Quantification of Human Oral and Fecal Streptococcus parasanguinis by Use of Quantitative Real-Time PCR Targeting the groEL Gene
Quantification of Human Oral and Fecal Streptococcus parasanguinis by Use of Quantitative Real-Time PCR Targeting the groEL Gene
复制标题
使用针对 groEL 基因的定量实时 PCR 对人类口腔和粪便副血链球菌进行定量
DOI：
10.3389/fmicb.2019.02910
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发表时间：
2019-12-20
影响因子：
5.2
通讯作者：
Shen, Jian
中科院分区：
文献类型：
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作者：
Chen, Qiurong;Wu, Guojun;Shen, Jian
针对管家基因groEL设计了两对种特异性PCR引物Spa 146 f-Spa 525 r和Spa 93 f-Spa 525 r，用于人口腔和粪便中副血链球菌的定量检测。与NCBI核苷酸数据库和伴侣蛋白序列数据库中的参考序列进行比对分析，结果表明，正向引物Spa 146 f和Spa 93 f仅与S.模拟PCR算法显示两个引物对仅命中S. parasanguinis groEL基因在伴侣蛋白序列数据库中的序列。用这两对引物分别对6名受试者的唾液DNA进行PCR反应，并克隆每个PCR反应的扩增产物。系统发育分析表明，克隆序列均属于S。这进一步验证了两对引物的特异性，并且个体受试者具有多个S.唾液中的副血吸虫通过对S.在将副血吸虫的DNA引入人粪便样品中时，我们发现两种引物对的定量实时PCR（qPCR）测定的定量限为5-6 log（10）groEL拷贝/g粪便。人粪便沙门氏菌使用两个引物对中的每一个用qPCR定量的副血吸虫量与用宏基因组测序确定的量良好相关。qPCR结果显示牙周炎患者唾液中S含量明显低于对照组。parasanguinis比healthy健康people.在粪便和唾液中，S.用两个引物对定量的副血吸虫丰度表现出强而显著的相关性。结果表明，两个S.可使用副猪链球菌特异性引物对来定量和分析人唾液和粪便S。副血吸虫
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.