Quantification of Human Oral and Fecal by Use of Quantitative Real-Time PCR Targeting the Gene.

Two pairs of species-specific PCR primers targeting the housekeeping gene, Spa146f-Spa525r and Spa93f-Spa525r, were designed to quantify human oral and fecal . Blast analysis against reference sequences of NCBI nucleotide collection database and the Chaperonin Sequence Database showed the forward primers Spa146f and Spa93f 100% matched only with , and the Simulated PCR algorithm showed both primer pairs hit only gene in Chaperonin Sequence Database. The two primer pairs were respectively used to perform PCR with saliva DNA of each of 6 human subjects, and the amplicons of individual PCR reactions were cloned. The phylogenetic analysis showed cloned sequences were all affiliated to , which further validates the specificity of two primer pairs, and that individual subjects harbored multiple genotypes of in saliva. By spiking into human fecal samples, we found the quantification limit of quantitative real-time PCR (qPCR) assays for both primer pairs was 5-6 log copies/g feces. Human fecal amounts quantified with qPCR using each of the two primer pairs correlated well with those determined with metagenomic sequencing. qPCR with either primer pair showed periodontitis patients had significantly lower level of saliva than healthy people. In both feces and saliva, the abundances quantified with two primer pairs exhibited strong and significant correlation. Our results show that the two -specific primer pairs can be used to quantify and profile human saliva and fecal .