Dataset Information

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 log10 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.

SUBMITTER: Chen Q

PROVIDER: S-EPMC6933288 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Publications

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

Chen Qiurong Q Wu Guojun G Chen Hui H Li Hui H Li Shuo S Zhang Chenhong C Pang Xiaoyan X Wang Linghua L Zhao Liping L Shen Jian J

Frontiers in microbiology 20191220

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 on ...[more]

PMID: 31921079

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Project description:Clostridium perfringens is a widespread pathogen, but the precise quantification of this subdominant gut microbe remains difficult due to its low fecal count (particularly in asymptomatic subjects) and also due to the presence of abundant polymerase-inhibitory substances in human feces. Also, information on the intestinal carriage of toxigenic C. perfringens strains in healthy subjects is sparse. Therefore, we developed a sensitive quantitative real-time PCR assays for quantification of C. perfringens in human feces by targeting its ?-toxin and enterotoxin genes. To validate the assays, we finally observed the occurrence of ?-toxigenic and enterotoxigenic C. perfringens in the fecal microbiota of healthy Japanese infants and young adults.The plc-specific qPCR assay was newly validated, while primers for 16S rRNA and cpe genes were retrieved from literature. The assays were validated for specificity and sensitivity in pre-inoculated fecal samples, and were finally applied to quantify C. perfringens in stool samples from apparently healthy infants (n 124) and young adults (n 221).The qPCR assays were highly specific and sensitive, with a minimum detection limit of 10(3) bacterial cells/g feces. Alpha-toxigenic C. perfringens was detected in 36% infants and 33% adults, with counts ranging widely (10(3)-10(7) bacterial cells/g). Intriguingly, the mean count of ?-toxigenic C. perfringens was significantly higher in infants (6.0±1.5 log10 bacterial cells/g), as compared to that in adults (4.8±1.2). Moreover, the prevalence of enterotoxigenic C. perfringens was also found to be significantly higher in infants, as compared to that in adults. The mean enterotoxigenic C. perfringens count was 5.9±1.9 and 4.8±0.8 log10 bacterial cells/g in infants and adults, respectively.These data indicate that some healthy infants and young adults carry ?-toxigenic and enterotoxigenic C. perfringens at significant levels, and may be predisposed to related diseases. Thus, high fecal carriage of toxigenic C. perfringens in healthy children warrants further investigation on its potential sources and clinical significance in these subjects. In summary, we present a novel qPCR assay for sensitive and accurate quantification of ?-toxigenic and enterotoxigenic C. perfringens in human feces, which should facilitate prospective studies of the gut microbiota.

Dataset Information

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

Publications

Quantification of Human Oral and Fecal <i>Streptococcus parasanguinis</i> by Use of Quantitative Real-Time PCR Targeting the <i>groEL</i> Gene.

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