Development of a multiplex real-time PCR assay for the simultaneous detection of four bacterial pathogens causing pneumonia

被引:17
作者
Lim, Ho Jae [1 ,2 ]
Kang, Eun-Rim [1 ]
Park, Min Young [1 ]
Kim, Bo Kyung [3 ]
Kim, Min Jin [3 ]
Jung, Sunkyung [1 ]
Roh, Kyoung Ho [4 ]
Sung, Nackmoon [5 ]
Yang, Jae-Hyun [6 ]
Lee, Min-Woo [7 ,8 ]
Lee, Sun-Hwa [1 ,3 ]
Yang, Yong-Jin [1 ]
机构
[1] Seegene Med Fdn, Dept Mol Diagnost, Seoul, South Korea
[2] Chosun Univ, Dept Integrat Biol Sci, Gwangju, South Korea
[3] Seegene Med Fdn, Dept Lab Med, Seoul, South Korea
[4] Ilsan Hosp, Dept Lab Med, Natl Hlth Insurance Serv, Goyang, Gyeonggi, South Korea
[5] Seegene Med Fdn, Clin Res Inst, Seoul, South Korea
[6] Harvard Med Sch, Dept Genet, Blavatnik Inst, Paul F Glenn Ctr Biol Aging Res, Boston, MA USA
[7] Soonchunhyang Univ, Soonchunhyang Inst Medi Bio Sci SIMS, Cheonan Si, South Korea
[8] Soonchunhyang Univ, Dept Integrated Biomed Sci, Cheonan Si, South Korea
关键词
COMMUNITY-ACQUIRED PNEUMONIA; RAPID DETECTION; RESISTANCE; SPUTUM; GENES;
D O I
10.1371/journal.pone.0253402
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
Classification of clinical symptoms and diagnostic microbiology are essential to effectively employ antimicrobial therapy for lower respiratory tract infections (LRTIs) in a timely manner. Empirical antibiotic treatment without microbial identification hinders the selective use of narrow-spectrum antibiotics and effective patient treatment. Thus, the development of rapid and accurate diagnostic procedures that can be readily adopted by the clinic is necessary to minimize non-essential or excessive use of antibiotics and accelerate patient recovery from LRTI-induced damage. We developed and validated a multiplex real-time polymerase chain reaction (mRT-PCR) assay with good analytical performance and high specificity to simultaneously detect four bacterial pathogens causing pneumonia: Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and Moraxella catarrhalis. The analytical performance of mRT-PCR against target pathogens was evaluated by the limit of detection (LOD), specificity, and repeatability. Two hundred and ten clinical specimens from pneumonia patients were processed using an automatic nucleic acid extraction system for the "respiratory bacteria four" (RB4) mRT-PCR assay, and the results were directly compared to references from bacterial culture and/or Sanger sequencing. The RB4 mRT-PCR assay detected all target pathogens from sputum specimens with a coefficient of variation ranging from 0.29 to 1.71 and conservative LOD of DNA corresponding to 5 x 10(2) copies/reaction. The concordance of the assay with reference-positive specimens was 100%, and additional bacterial infections were detected from reference-negative specimens. Overall, the RB4 mRT-PCR assay showed a more rapid turnaround time and higher performance that those of reference assays. The RB4 mRT-PCR assay is a high-throughput and reliable tool that assists decision-making assessment and outperforms other standard methods. This tool supports patient management by considerably reducing the inappropriate use of antibiotics.
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页数:12
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