Simultaneous detection of pathogens in clinical samples from patients with community-acquired pneumonia by real-time PCR with pathogen-specific molecular beacon probes
Simultaneous detection of pathogens in clinical samples from patients with community-acquired pneumonia by real-time PCR with pathogen-specific molecular beacon probes
复制标题
DOI：
10.1128/jcm.44.4.1440-1446.2006
复制
发表时间：
2006-04-01
影响因子：
9.4
通讯作者：
Ubukata, K
中科院分区：
文献类型：
--
作者：
Morozumi, M;Nakayama, E;Ubukata, K
本研究采用病原体特异性分子信标（MB）和引物实时荧光定量PCR技术预测社区获得性肺炎（CAP）病原，同时检测出肺炎链球菌、流感嗜血杆菌、肺炎支原体、肺炎衣原体、嗜肺军团菌和化脓性链球菌6种主要的CAP病原。对来自婴儿和儿童（n = 389）和成人（n = 40）的新鲜临床标本进行PCR检测。MB探针和引物都是病原体特异性的，即肺炎链球菌的lytA基因，嗜肺链球菌的inip基因，其余四种生物的16S rRNA基因。使用市售EXTRAGEN II试剂盒提取临床标本的DNA，使用Stratagene Mx3000P进行扩增。这些病原体的检出限从2拷贝到18拷贝不等。从DNA提取到分析的整个过程在2 h内完成。与常规培养相比，本实时PCR研究的灵敏度和特异性分别为：肺炎链球菌96.2%和93.2%，流感嗜血杆菌95.8%和95.4%，化脓性链球菌100%和100%，肺炎支原体100%和95.4%。与血清学检测相比，肺炎支原体的敏感性和特异性分别为90.2%和97.9%。在6例肺炎原体临床样本中，实时荧光定量PCR结果均为阳性预测值，并且在这些病例中，还观察到滴度值升高。总之，我们证明了病原体特异性MB的实时PCR检测有助于快速识别CAP病原体，并及时检查经验性化疗的临床过程，支持传统的培养方法。
In this study, real-time PCR with pathogen-specific molecular beacons (MB) and primers was evaluated for prediction of community-acquired pneumonia (CAP) causative agents, detecting six main CAP agents, Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, and Streptococcus pyogenes, simultaneously. The PCR assay was evaluated for fresh clinical specimens from infants and children (n = 389) and from adults (it = 40). The MB probes and primers are both pathogen specific, namely, the lytA gene for S. pneumoniae, the inip gene for L. pneumophila, and 16S rRNA genes for the remaining four organisms. DNA extraction of clinical specimens was performed with a commercially available EXTRAGEN II kit, and amplification was performed with Stratagene Mx3000P. The limit of detection for these pathogens ranged from 2 copies to 18 copies. The whole process from DNA extraction to the analysis was finished in less than 2 h. The obtained sensitivity and specificity of this real-time PCR study relative to those of conventional cultures were as follows: 96.2% and 93.2% for S. pneumoniae, 95.8% and 95.4% for H. influenzae, 100% and 100% for S. pyogenes, and 100% and 95.4% for M. pneumoniae, respectively. The sensitivity and specificity for M. pneumoniae relative to those of a serologic assay were 90.2% and 97.9%, respectively. In six clinical samples of C. pneumoniae, the real-time PCR gave positive predictable values, and in those cases, elevation of the titer value was also observed. In conclusion, we demonstrated that a real-time PCR assay with pathogen-specific MB is useful in identifying CAP causative agents rapidly and in examining the clinical course of empirical chemotherapy in a timely manner, supporting conventional culture methods.