Browse
Submit Data
Databases
API
Help

Dataset Information

24 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Multiplex real-time PCR assay and melting curve analysis for identifying Mycobacterium tuberculosis complex and nontuberculous mycobacteria.

ABSTRACT: A multiplex real-time PCR assay and melting curve analysis for identifying 23 mycobacterial species was developed and evaluated using 77 reference strains and 369 clinical isolates. Concordant results were obtained for all 189 (100%) isolates of the Mycobacterium tuberculosis complex and 169 (93.9%) isolates of nontuberculous mycobacteria. Our results showed that this multiplex real-time PCR assay is an effective tool for the mycobacterial identification from cultures.

SUBMITTER: Kim JU

PROVIDER: S-EPMC3264165 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Multiplex real-time PCR assay and melting curve analysis for identifying Mycobacterium tuberculosis complex and nontuberculous mycobacteria.

Kim Jeong-Uk JU Cha Choong-Hwan CH An Hae-Kyong HK

Journal of clinical microbiology 20111207 2

A multiplex real-time PCR assay and melting curve analysis for identifying 23 mycobacterial species was developed and evaluated using 77 reference strains and 369 clinical isolates. Concordant results were obtained for all 189 (100%) isolates of the Mycobacterium tuberculosis complex and 169 (93.9%) isolates of nontuberculous mycobacteria. Our results showed that this multiplex real-time PCR assay is an effective tool for the mycobacterial identification from cultures. ...[more]

PMID: 22162553

Similar Datasets

Diversity of Nontuberculous Mycobacteria in Kuwait: Rapid Identification and Differentiation of Mycobacterium Species by Multiplex PCR, INNO-LiPA Mycobacteria v2 Assay and PCR Sequencing of rDNA.

Project description:ObjectiveNontuberculous mycobacteria (NTM) often cause disease that is clinically indistinguishable from tuberculosis. Specific identification is important as treatment varies according to Mycobacterium species causing the infection. This study used multiplex PCR (mPCR) assay for rapid differentiation of mycobacterial growth indicator tube 960 system (MGIT) cultures as Mycobacterium tuberculosis (MTB) or NTM together with INNO LiPA Mycobacteria v2 assay (LiPA) and/or PCR sequencing of rDNA for species-specific identification of selected MTB and all NTM isolates in Kuwait.Materials and methodsDNA was extracted from MGIT cultures (n = 1,033) grown from 664 pulmonary and 369 extrapulmonary specimens from 1,033 suspected tuberculosis patients. mPCR was performed to differentiate MTB from NTM. LiPA was performed and results were interpreted according to kit instructions. rDNA was amplified and sequenced by using panmycobacterial primers.ResultsmPCR identified 979 isolates as MTB, 53 as NTM and 1 isolate as mixed culture. LiPA and/or PCR sequencing confirmed 112 of 979 selected isolates as MTB. Mixed culture contained M. tuberculosis and M. fortuitum. LiPA yielded 12 patterns and identified 10 species/species complexes among 47 NTM, M. kansasii + M. scrofulaceum in one culture and 5 isolates only at genus level. PCR sequencing yielded more specific identification for 22 isolates at the species/subspecies level.ConclusionsmPCR rapidly differentiated MTB from NTM. LiPA identified 44 of 52 NTM isolates at the species/species complex level and 2 mixed cultures. PCR sequencing yielded more specific identification at the species/subspecies level. Rapid differentiation as MTB or NTM by mPCR, followed by species-specific NTM identification by LiPA/PCR sequencing is suitable for the proper management of mycobacterial infections in Kuwait.

| S-EPMC6597937 | biostudies-literature

Mycobacteria mobility shift assay: a method for the rapid identification of Mycobacterium tuberculosis and nontuberculous mycobacteria.

Project description:The identification of mycobacteria is essential because tuberculosis (TB) and mycobacteriosis are clinically indistinguishable and require different therapeutic regimens. The traditional phenotypic method is time consuming and may last up to 60 days. Indeed, rapid, affordable, specific and easy-to-perform identification methods are needed. We have previously described a polymerase chain reaction-based method called a mycobacteria mobility shift assay (MMSA) that was designed for Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) species identification. The aim of this study was to assess the MMSA for the identification of MTC and NTM clinical isolates and to compare its performance with that of the PRA-hsp65 method. A total of 204 clinical isolates (102 NTM and 102 MTC) were identified by the MMSA and PRA-hsp65. For isolates for which these methods gave discordant results, definitive species identification was obtained by sequencing fragments of the 16S rRNA and hsp65 genes. Both methods correctly identified all MTC isolates. Among the NTM isolates, the MMSA alone assigned 94 (92.2%) to a complex or species, whereas the PRA-hsp65 method assigned 100% to a species. A 91.5% agreement was observed for the 94 NTM isolates identified by both methods. The MMSA provided correct identification for 96.8% of the NTM isolates compared with 94.7% for PRA-hsp65. The MMSA is a suitable auxiliary method for routine use for the rapid identification of mycobacteria.

| S-EPMC4131790 | biostudies-literature

Differential identification of Mycobacterium tuberculosis complex and nontuberculous mycobacteria by duplex PCR assay using the RNA polymerase gene (rpoB).

Project description:A novel duplex PCR method that can amplify the 235- and 136-bp rpoB DNAs of Mycobacterium tuberculosis complex and nontuberculous mycobacteria (NTM), respectively, with two different sets of primers was used to differentially identify 44 reference strains and 379 clinical isolates of mycobacteria in a single-step assay. Showing 100% sensitivity and specificity, the duplex PCR method could clearly differentiate M. tuberculosis complex and NTM strains. In addition, restriction fragment length polymorphism analysis and direct sequencing of the amplicon of NTM could be used to supplement species identification.

| S-EPMC356837 | biostudies-literature

Development and clinical evaluation of a new multiplex PCR assay for a simultaneous diagnosis of tuberculous and nontuberculous mycobacteria.

Project description:BackgroundThe prevalence of non-tuberculous mycobacteria (NTM) has been increasing worldwide in both developed and developing countries. NTM infection is clinically indistinguishable from tuberculosis and therefore poses significant challenges in patient management, especially in patients chronically treated for pulmonary TB. In this study, we evaluated a new highly sensitive Multiplex MTB/NTM assay that can differentiate M. tuberculosis complex (MTBC) from all NTM, including the treatable and most common NTM, M. avium complex (MAC).MethodsWe developed and optimized a new open- Multiplex MTB/NTM assay with two gene-targets for MTBC (IS6110/senX3-regX3) and two targets for MAC (IS1311/DT1) with samples spiked with stored strains and testing 20 replicates. Patients with presumptive TB and NTM were enrolled at the Respiratory Disease Department of The University Teaching Hospital of Point G, in Mali.FindingsIn the development stage, the new assay showed a high analytic performance with 100% detections of MTBC and MAC at only 5 colony forming units (CFUs). Overall, without the treatment failure cases, the Multiplex assay and the Xpert showed a sensitivity, specificity, PPV and NPV of 83·3% [66·4-92·6], 96·6% [88·6-99·0], 92·5% [82·3-96·5] and 92·2% [82·7-96·5] and the Xpert had values of 96·7% [83·3-99·4], 80·0% [68·2-88·1], 70·7 [55·5-82·3] and 97·9% [89·3-99·6], respectively. The Multiplex assay successfully detected all (5/5) the MAC cases.InterpretationOur new Multiplex assay demonstrates better specificity than Xpert for all group studied, in addition to detecting potential NTM cases. The assay could therefore complement the widely used Xpert assay and enhance discrimination of TB and NTM infections.FundingThis work was supported by the National Institutes of Health (R03AI137674, U54EB027049, D43TW010350 and UM1AI069471) and Northwestern University's Institute for Global Health Catalyzer Fund.

| S-EPMC8365364 | biostudies-literature

Novel multiplex real-time PCR diagnostic assay for identification and differentiation of Mycobacterium tuberculosis, Mycobacterium canettii, and Mycobacterium tuberculosis complex strains.

Project description:Tuberculosis (TB) in humans is caused by members of the Mycobacterium tuberculosis complex (MTC). Rapid detection of the MTC is necessary for the timely initiation of antibiotic treatment, while differentiation between members of the complex may be important to guide the appropriate antibiotic treatment and provide epidemiological information. In this study, a multiplex real-time PCR diagnostics assay using novel molecular targets was designed to identify the MTC while simultaneously differentiating between M. tuberculosis and M. canettii. The lepA gene was targeted for the detection of members of the MTC, the wbbl1 gene was used for the differentiation of M. tuberculosis and M. canettii from the remainder of the complex, and a unique region of the M. canettii genome, a possible novel region of difference (RD), was targeted for the specific identification of M. canettii. The multiplex real-time PCR assay was tested using 125 bacterial strains (64 MTC isolates, 44 nontuberculosis mycobacteria [NTM], and 17 other bacteria). The assay was determined to be 100% specific for the mycobacteria tested. Limits of detection of 2.2, 2.17, and 0.73 cell equivalents were determined for M. tuberculosis/M. canettii, the MTC, and M. canettii, respectively, using probit regression analysis. Further validation of this diagnostics assay, using clinical samples, should demonstrate its potential for the rapid, accurate, and sensitive diagnosis of TB caused by M. tuberculosis, M. canettii, and the other members of the MTC.

| S-EPMC3043491 | biostudies-literature

Detection of mycobacteria, Mycobacterium avium subspecies, and Mycobacterium tuberculosis complex by a novel tetraplex real-time PCR assay.

Project description:Mycobacterium tuberculosis complex, Mycobacterium avium, and many other nontuberculous mycobacteria are worldwide distributed microorganisms of major medical and veterinary importance. Considering the growing epidemiologic significance of wildlife-livestock-human interrelation, developing rapid detection tools of high specificity and sensitivity is vital to assess their presence and accelerate the process of diagnosing mycobacteriosis. Here we describe the development and evaluation of a novel tetraplex real-time PCR for simultaneous detection of Mycobacterium genus, M. avium subspecies, and M. tuberculosis complex in an internally monitored single assay. The method was evaluated using DNA from mycobacterial (n = 38) and nonmycobacterial (n = 28) strains, tissues spiked with different CFU amounts of three mycobacterial species (n = 57), archival clinical samples (n = 233), and strains isolated from various hosts (n = 147). The minimum detectable DNA amount per reaction was 50 fg for M. bovis BCG and M. kansasii and 5 fg for M. avium subsp. hominissuis. When spiked samples were analyzed, the method consistently detected as few as 100 to 1,000 mycobacterial CFU per gram. The sensitivity and specificity values for the panel of clinical samples were 97.5 and 100% using a verified culture-based method as the reference method. The assays performed on clinical isolates confirmed these results. This PCR was able to identify M. avium and M. tuberculosis complex in the same sample in one reaction. In conclusion, the tetraplex real-time PCR we designed represents a highly specific and sensitive tool for the detection and identification of mycobacteria in routine laboratory diagnosis with potential additional uses.

| S-EPMC4390653 | biostudies-literature

Rapid detection of isoniazid resistance in Mycobacterium tuberculosis isolates by use of real-time-PCR-based melting curve analysis.

Project description:The MeltPro TB/INH assay, recently approved by the Chinese Food and Drug Administration, is a closed-tube, dual-color, melting curve analysis-based, real-time PCR test specially designed to detect 30 isoniazid (INH) resistance mutations in katG position 315 (katG 315), the inhA promoter (positions -17 to -8), inhA position 94, and the ahpC promoter (positions -44 to -30 and -15 to 3) of Mycobacterium tuberculosis. Here we evaluated both the analytical performance and clinical performance of this assay. Analytical studies with corresponding panels demonstrated that the accuracy for detection of different mutation types (10 wild-type samples and 12 mutant type samples), the limit of detection (2×10(3) to 2×10(4) bacilli/ml), reproducibility (standard deviation [SD], <0.4°C), and the lowest heteroresistance level (40%) all met the parameters preset by the kit. The assay could be run on five types of real-time PCR machines, with the shortest running time (105 min) obtained with the LightCycler 480 II. Clinical studies enrolled 1,096 clinical isolates collected from three geographically different tuberculosis centers, including 437 INH-resistant isolates and 659 INH-susceptible isolates characterized by traditional drug susceptibility testing on Löwenstein-Jensen solid medium. The clinical sensitivity and specificity of the MeltPro TB/INH assay were 90.8% and 96.4%, respectively. DNA sequencing analysis showed that, except for the 5 mutants outside the detection range of the MeltPro assay, a concordance rate between the two methods of 99.1% (457/461) was obtained. Among the 26 mutation types detected, katG S315T (AGC?ACC), inhA -15C?T, katG S315N (AGC?AAC), and ahpC promoter -10C?T accounted for more than 90%. Overall, the MeltPro TB/INH assay represents a reliable and rapid tool for the detection of INH resistance in clinical isolates.

| S-EPMC3993622 | biostudies-literature

Rapid identification of mycobacteria and drug-resistant Mycobacterium tuberculosis by use of a single multiplex PCR and DNA sequencing.

Project description:Tuberculosis (TB) remains a significant global health problem for which rapid diagnosis is critical to both treatment and control. This report describes a multiplex PCR method, the Mycobacterial IDentification and Drug Resistance Screen (MID-DRS) assay, which allows identification of members of the Mycobacterium tuberculosis complex (MTBC) and the simultaneous amplification of targets for sequencing-based drug resistance screening of rifampin-resistant (rifampin(r)), isoniazid(r), and pyrazinamide(r) TB. Additionally, the same multiplex reaction amplifies a specific 16S rRNA gene target for rapid identification of M. avium complex (MAC) and a region of the heat shock protein 65 gene (hsp65) for further DNA sequencing-based confirmation or identification of other mycobacterial species. Comparison of preliminary results generated with MID-DRS versus culture-based methods for a total of 188 bacterial isolates demonstrated MID-DRS sensitivity and specificity as 100% and 96.8% for MTBC identification; 100% and 98.3% for MAC identification; 97.4% and 98.7% for rifampin(r) TB identification; 60.6% and 100% for isoniazid(r) TB identification; and 75.0% and 98.1% for pyrazinamide(r) TB identification. The performance of the MID-DRS was also tested on acid-fast-bacterium (AFB)-positive clinical specimens, resulting in sensitivity and specificity of 100% and 78.6% for detection of MTBC and 100% and 97.8% for detection of MAC. In conclusion, use of the MID-DRS reduces the time necessary for initial identification and drug resistance screening of TB specimens to as little as 2 days. Since all targets needed for completing the assay are included in a single PCR amplification step, assay costs, preparation time, and risks due to user errors are also reduced.

| S-EPMC3264146 | biostudies-literature

"Multidrug-resistant tuberculosis" may be nontuberculous mycobacteria.

Project description:IntroductionMultidrug resistant tuberculosis (MDR-TB) presents a great challenge to public health, especially for developing countries. Some nontuberculous mycobacteria (NTM) cause the similar clinical and radiological characteristics with tuberculosis. We aimed to identify the frequency of NTM infections among subjects who were suspected to have MDR-TB due to lack of response to anti-TB treatment.MethodsThis retrospective study evaluated patients with suspected MDR-TB due to lack of sputum conversion after 2-3 month therapy with first line anti-TB treatment from 2009 through 2014. Cultures for mycobacteria were performed and identification was done to species level by phenotypic and molecular tests. The outcome of the patients with NTM disease and related risk factors for poor outcome were evaluated.ResultsOut of 117 consecutive strains isolated from suspected MDR-TB subjects, 35 (30%) strains were identified as NTM by using conventional and molecular approaches. Of these patients with positive NTM cultures, 32 (27%) patients met ATS/IDSA diagnostic criteria. Out of 32, 29 (90%) individuals with confirmed NTM diseases had underlying disorders including 8 subjects with malignancy, 5 with organ transplantations, and 4 with the human immunodeficiency virus. No known underlying disorder was found in 3 (9%) subjects. Treatment outcomes were available for 27 subjects, 17 (63%) of whom were cured and 10 (37%) had poor outcome including 6 (60%) who failed and 4 (40%) who died during treatment.ConclusionThe high costs to the patient and society should lead health care providers to consider NTM in all patients suspected of having TB.

| S-EPMC4414892 | biostudies-literature

A Melting Curve-Based Multiplex RT-qPCR Assay for Simultaneous Detection of Four Human Coronaviruses.

Project description:Human coronaviruses HCoV-OC43, HCoV-229E, HCoV-NL63 and HCoV-HKU1 are common respiratory viruses associated with acute respiratory infection. They have a global distribution. Rapid and accurate diagnosis of HCoV infection is important for the management and treatment of hospitalized patients with HCoV infection. Here, we developed a melting curve-based multiplex RT-qPCR assay for simultaneous detection of the four HCoVs. In the assay, SYTO 9 was used to replace SYBR Green I as the fluorescent dye, and GC-modified primers were designed to improve the melting temperature (Tm) of the specific amplicon. The four HCoVs were clearly distinguished by characteristic melting peaks in melting curve analysis. The detection sensitivity of the assay was 3 × 10² copies for HCoV-OC43, and 3 × 10¹ copies for HCoV-NL63, HCoV-229E and HCoV-HKU1 per 30 μL reaction. Clinical evaluation and sequencing confirmation demonstrated that the assay was specific and reliable. The assay represents a sensitive and reliable method for diagnosis of HCoV infection in clinical samples.

| S-EPMC5133880 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

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