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

Abstract

Two pairs of species-specific PCR primers targeting the housekeeping groEL gene, Spa146f-Spa525r and Spa93f-Spa525r, were designed to quantify human oral and fecal Streptococcus parasanguinis. 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 S. parasanguinis, and the in silico Simulated PCR algorithm showed both primer pairs hit only S. parasanguinis groEL 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 S. parasanguinis, which further validates the specificity of two primer pairs, and that individual subjects harbored multiple genotypes of S. parasanguinis in saliva. By spiking S. parasanguinis into human fecal samples, we found the quantification limit of quantitative real-time PCR (qPCR) assays for both primer pairs was 5-6 log 10 groEL copies/g feces. Human fecal S. parasanguinis 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 S. parasanguinis than healthy people. In both feces and saliva, the S. parasanguinis abundances quantified with two primer pairs exhibited strong and significant correlation. Our results show that the two S. parasanguinis-specific primer pairs can be used to quantify and profile human saliva and fecal S. parasanguinis.

Resources to Facilitate Use of the Altered Schaedler Flora (ASF) Mouse Model to Study Microbiome Function

“…For this, primer pairs were selected for the groEL ( cpn ) gene specific to each of the ASF ( Table 2 ). groEL is a highly conserved gene found throughout Eubacteria , and sequence differences have been exploited for taxon-specific PCR primers ( 69 – 71 ). In contrast to 16S rRNA genes, targeting groEL simplifies the accurate determination of bacterial abundance as the gene is present in a single copy in the ASF members.…”

Blood Meals With Active and Heat-Inactivated Serum Modifies the Gene Expression and Microbiome of Aedes albopictus

“…The search revealed alterations in abundancy of Wolbachia reads abundance ( Figure 7A ). To validate the observed results, a PCR-based assay was performed to measure transcripts levels of the Wolbachia groEL gene, a single copy gene constitutively expressed ( Chen et al, 2019 ). The relative levels of Wolbachia transcripts were normalized with respect to the Ae.…”

Trial Proteomic Qualitative and Quantitative Analysis of the Protein Matrix of Submandibular Sialoliths

“…The following and most numerous groups of bacteria present in sialoliths are Gram-positive and nonmotile streptococci. Based on the division into types, we identified the pyogenic type (S. porcinus [37] and S. pseudoporcinus [38]), the mitis type (S. pneumoniae, S. mitis, S. oralis and S. parasanguinis [39]) [40], the sanguinis type (S. sanguinis [41], S. gordoni [42], S. cristatus [43]), and the sinensis type (S. sinensis [44]). S. porcinus and S. pseudoporcinus are rarely identified in humans, and if anything, their presence is associated with the occupation of the rectum and vagina in women of reproductive age.…”