Development and evaluation of a novel quenching probe PCR (GENECUBE) assay for rapidly detecting and distinguishing between Chlamydia pneumoniae and Chlamydia psittaci
Development and evaluation of a novel quenching probe PCR (GENECUBE) assay for rapidly detecting and distinguishing between Chlamydia pneumoniae and Chlamydia psittaci
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DOI：
10.1016/j.mimet.2021.106212
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发表时间：
2021-04-01
影响因子：
2.2
通讯作者：
Kimura, Hideki
中科院分区：
文献类型：
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作者：
Hisada, Kyoko;Hida, Yukio;Kimura, Hideki
及早发现衣原体科病原体，对全球迅速有效地控制非典型肺炎至关重要。GENECUBE（TOYOBO）是一种新型的全自动基因分析仪，能够在50 min内扩增和检测目标DNA。在这项研究中，我们开发了一种新的PCR检测与特异性淬灭探针（PCR-QC assay），用于快速区分肺炎衣原体（CPN）和鹦鹉热衣原体（CPS）。PCR-QC分析使我们能够通过确定独特的解链温度，精确地同时检测2种不同类型的DNA片段，即使在混合样品中。接下来，我们使用PCR-QC分析和细胞培养方法作为金标准，检查了总计300份来自呼吸道感染患者的冷冻样本。PCR-QC检测和培养方法之间一致性的Kappa指数为0.43（95%置信区间（CI）：0.08？0.78）。PCR-QC检测的敏感性和特异性分别为36.3%（4/ 11; 95%CI：10.9？69.2%）和99.0%（286/289; 95% CI：97.0？99.8%）。还通过常规PCR TaqMan试验检查了任一方法检测的CPN（n = 13）或CPS（n = 1）阳性样本，其结果与PCR-QC试验结果相同。此外，与传统的PCR TaqMan检测相比，使用GENECUBE的PCR-QC检测缩短了CPN或CPS的完整检测时间（50分钟内vs.超过2至3小时）。因此，新的PCR-QC检测系统配备GENECUBE是有用的快速检测CPN或CPS病原体在临床实验室，并可能提高非典型肺炎的管理。
Early detection of the family Chlamydiaceae as pathogens is essential worldwide for the rapid and sufficient management of atypical pneumonia. GENECUBE (TOYOBO) is a novel fully automated gene analyzer capable of amplifying and detecting target DNAs within 50 min. In this study, we developed a new PCR assay with a specific quenching probe (PCR-QC assay) for rapidly distinguishing between Chlamydia pneumoniae (CPN) and Chlamydia psittaci (CPS). The PCR-QC assay enabled us to precisely and simultaneously detect the 2 different types of DNA fragments even in a mixed sample by identifying unique melting temperatures. Next, we examined a total of 300 frozen samples from patients with respiratory tract infection using the PCR-QC assay and the cell culture method as the gold standard. Kappa index for agreement between the PCR-QC assay and the culture method was 0.43 (95% confidential interval (CI): 0.08?0.78). The sensitivity and specificity of the PCR-QC assay were 36.3% (4/ 11; 95% CI: 10.9?69.2%)) and 99.0% (286/289; 95% CI: 97.0?99.8%), respectively. The samples positive for CPN (n = 13) or CPS (n = 1) by either method were also examined by a conventional PCR TaqMan assay, which produced the same results as those from the PCR-QC assay. Furthermore, the PCR-QC assay using GENECUBE shortened the full detection time for CPN or CPS (within 50 min vs. more than 2 to 3 h) compared with conventional PCR TaqMan assays. Therefore, the new PCR-QC assay system equipped with GENECUBE is useful for rapidly detecting CPN or CPS pathogens in clinical laboratory, and may improve the management of atypical pneumonia.