EurekaMag
+ Translate
+ Most Popular
The pigeon tick (Argas reflexus): its biology, ecology, and epidemiological aspects
Prevalence of hemoglobin abnormalities in Kebili (Tunisian South)
Lipogranuloma: a preventable complication of dacryocystorhinostomy
Value of basal plasma cortisol assays in the assessment of pituitary-adrenal insufficiency
Bees from the Belgian Congo. The acraensis group of Anthophora
Placing gingival retraction cord
Total serum IgE, allergy skin testing, and the radioallergosorbent test for the diagnosis of allergy in asthmatic children
Acariens plumicoles Analgesoidea parasites des oiseaux du Maroc
Injuries of terminal phalanges of the fingers in children
Biology of flowering and nectar production in pear (Pyrus communis)
Das Reliktvorkommen der Aspisviper (Vipera aspis L.) im Schwarzwald
Hydrological modelling of drained blanket peatland
Pathologic morphology and clinical significance of the anomalous origin of the left circumflex coronary artery from the right coronary artery. General review and autopsy analysis of 30 cases
Cyto genetic analyses of lymphocyte cultures after exposure to calcium cyclamate
Axelrodia riesei, a new characoid fish from Upper Rio Meta in Colombia With remarks concerning the genus Axelrodia and description of a similar, sympatric, Hyphessobrycon-species
Favorable evolution of a case of tuberculosis of pancreas under antibiotic action
RIFM fragrance ingredient safety assessment, Valencene, CAS Registry Number 4630-07-3
Parenteral microemulsions: an overview
Temperate pasture: management for grazing and conservation
Evaluation of a new coprocessed compound based on lactose and maize starch for tablet formulation
Thermal expansion and cracking of three confined water-saturated igneous rocks to 800C
Revision of the genera of the tribe Stigmoderini (Coleoptera: Buprestidae) a discussion of phylogenetic relationships
Anal tuberculosis. Report of a case
Gastric tuberculosis in the past and present
Adaptive responses of the cardiovascular system to prolonged spaceflight conditions: assessment with Holter monitoring

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


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



Japanese Journal of Infectious Diseases 63(3): 173-180



ISSN/ISBN: 1344-6304

PMID: 20495268

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.

Please choose payment method:






(PDF emailed within 1 workday: $29.90)

Accession: 052566088

Download citation: RISBibTeXText

Related references

Single tube real time PCR for detection of Streptococcus pneumoniae, Mycoplasma pneumoniae, Chlamydophila pneumoniae and Legionella pneumophila from clinical samples of CAP. Acta Microbiologica et Immunologica Hungarica 59(2): 171-184, 2012

Development of real-time multiplex nucleic acid sequence-based amplification for detection of Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella spp. in respiratory specimens. Journal of Clinical Microbiology 46(1): 185-191, 2008

The pneumoplex assays, a multiplex PCR-enzyme hybridization assay that allows simultaneous detection of five organisms, Mycoplasma pneumoniae, Chlamydia (Chlamydophila) pneumoniae, Legionella pneumophila, Legionella micdadei, and Bordetella pertussis, and its real-time counterpart. Journal of Clinical Microbiology 43(2): 565-571, 2005

Evaluation of the NucliSens miniMAG RNA extraction and real-time NASBA applications for the detection of Mycoplasma pneumoniae and Chlamydophila pneumoniae in throat swabs. Journal of Microbiological Methods 72(2): 217-219, 2008

Development of a multiplex real-time quantitative PCR assay to detect Chlamydia pneumoniae, Legionella pneumophila and Mycoplasma pneumoniae in respiratory tract secretions. Diagnostic Microbiology and Infectious Disease 45(2): 85-95, 2003

Simultaneous detection of Chlamydophila pneumoniae and Mycoplasma pneumoniae by use of molecular beacons in a duplex real-time PCR. Journal of Clinical Microbiology 46(2): 727-731, 2008

Analysis of children with Chlamydophila (Chlamydia) pneumoniae and Mycoplasma pneumoniae respiratoryinfections by real-time PCR assay and serological tests. Apmis: Acta Pathologica Microbiologica et Immunologica Scandinavica 116(6): 477-483, 2008

Development and evaluation of Chlamylege, a new commercial test allowing simultaneous detection and identification of Legionella, Chlamydophila pneumoniae, and Mycoplasma pneumoniae in clinical respiratory specimens by multiplex PCR. Journal of Clinical Microbiology 43(7): 3247-3254, 2005

Development of a multiplex real-time PCR assay for detection of Mycoplasma pneumoniae, Chlamydia pneumoniae and mutations associated with macrolide resistance in Mycoplasma pneumoniae from respiratory clinical specimens. Springerplus 4: 684, 2015

Detection of Mycoplasma pneumoniae and Chlamydophila pneumoniae directly from respiratory clinical specimens using a rapid real-time polymerase chain reaction assay. Diagnostic Microbiology and Infectious Disease 73(3): 278-280, 2012

A multiplex PCR for detection of Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, and Bordetella pertussis in clinical specimens. Molecular and Cellular Probes 19(5): 314-322, 2005

Detection of Mycoplasma pneumoniae, Chlamydia pneumoniae, and Legionella spp. in clinical specimens using a single-tube multiplex real-time PCR assay. Diagnostic Microbiology and Infectious Disease 70(1): 1-9, 2011

Comparison of sputum and nasopharyngeal swab specimens for molecular diagnosis of Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella pneumophila. Annals of Laboratory Medicine 32(2): 133-138, 2012

Evaluation of a new commercial kit for the detection of Chlamydophila pneumoniae, Mycoplasma pneumoniae and Legionella species in respiratory specimens through a prospective pediatric clinical study. Abstracts of the General Meeting of the American Society for 105): 121, 2005

Development of real-time PCR systems based on SYBR Green for the specific detection and quantification of Klebsiella pneumoniae in infant formula. Food Control: 4, 487-491, 2010