Development of triplex SYBR green real-time PCR for detecting Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella spp. without extraction of DNA

Anusak Kerdsin, Ryuichi Uchida, Chris Verathamjamrus, Parichart Puangpatra, Kazuyoshi Kawakami, Pollert Puntanakul, Sorasak Lochindarat, Thanyanut Bunnag, Pathom Sawanpanyalert, Surang Dejsirilert, Kazunori Oishi

研究成果: Article査読

8 被引用数 (Scopus)

抄録

Although Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella spp. are prevalent causes of community-acquired pneumonia, rapid and sensitive diagnosis is difficult. Realtime 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® 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.

本文言語English
ページ(範囲)173-180
ページ数8
ジャーナルJapanese journal of infectious diseases
63
3
出版ステータスPublished - 2010
外部発表はい

ASJC Scopus subject areas

  • Microbiology (medical)
  • Infectious Diseases

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