Browse
Submit Data
Databases
API
Help

Dataset Information

33 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Sensitive detection of Mycoplasma pneumoniae in human respiratory tract samples by optimized real-time PCR approach.

ABSTRACT: To enhance the sensitivity of the available real-time PCR systems for the detection of Mycoplasma pneumoniae, we established a method to amplify copies of the repetitive element repMp1. In a study of respiratory tract samples, we found that, compared to the use of the conserved part of the P1 adhesin gene as a monocopy target, the use of the repMp1-PCR showed an increase in the detected genome equivalents by a factor of 22.

SUBMITTER: Dumke R

PROVIDER: S-EPMC1951254 | biostudies-literature | 2007 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Sensitive detection of Mycoplasma pneumoniae in human respiratory tract samples by optimized real-time PCR approach.

Dumke Roger R Schurwanz Nicol N Lenz Matthias M Schuppler Markus M Lück Christian C Jacobs Enno E

Journal of clinical microbiology 20070530 8

To enhance the sensitivity of the available real-time PCR systems for the detection of Mycoplasma pneumoniae, we established a method to amplify copies of the repetitive element repMp1. In a study of respiratory tract samples, we found that, compared to the use of the conserved part of the P1 adhesin gene as a monocopy target, the use of the repMp1-PCR showed an increase in the detected genome equivalents by a factor of 22. ...[more]

PMID: 17537933

Similar Datasets

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.

Project description:The aim of this study was to improve detection of Mycoplasma pneumoniae and Chlamydia pneumoniae in clinical specimens by developing a multiplex real-time PCR assay that includes identification of macrolide-resistant M. pneumoniae. Novel assays targeting a M. pneumoniae conserved hypothetical protein gene, M. pneumoniae 23S rRNA gene mutations associated with macrolide resistance and human ?-globin gene (an endogenous internal control) were designed and combined with a previously published C. pneumoniae PCR targeting ompA gene. The resulting quadraplex PCR was validated with a panel of clinical specimens supplemented with external quality assessment specimens, simulated specimens and various bacterial and viral strains. The obtained results were compared to those obtained by reference PCRs or confirmed by sequencing (typing of macrolide resistance). The novel multiplex PCR assay was in 100 % agreement with reference PCRs. Four M. pneumoniae strains with macrolide resistance-associated mutations were identified among 42 strains, which comprises 9.5 % of the study material. Amplification of an internal control excluded sample-derived inhibition possibly leading to false-negative reporting. In conclusion, we have developed a resources conserving multiplex real-time PCR assay for simultaneous detection of M. pneumoniae, C. pneumoniae and the most common mutations leading to macrolide resistance in M. pneumoniae. The assay is a widely useful tool for detection of these respiratory pathogens and will also shed light on the occurrence of macrolide resistance in M. pneumoniae.

| S-EPMC4641141 | biostudies-literature

Allele-specific real-time PCR testing for minor macrolide-resistant Mycoplasma Pneumoniae.

Project description:BACKGROUND:The point mutations in 23S rRNA gene of Mycoplasma pneumoniae (M. pneumoniae) can lead to high-level resistance to macrolides. This study aimed to evaluate allele-specific real-time PCR (ASPCR) to detect the resistance-related mutations located at positions A2063G and A2064G of 23S rRNA gene. METHODS:We detected 178 pharyngeal swab specimens and calculated the proportions of resistant and sensitive quasispecies using ASPCR assays. ASPCR assays can detect down to 10 copies of 23S rRNA gene and achieved sensitivities of <?0.1% for A2063G and A2064G. We also compared the findings of ASPCR with the results of nested PCR with sequencing. RESULTS:Of 178 samples, 164 were found to have M. pneumoniae including 90.85% (149/164) samples with macrolide-resistant M. pneumoniae (MRMP) quasispecies by ASPCR, while 153 were found to be M. pneumoniae-positive including 71.90% (110/153) samples with MRMP quasispecies by nested PCR with sequencing. Of the 164?M. pneumoniae-positive samples, 61.59% (101/164) had the mixed population of wild-type and mutant M. pneumoniae, and 56.44% (57/101) of the latter contained the mutations at low frequency (?50%). CONCLUSION:ASPCR indicated that sensitive and resistant quasispecies coexisted in most of the M. pneumoniae positive samples. The ASPCR was a highly sensitive, accurate and rapid method for detecting the macrolide resistance-associated mutations and it could provide earlier and more drug-resistant information for M. pneumoniae research and the clinical therapy.

| S-EPMC6626384 | biostudies-literature

Comparison and evaluation of real-time PCR, real-time nucleic acid sequence-based amplification, conventional PCR, and serology for diagnosis of Mycoplasma pneumoniae.

Project description:Mycoplasma pneumoniae is a common cause of community-acquired pneumonia and lower-respiratory-tract infections. Diagnosis has traditionally been obtained by serological diagnosis, but increasingly, molecular techniques have been applied. However, the number of studies actually comparing these assays is limited. The development of a novel duplex real-time PCR assay for detection of M. pneumoniae in the presence of an internal control real-time PCR is described. In addition, real-time nucleic acid sequence-based amplification (NASBA) on an iCycler apparatus is evaluated. These assays were compared to serology and a conventional PCR assay for 106 clinical samples from patients with lower-respiratory-tract infection. Of the 106 samples, 12 (11.3%) were positive by all the molecular methods whereas serology with acute sample and convalescent samples detected 6 (5.6%) and 9 (8.5%), respectively. Clinical symptoms of the patients with Mycoplasma-positive results were compared to those of the other patients with lower-respiratory-tract infections, and it was found that the results for mean lower age numbers as well as the presence of chills, increased erythrocyte sedimentation rate, and raised C-reactive protein levels showed significant differences. Molecular methods are superior for diagnosis of M. pneumoniae, providing more timely diagnosis. In addition, using real-time methods involves less hands-on time and affords the ability to monitor the reaction in the same tube.

| S-EPMC193789 | biostudies-literature

Simultaneous detection of Chlamydophila pneumoniae and Mycoplasma pneumoniae by use of molecular beacons in a duplex real-time PCR.

Project description:A real-time PCR was designed for detection of Chlamydophila pneumoniae and Mycoplasma pneumoniae such that each pathogen could be detected in a single tube and differentiated using molecular beacons marked with different fluorochromes. This duplex PCR, targeting the P1 adhesion gene for M. pneumoniae and the ompA gene for C. pneumoniae, was compared with two conventional PCR assays targeting the 16S rRNA gene and the ompA gene. A total of 120 clinical throat and nasopharyngeal swab samples were tested. DNA extraction was performed using an alkali denaturation/neutralization method, and real-time amplification, detection, and data analysis were performed using a Rotor-Gene 2000 real-time rotary analyzer (Corbett Life Science, Sydney, Australia). Using conventional PCR as a reference in an analysis of 120 samples, 13 of 14 samples positive for C. pneumoniae were detected by the novel real-time PCR. In an analysis of M. pneumoniae, 22 samples were positive in the conventional PCR and the novel assay detected 24 positive samples. When using the conventional PCR as a reference, sensitivity and specificity were 93% and 100%, respectively, for C. pneumoniae and 100% and 98%, respectively, for M. pneumoniae. With an overall agreement of 98.8%, this suggests that performance of the new duplex real-time PCR is comparable to that of conventional PCR.

| S-EPMC2238133 | biostudies-literature

Detection of macrolide resistance in Mycoplasma pneumoniae by real-time PCR and high-resolution melt analysis.

Project description:Mycoplasma pneumoniae is a significant cause of community-acquired pneumonia, which is often empirically treated with macrolides or azalides such as erythromycin or azithromycin. Recent studies have discovered the existence of macrolide-resistant strains within the population that have been mapped to mutations within the domain V region of the 23S rRNA gene. Currently, identification of these resistant strains relies on time-consuming and labor-intensive procedures such as restriction fragment length polymorphism, MIC studies, and sequence analysis. The current study reports two distinct real-time PCR assays that can detect the A2063G or A2064G base mutation (A2058G or A2059G by Escherichia coli numbering) conferring macrolide resistance. By subjecting the amplicon of the targeted domain V region of the 23S rRNA gene to a high-resolution melt curve analysis, macrolide-resistant strains can quickly be separated from susceptible strains. Utilizing this method, we screened 100 clinical isolates and found 5 strains to possess mutations conferring resistance. These findings were concordant with both sequencing and MIC data. This procedure was also used successfully to identify both susceptible and resistant genotypes in 23 patient specimens. These patient specimens tested positive for the presence of M. pneumoniae by a separate real-time PCR assay, although the bacteria could not be isolated by culture. This is the first report of a real-time PCR assay capable of detecting the dominant mutations that confer macrolide resistance on M. pneumoniae, and these assays may have utility in detecting resistant strains of other infectious agents. These assays may also allow for clinicians to select appropriate treatment options more rapidly and may provide a convenient method to conduct surveillance for genetic mutations conferring antibiotic resistance.

| S-EPMC2565909 | biostudies-literature

Evaluation of three real-time PCR assays for detection of Mycoplasma pneumoniae in an outbreak investigation.

Project description:We compared the performances of three recently optimized real-time PCR assays derived from distinct genomic regions of Mycoplasma pneumoniae during an outbreak. Comprehensive evaluation established that a newly described toxin gene represents a superior target for detecting M. pneumoniae DNA in clinical specimens, although use of multiple targets may increase testing confidence.

| S-EPMC2546712 | biostudies-literature

Detection of Mycoplasma pneumoniae, Chlamydia pneumoniae, and Legionella spp. in clinical specimens using a single-tube multiplex real-time PCR assay.

Project description:A multiplex real-time PCR assay for the detection of Mycoplasma pneumoniae (MP181), Chlamydia (Chlamydophila) pneumoniae (CP-Arg), Legionella spp. (Pan-Leg), and the human RNase P (RNase P) gene was developed for rapid testing of atypical bacterial respiratory pathogens in clinical specimens. This method uses 4 distinct hydrolysis probes to detect 3 leading causes of community-acquired pneumonia. The assay was evaluated for specificity and sensitivity by testing against 35 related organisms, a dilution series of each specific target and 197 clinical specimens. Specificity testing demonstrated no cross-reactivity. A comparison to previously validated singleplex real-time PCR assays for each agent was also performed. The analytical sensitivity for specific pathogen targets in both the singleplex and multiplex was identical (50 fg), while efficiencies ranged from 82% to 97% for the singleplex assays and from 90% to 100% for the multiplex assay. The clinical sensitivity of the multiplex assay was improved for the Pan-Leg and CP-Arg targets when compared to the singleplex. The MP181 assay displayed equivalent performance. This multiplex assay provides an overall improvement in the diagnostic capability for these agents by demonstrating a sensitive, high-throughput and rapid method. This procedure may allow for a practical and efficient means to test respiratory clinical specimens for atypical pneumonia agents in health care settings and facilitate an appropriate public health response to outbreaks.

| S-EPMC7126442 | biostudies-literature

A sensitive real-time PCR for detection and subgrouping of human respiratory syncytial virus.

Project description:Improved diagnostic tools for rapid detection, quantitation, and subgrouping of human respiratory syncytial virus (RSV) are needed to aid the development and evaluation of novel intervention strategies. A quantitative real-time RT-PCR using specific locked nucleic acid (LNA) probes was developed to identify RSV and to distinguish RSV subgroups A and B (RSV LNA assay). RSV subgroup diversity and the relationship between viral load and disease severity in confirmed RSV infections were also explored. 264 archived respiratory specimens from pediatric patients were tested in parallel using the commercial multiplex Seeplex™ RV detection kit (Seegene) and the novel RSV LNA assay. The LNA assay demonstrated a significantly higher sensitivity than Seeplex, improving overall detection rates from 24% (64/264) to 32% (84/264). Detection limits of 9.0×10(1) and 6.0×10(2)copies/mL were observed for RSV A and B, respectively. RSV A was detected in 53/84 (63%) cases, and 31/84 (37%) were positive for RSV B. This novel method offers a rapid, quantitative, highly specific and sensitive approach to laboratory diagnosis of RSV.

| S-EPMC3405522 | biostudies-literature

Development of real-time PCR for detection of Mycoplasma hominis.

Project description:BACKGROUND: Mycoplasma hominis is associated with pelvic inflammatory disease, bacterial vaginosis, post partum fever, sepsis and infections of the central nervous system often leading to serious conditions. Association with development of female infertility has also been suggested, but different publications present different results. We developed a sensitive and fast diagnostic real-time PCR to test clinical samples from women undergoing laparoscopic examination before fertility treatment. To develop a test for the detection and quantification of M. hominis we selected a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (gap), as a target. RESULTS: Real-time PCR was optimized to detect 10 copies of M. hominis PG21 genomic DNA. A fluorescence signal was measured for all 20 other M. hominis isolates, and melting curves analysis showed variations in the melting temperature in agreement with sequence variation in the region of the probes. There was no amplification of other mycoplasmal DNA and human DNA. Eighty-three patient cervical swab samples from infertile women were cultured for M. hominis in the BEa medium. Two of the samples (2.4%) were positive after 48 hours of incubation. The real-time PCR detected the same two samples positive, and the DNA concentrations in the clinical specimens were calculated to 37.000 copies/ml and 88.500 copies/ml, respectively. CONCLUSION: The results demonstrate that real-time PCR may prove to be a rapid alternative to the traditional cultivation method. Information on bacterial load in genital swabs can be obtained. The assay allowed detection of M. hominis in a closed system reducing the risk of contamination by amplicon carry-over.

| S-EPMC518963 | biostudies-literature

Clinical evaluation of a multiplex real-time RT-PCR assay for detection of SARS-CoV-2 in individual and pooled upper respiratory tract samples.

Project description:The aim of this study was to identify and validate a sensitive, high-throughput, and cost-effective SARS-CoV-2 real-time RT-PCR assay to be used as a surveillance and diagnostic tool for SARS-CoV-2 in a university surveillance program. We conducted a side-by-side clinical evaluation of a newly developed SARS-CoV-2 multiplex assay (EZ-SARS-CoV-2 Real-Time RT-PCR) with the commercial TaqPath COVID-19 Combo Kit, which has an Emergency Use Authorization from the FDA. The EZ-SARS-CoV-2 RT-PCR incorporates two assays targeting the SARS-CoV-2 N gene, an internal control targeting the human RNase P gene, and a PCR inhibition control in a single reaction. Nasopharyngeal (NP) and anterior nares (AN) swabs were tested as individuals and pools with both assays and in the ABI 7500 Fast and the QuantStudio 5 detection platforms. The analytical sensitivity of the EZ-SARS-CoV-2 RT-PCR assay was 250 copies/ml or approximately 1.75 genome copy equivalents per reaction. The clinical performance of the EZ-SARS-CoV-2 assay was evaluated using NP and AN samples tested in other laboratories. The diagnostic sensitivity of the assay ranged between 94 and 96% across the detection platforms, and the diagnostic specificity was 94.06%. The positive predictive value was 94%, and the negative predictive value ranged from 94 to 96%. Pooling five NP or AN specimens yielded 93% diagnostic sensitivity. The overall agreement between these SARS-CoV-2 RT-PCR assays was high, supported by a Cohen's kappa value of 0.93. The EZ-SARS-CoV-2 RT-PCR assay performance attributes of high sensitivity and specificity with AN sample matrix and pooled upper respiratory samples support its use in a high-throughput surveillance testing program.

| S-EPMC8278812 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data