title: Development of triplex SYBR green real-time PCR for detecting Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella spp. without extraction of DNA.
abstract: Although Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella spp. are prevalent causes of community-acquired pneumonia, rapid and sensitive diagnosis is difficult. Real-time PCR provides rapid and sensitive diagnosis, however, DNA extraction is still required, which is time-consuming, costly and includes a risk of contamination. Therefore, we aimed to develop triplex real-time PCR without DNA extraction. AmpDirect(R) Plus which inhibits PCR inhibitors was used as the PCR buffer. Melting temperatures of the PCR products for the three bacteria were analyzed by SYBR green triplex real-time PCR and were found to be significantly different. Detection limits of bacteria cells diluted in nasopharyngeal aspirates (NPAs) were comparable with the detection limits of previously reported real-time PCR. Our PCR without DNA extraction and probe real-time PCR with DNA extraction showed identical results for the detection of the three bacteria from 38 respiratory specimens (sputum, endotracheal aspirates, and NPAs) collected from patients with pneumonia. No cross-reaction with other bacteria was observed. Our triplex real-time PCR successfully detected and differentiated the three bacteria. Although further field tests are required, our assay is a promising method for the rapid and cost-effective detection of the three bacteria.
authors.affiliation: National Institute of Health, Department of Medical Sciences, Ministry of Public Health, Nonthaburi, Thailand.
authors.forename: Anusak
authors.initials: A
authors.lastname: Kerdsin
authors.forename: Ryuichi
authors.initials: R
authors.lastname: Uchida
authors.forename: Chris
authors.initials: C
authors.lastname: Verathamjamrus
authors.forename: Parichart
authors.initials: P
authors.lastname: Puangpatra
authors.forename: Kazuyoshi
authors.initials: K
authors.lastname: Kawakami
authors.forename: Pollert
authors.initials: P
authors.lastname: Puntanakul
authors.forename: Sorasak
authors.initials: S
authors.lastname: Lochindarat
authors.forename: Thanyanut
authors.initials: T
authors.lastname: Bunnag
authors.forename: Pathom
authors.initials: P
authors.lastname: Sawanpanyalert
authors.forename: Surang
authors.initials: S
authors.lastname: Dejsirilert
authors.forename: Kazunori
authors.initials: K
authors.lastname: Oishi
mesh_terms: D000704:Analysis of Variance; D015373:Bacterial Typing Techniques / Q000379:methods; D052160:Benzothiazoles; D016993:Chlamydophila pneumoniae / Q000235:genetics*; D017714:Community-Acquired Infections / Q000382:microbiology; D004269:DNA, Bacterial / Q000032:analysis; D003959:Diamines; D004587:Electrophoresis, Agar Gel; D006801:Humans; D007875:Legionella / Q000235:genetics*; D009930:Organic Chemicals / Q000737:chemistry*; D018410:Pneumonia, Bacterial / Q000382:microbiology; D011019:Pneumonia, Mycoplasma / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D013045:Species Specificity; D044366:Transition Temperature
chemical_list: D052160:Benzothiazoles; D004269:DNA, Bacterial; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I