Laboratory of Molecular Microbiology of Human Pathogens, Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa (ITQB-NOVA), Oeiras, Portugal.
Laboratory of Molecular Genetics, ITQB-NOVA, Oeiras, Portugal.
Sci Rep. 2019 Mar 1;9(1):3285. doi: 10.1038/s41598-019-39791-1.
Real-time PCR targeting lytA (the major autolysin gene) and piaB (permease gene of the pia ABC transporter) are currently used as the gold-standard culture-independent assays for Streptococcus pneumoniae identification. We evaluated the performance of a new real-time PCR assay - targeting SP2020 (putative transcriptional regulator gene) - and compared its performance with the assays previously described. A collection of 150 pneumococci, 433 non-pneumococci and 240 polymicrobial samples (obtained from nasopharynx, oropharynx, and saliva; 80 from each site) was tested. SP2020 and lytA-CDC assays had the best performance (sensitivity of 100% for each compared to 95.3% for piaB). The specificity for lytA and piaB was 99.5% and for SP2020 was 99.8%. Misidentifications occurred for the three genes: lytA, piaB and SP2020 were found in non-pneumococcal strains; piaB was absent in some pneumococci including a serotype 6B strain. Combining lytA and SP2020 assays resulted in no misidentifications. Most polymicrobial samples (88.8%) yielded concordant results for the three molecular targets. The remaining samples seemed to contain non-typeable pneumococci (0.8%), and non-pneumococci positive for lytA (1.7%) or SP2020 (8.7%). We propose that combined detection of both lytA-CDC and SP2020 is a powerful strategy for the identification of pneumococcus either in pure cultures or in polymicrobial samples.
实时 PCR 针对 lytA（主要自溶素基因）和 piaB（pia ABC 转运蛋白的渗透酶基因）目前被用作肺炎链球菌鉴定的无培养依赖性金标准检测方法。我们评估了一种新的实时 PCR 检测方法——针对 SP2020（假定的转录调节基因）——的性能，并将其与之前描述的检测方法进行了比较。我们对 150 株肺炎球菌、433 株非肺炎球菌和 240 株混合微生物样本（分别从鼻咽、口咽和唾液中获得，每个部位 80 个样本）进行了检测。SP2020 和 lytA-CDC 检测方法的性能最佳（与 piaB 的敏感性为 95.3%相比，每种方法的敏感性均为 100%）。lytA 和 piaB 的特异性为 99.5%，SP2020 的特异性为 99.8%。三种基因都存在误识别：非肺炎球菌菌株中存在 lytA 和 piaB 基因；某些肺炎球菌中不存在 piaB 基因，包括 6B 血清型菌株。lytA 和 SP2020 检测方法的组合未导致误识别。三种分子靶标对大多数混合微生物样本（88.8%）的检测结果一致。其余样本似乎含有无法分型的肺炎球菌（0.8%）和 lytA（1.7%）或 SP2020（8.7%）阳性的非肺炎球菌。我们建议，lytA-CDC 和 SP2020 的联合检测是一种强大的策略，无论是在纯培养物还是混合微生物样本中，都可以用于肺炎链球菌的鉴定。
