Development and evaluation of a novel quenching probe PCR (GENECUBE) assay for rapidly detecting and distinguishing between Chlamydia pneumoniae and Chlamydia psittaci

• Published in: Journal of microbiological methods 2021-05, Vol.184, p.106212-106212, Article 106212
• Main Authors: Hisada, Kyoko, Hida, Yukio, Kawabata, Naoki, Kawashima, Yosuke, Soya, Yoshihiro, Shimada, Akihiro, Iwano, Masayuki, Kimura, Hideki
• Format: Article
• Language: English
• Subjects: Atypical pneumonia; Chlamydia pneumoniae; Chlamydia psittaci; GENECUBE; Quenching probe; Rapid PCR assay
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2021.106212

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. •A newly developed PCR assay rapidly distinguished between C. pneumonia and C psittaci.•The assay with GENECUBE is a simple method well adapted for clinical routine use.•The assay made the full detection time of the two organisms less than 50 min.•The kappa index for agreement between the assay and the culture method was 0.43.•The assay may be useful for excluding atypical pneumonia by C. pneumonia and C. psittaci.