Peptide Nucleic Acid-Mediated Competitive PCR Clamping for Detection of Rifampin-Resistant
            <i>Mycobacterium tuberculosis</i>

Iwamoto, Tomotada; Sonobe, Toshiaki

doi:10.1128/aac.48.10.4023-4026.2004

Cited by 10 publications

(4 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…2. In 103 strains the specific mutations could be identified directly by hybridization to the oligonucleotide targeting the mutated sequence, and in 30 strains they could be identified indirectly by the absence of specific wild-type signals (Table 2) As expected, the test recognized as wild type a strain that had a duplication at codon 514 (15,16). In six cases the test was able to identify correctly the mutation in a subregion within hot-spot region but did not classify the amino acid substitution despite the presence of a specific probe targeting that mutation (three cases for H526D, one case for H526Y, and two cases for S531L).…”

Section: Strain Analysismentioning

confidence: 99%

Use of Genotype MTBDR Assay for Molecular Detection of Rifampin and Isoniazid Resistance in Mycobacterium tuberculosis Clinical Strains Isolated in Italy

Miotto

Piana

Penati³

et al. 2006

J Clin Microbiol

105

View full text Add to dashboard Cite

Drug-resistant Mycobacterium tuberculosis strains are a real threat to tuberculosis (TB) control worldwide, as strains resistant to major anti-TB drugs have emerged. Multidrug-resistant TB (MDR-TB; resistant at least to rifampin and isoniazid) requires prolonged and expensive chemotherapy, with low cure and high fatality rates (8). In several Eastern European countries economic crisis and health system weaknesses have led to an increase in the numbers of TB cases, together with the establishment of hot spots for MDR-TB. Laboratory surveillance of drug resistance and early identification of resistant strains are critical steps for the beginning of appropriate treatment. Any delay in resistant strain identification jeopardizes the efforts to control the transmission of the disease.Rifampin (RIF) resistance is due to mutation in a relatively small fragment (81 bp) of the rpoB gene encoding for the ␤-subunit of the RNA polymerase (12, 29, 35); isoniazid (INH) resistance is caused by mutations in one of several regions of the katG gene, the inhA regulatory and coding region, and the ahpC-oxyR, ndh, and kasA genes (3,4,18,19,26,29,32,33,36,39). Analysis of strains collected in different countries shows different prevalences of the mutations (24).Easy-to-perform, rapid, and cost-effective assays based on molecular techniques that are suitable for application in clinical mycobacteriology laboratories are necessary to evaluate the presence of genomic mutations conferring resistance. Detection of resistance by conventional methods is inadequate due to the slow growth rate of M. tuberculosis; in addition, direct detection of known mutations could be more reliable in predicting the response to therapy.The Genotype MTBDR (Hain Lifescience, Nehren, Germany) is a new commercial and easy-to-perform assay developed for the detection of RIF and/or INH resistance in TB strains. The test is based on reverse hybridization between amplicons derived from a multiplex PCR and nitrocellulosebound wild-type and mutated probes for the mutations of interest.The aim of the present study was to evaluate Genotype MTBDR as a rapid diagnostic tool to detect rpoB and katG gene mutations that are associated with RIF and INH resistance, respectively, in TB isolates.We tested 206 M. tuberculosis strains isolated in Italy during the past 3 years, including 139 MDR strains and 30 fully susceptible ones. A total of 69 strains were collected from Italian patients, and 137 were obtained from foreign-born people. In order to show that the test is suitable for the direct detection on clinical specimens, we also performed the test on respiratory samples collected from active TB patients. (Table 1). A total of 85% of the MDR strains were isolated from retreatment cases. MATERIALS AND METHODS Strains

show abstract

Section: Strain Analysismentioning

confidence: 99%

Use of Genotype MTBDR Assay for Molecular Detection of Rifampin and Isoniazid Resistance in Mycobacterium tuberculosis Clinical Strains Isolated in Italy

Miotto

Piana

Penati³

et al. 2006

J Clin Microbiol

105

View full text Add to dashboard Cite

show abstract

“…Afterwards, the mixture was heated to 65°C and cooled down to 25°C during 100 min. Addition of cold oligonucleotide 1 results in a 1.4-fold increase in fluorescence intensity whereas all other cold oligonucleotides ( 1 – 5 ) generate a smaller increase in the range of 1.1–1.2 fold. Upon simultaneous addition of all cold oligonucleotides 1–6, a 2.0-fold increase in fluorescence intensity was measured.…”

Section: Resultsmentioning

confidence: 99%

“…Today, molecular methods already play an important role in early-stage diagnosis of cancer diseases, diagnostic of viral diseases (e.g. HIV infections), laboratory test for the confirmation of tuberculosis and fast detection of antimicrobial resistance mechanisms ( 1 – 5 ). The molecular methods currently available are, however, poorly standardized (many of them being in house methods).…”

Section: Introductionmentioning

confidence: 99%

Identification of single-point mutations in mycobacterial 16S rRNA sequences by confocal single-molecule fluorescence spectroscopy

Marmé¹

2006

Nucleic Acids Research

View full text Add to dashboard Cite

We demonstrate the specific identification of single nucleotide polymorphism (SNP) responsible for rifampicin resistance of Mycobacterium tuberculosis applying fluorescently labeled DNA-hairpin structures (smart probes) in combination with single-molecule fluorescence spectroscopy. Smart probes are singly labeled hairpin-shaped oligonucleotides bearing a fluorescent dye at the 5′ end that is quenched by guanosine residues in the complementary stem. Upon hybridization to target sequences, a conformational change occurs, reflected in a strong increase in fluorescence intensity. An excess of unlabeled (‘cold’) oligonucleotides was used to prevent the formation of secondary structures in the target sequence and thus facilitates hybridization of smart probes. Applying standard ensemble fluorescence spectroscopy we demonstrate the identification of SNPs in PCR amplicons of mycobacterial rpoB gene fragments with a detection sensitivity of 10−8 M. To increase the detection sensitivity, confocal fluorescence microscopy was used to observe fluorescence bursts of individual smart probes freely diffusing through the detection volume. By measuring burst size, burst duration and fluorescence lifetime for each fluorescence burst the discrimination accuracy between closed and open (hybridized) smart probes could be substantially increased. The developed technique enables the identification of SNPs in 10−11 M solutions of PCR amplicons from M.tuberculosis in only 100 s.

show abstract

“…In their approach, PNA clamping effectively prevented off-target amplification despite the similarity of the two stains (Takiya et al, 2004). Similarly, Iwamoto and co-workers demonstrated that PNA-based clamping is useful for the detection of rifampin resistance in the rpoB gene in M. tuberculosis (Iwamoto & Sonobe, 2004). This study demonstrated that using an amplification and clamping protocol, they were able to reach a limit of detection of approximately 100 fg of genomic DNA (approximately 25 cells) and were able to suppress amplification from as much as 100 ng of off-target genomic DNA (approximately 2.5 Â 10 7 cells).…”

Section: Elongation Arrestmentioning

confidence: 94%

Artificial Nucleic Acid Probes and Their Applications in Clinical Microbiology

Singer

Tang

2015

Methods in Microbiology

View full text Add to dashboard Cite

Peptide Nucleic Acid-Mediated Competitive PCR Clamping for Detection of Rifampin-Resistant Mycobacterium tuberculosis

Cited by 10 publications

References 17 publications

Use of Genotype MTBDR Assay for Molecular Detection of Rifampin and Isoniazid Resistance in Mycobacterium tuberculosis Clinical Strains Isolated in Italy

Use of Genotype MTBDR Assay for Molecular Detection of Rifampin and Isoniazid Resistance in Mycobacterium tuberculosis Clinical Strains Isolated in Italy

Identification of single-point mutations in mycobacterial 16S rRNA sequences by confocal single-molecule fluorescence spectroscopy

Artificial Nucleic Acid Probes and Their Applications in Clinical Microbiology

Contact Info

Product

Resources

About