Development of triplex SYBR green real-time PCR for detecting Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella spp. without extraction of DNA.
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.
Kerdsin
Anusak
A
National Institute of Health, Department of Medical Sciences, Ministry of Public Health, Nonthaburi, Thailand.
Uchida
Ryuichi
R
Verathamjamrus
Chris
C
Puangpatra
Parichart
P
Kawakami
Kazuyoshi
K
Puntanakul
Pollert
P
Lochindarat
Sorasak
S
Bunnag
Thanyanut
T
Sawanpanyalert
Pathom
P
Dejsirilert
Surang
S
Oishi
Kazunori
K
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
D052160:Benzothiazoles; D004269:DNA, Bacterial; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I