimating total bacterial load: relative qPCR normalisation of bacterial load with associated clinical implications

Abstract

Relative microorganism abundance is a parameter describing biodiversity, referring to how common a bacterial species is within the total bacterial flora. Anal, rectal, skin, mucal, and respiratory swabs are typical clinical samples where knowledge of relative bacterial abundance might make distinction between asymptomatic carriers and symptomatic cases. Assays trying to measure total bacterial load are usually based on the amplification of universal segments of 16S rRNA genes. Previous assays were not adoptable to “direct” PCR protocols, and/or they were not compatible with hydrolysis-based detection. Using the latest summary of universal 16S sequence motifs present in literature and testing our design with 500 liquid and 50 formed stool samples, we illustrate the performance characteristics of a new 16S quantitative PCR (qPCR) assay, which addresses well-known technical problems, including a) positive priming reaction in the absence of intended target due to self-priming and/or mispriming of unintended targets; b) amplification bias due to nonoptimal primer/probe coverage; and c) too large amplicons for clinical qPCR. Stool swabs ranked into bins of different bacterial loads show significant correlation with threshold cycle values of our new assay. To the best of our knowledge, this is the first description of qPCR assay measuring individual differences of total bacterial load present in human stool.