Use of a triplex polymerase chain reaction for the detection and differentiation of Mycoplasma pneumoniae and Mycoplasma genitalium in the presence of human DNA

Cadieux, Nathale; Lebel, Pierre; Brousseau, Roland

doi:10.1099/00221287-139-10-2431

Cited by 57 publications

(28 citation statements)

References 25 publications

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“…[20][21][22][23][24][25][26] Since the clinical features of atypical pneumonia caused by mycoplasma and chlamydia are very similar, 1 it is necessary to have an approach that can detect and diVerentiate all relevant organisms using the same sample and the same assay. Here we reported the successful development of a multiplex PCR for the simultaneous detection and diVerentiation of M pneumoniae, C pneumoniae, and C psittaci.…”

Section: Discussionmentioning

confidence: 99%

Multiplex polymerase chain reaction for the simultaneous detection of Mycoplasma pneumoniae, Chlamydia pneumoniae, and Chlamydia psittaci in respiratory samples.

Tong

Donnelly²,

Harvey³

et al. 1999

J Clin Pathol

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Aims-To develop a multiplex polymerase chain reaction (PCR) for the simultaneous detection of Mycoplasma pneumoniae, Chlamydia pneumoniae, and Chlamydia psittaci in respiratory samples. Methods-Oligonucleotide primers for the amplification of the DNA of these three organisms were optimised for use in combination in the same reaction. PCR products were detected by hybridisation with pooled internal probes using an enzyme linked immunosorbent assay. Those with positive signals were further diVerentiated using species specific probes. Quality of DNA extraction and PCR inhibition were controlled by amplification of a human mitochondrial gene. A panel of 53 respiratory samples with known results was evaluated blindly. This was followed by a retrospective study on sputa collected from 244 patients with suspected community acquired pneumonia. Results-The multiplex assay had a lower sensitivity than PCR with individual primers by about one log. The resultant sensitivity was considered acceptable for diagnostic use. Of the panel of 53 samples, nine of 11 M pneumoniae, 11 of 11 C pneumoniae, six of seven C psittaci, and 24 of 24 negative samples were correctly identified. Of the 244 patients with pneumonia, seven (2.9%) had detectable M pneumoniae, six (2.5%) had C pneumoniae, and one (0.4%) had C psittaci. The case notes from 11 patients were studied. The PCR finding was of possible significance in at least eight of these patients. Conclusions-This multiplex PCR assay has the potential to be used as a diagnostic and epidemiological tool. Further prospective studies are needed to establish its clinical value. (J Clin Pathol 1999;52:257-263) Keywords: atypical pneumonia; polymerase chain reaction; Mycoplasma pneumoniae; Chlamydia pneumoniae; Chlamydia psittaci Mycoplasma and chlamydia are major causes of community acquired atypical pneumonia.

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Section: Discussionmentioning

confidence: 99%

Multiplex polymerase chain reaction for the simultaneous detection of Mycoplasma pneumoniae, Chlamydia pneumoniae, and Chlamydia psittaci in respiratory samples.

Tong

Donnelly²,

Harvey³

et al. 1999

J Clin Pathol

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“…DNA was extracted from standard strains (U.urealyticum serotype VIII and M. hominis PG 21 were kindly provided by Statns Serum Institut of Copenhagen, Denmark) and clinical samples as described previously by Cadieux et al (17). Briefly, 1ml of the sample was centrifuged at 12000 ×g for 10 min.…”

Section: Dna Extraction From Specimensmentioning

confidence: 99%

Comparison of Culture With the Polymerase Chain Reaction for Detection of Gennital Mycoplasma

Peerayeh

Samimi

2008

ELECTRON J GEN MED

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“…If this second specimen also yielded a negative result in this assay, the specimen was scored as unsuitable for further testing. The HMCO assay is known to be an extremely robust PCR (Cadieux et al, 1993;D. Pitcher, personal communication) and thus amplicons are normally still visible even in the presence of PCR inhibitors, due to the large amount of human DNA in clinical samples compared to microbial DNA.…”

Section: Methodsmentioning

confidence: 99%

“…25 U AmpliTaq Gold (PE Applied Biosystems). Thermal cycling conditions were adapted from those described by Cadieux et al (1993); pre-denaturation for 10 min at 95 8C, followed by 30 cycles of denaturation for 1 min at 94 8C, annealing for 1 min at 65 8C and elongation for 1 min at 72 8C, with a final elongation of 5 min at 72 8C.…”

Section: Methodsmentioning

confidence: 99%

“…To assess the quality of the DNA extracts from clinical specimens, the presence or absence of human DNA was determined using a PCR assay targeting the human mitochondrial cytochrome oxidase (HMCO) gene. Primers (H6A and H6B; Table 2) were used to amplify an 823-bp product of the HMCO gene (Cadieux et al, 1993). Reaction mixtures were in a total volume of 50 ìl and contained 2 .…”

Section: Methodsmentioning

confidence: 99%

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Laboratory diagnosis of pertussis infections: the role of PCR and serology

Fry¹,

Tzivra²,

Li³

et al. 2004

Journal of Medical Microbiology

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This study reports on practical laboratory aspects of pertussis diagnosis. PCR assays were applied to respiratory specimens obtained during a large study of infants (less than 5 months old) admitted to paediatric intensive care units (n ¼ 122), children (less than 15 years old) admitted to paediatric wards (n ¼ 16) and their household contacts (n ¼ 320). Estimation of antibodies to pertussis toxin and culture for Bordetella pertussis were attempted on specimens from the same patients, where available, and the overall utility of the diagnostic PCR assays was assessed by comparison to these results. A PCR assay for the human mitochondrial cytochrome oxidase (HMCO) gene was used for quality control of the extracted samples and an internal process control (IPC) was included in each sample to test for PCR inhibition. Four of 458 samples were considered unsuitable (three HMCO negative, one IPC negative) and excluded from further analyses. Positive PCR results were considered valid if they were either (i) positive for both of two B. pertussis gene targets (pertussis toxin S1 promoter and the insertion element IS481), i.e. consensus PCR positive, or (ii) repeatably positive in only one assay. Using these criteria, 52 of 454 (11 . 5 %) samples were considered as PCR positive for B. pertussis. Six of 356 samples were culture-positive for B. pertussis, 1/88 infants, 3/14 children and 2/254 contacts, giving an overall isolation rate of 1 . 7 %. Using these data, PCR gave an almost fivefold increase in diagnostic yield compared with culture (McNemar's test; P , 0 . 0001). Sera from 9/111 infants, 5/10 children and 14/210 contacts were positive. Serology and PCR results showed a high level of agreement (113/121) for infants and children. PCR demonstrated a significant improvement in diagnostic yield over culture. Serological testing also resulted in a significant increase in diagnostic yield compared to culture alone. PCR is a useful technique, but validity of results must be assured by careful control. Rapid diagnosis of B. pertussis infection particularly in infants by PCR, together with serological assays, can enhance surveillance systems for pertussis in all age groups.

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Use of a triplex polymerase chain reaction for the detection and differentiation of Mycoplasma pneumoniae and Mycoplasma genitalium in the presence of human DNA

Cited by 57 publications

References 25 publications

Multiplex polymerase chain reaction for the simultaneous detection of Mycoplasma pneumoniae, Chlamydia pneumoniae, and Chlamydia psittaci in respiratory samples.

Multiplex polymerase chain reaction for the simultaneous detection of Mycoplasma pneumoniae, Chlamydia pneumoniae, and Chlamydia psittaci in respiratory samples.

Comparison of Culture With the Polymerase Chain Reaction for Detection of Gennital Mycoplasma

Laboratory diagnosis of pertussis infections: the role of PCR and serology

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