Performance of the Roche LightCycler Real-Time PCR Assay for Diagnosing Extrapulmonary Tuberculosis

Gous, Natasha; Scott, Lesley; Wong, Emily; Touzani, Omar; Venter, François; Stevens, Wendy

doi:10.1128/jcm.00252-12

Cited by 12 publications

(8 citation statements)

References 24 publications

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“…We chose a wild type (WT) and seven sequences of rifampin-resistant strains that have been studied and characterized previously. [13] All the mutations were located in a characteristic 81-bp fragment of the rpoB gene. This fragment is known to contain 95 to 98% of all mutations that impart rifampin resistance to M.tb .…”

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confidence: 99%

“…This fragment is known to contain 95 to 98% of all mutations that impart rifampin resistance to M.tb . [13] We designed three fluorescent sensors, with a single universal MB probe reporter, targeting different segments of the 81-bp fragment of the rpoB gene. Each TX sensor hybridizes to a different region of the analyte due to the unique set of the adaptor stands.…”

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confidence: 99%

“…Therefore, the DMB-based differential receptor is promising for the distinction of drug-susceptible M.tb species from the drug-resistant ones, which is of great practical significance. [12,13] …”

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A Differential Fluorescent Receptor for Nucleic Acid Analysis

Bengtson

Kolpashchikov

2013

ChemBioChem

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Differential receptors use an array of sensors to recognize analytes. Each sensor in the array can recognize not one, but several analytes with different rates, so a single analyte triggers a response of several sensors in the array. The receptor thus produces a pattern of signals that is unique for each analyte, thereby enabling identification of a specific analyte by producing a “fingerprint” pattern. We applied this approach for the analysis of DNA sequences of Mycobacterium tuberculosis strains that differ by single nucleotide substitutions in the 81‐bp hot‐spot region that imparts rifampin resistance. The technology takes advantage of the new multicomponent, selfassembling sensor, which produces a fluorescent signal in the presence of specific DNA sequences. A differential fluorescent receptor (DFR) contained an array of three such sensors and differentiated at least eight DNA sequences. The approach requires only one molecular‐beacon‐like fluorescent reporter, which can be used by all three sensors. The DFR developed in this study represents a cost‐efficient alternative to molecular diagnostic technologies that use fluorescent hybridization probes.

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A Differential Fluorescent Receptor for Nucleic Acid Analysis

Bengtson

Kolpashchikov

2013

ChemBioChem

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“…Several assays exist to detect mutations responsible for antibiotic resistance 5. One of the most advanced commercial assays, Cepheid’s Expert MTB/RIF,5i–n takes advantage of real‐time PCR (rtPCR) and MB probes 6. MB probes, first introduced by Tyagi and Kramer,6a are stem–loop‐folded oligonucleotides with fluorophore and quencher dyes attached at opposite ends (Figure 1).…”

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Molecular Logic Gates for DNA Analysis: Detection of Rifampin Resistance in M. tuberculosis DNA

et al. 2012

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Molecular logic gates made of DNA have attracted significant attention because of biocompatibility, simple design and their ability to analyze and control biological systems. [1] To fuel further development of the field, applications of DNA-based gates to solve significant biological problems are required. Recently we characterized a set of DNA logic gates (YES, NOT, AND, and OR) and demonstrated their connectivity by designing ANDNOT and XOR operations. [1g,h] The gates used hybridization of DNA strands with a molecular beacon (MB) probe to produce a fluorescent output. Here we demonstrate how DNA logic gates can be applied to solve an important biomedical task of analysis of multiple DNA sequences containing a complex set of mutations.Mycobacterium tuberculosis (Mtb) infects approximately 2 billion people all over the world and is responsible for about 2 million deaths each year. [2] Approximately 10% of all patients are infected by strains of Mtb that are drug-resistant; these strains are primarily resistant to antibiotics rifampin (Rif) and isoniazid. [3] Currently, there is an urgent need for costeffective diagnostic tools that can detect Mtb in clinical samples and differentiate between drug-susceptible and drug-resistant Mtb strains. [2][3][4] Several assays exist to detect mutations responsible for antibiotic resistance. [5] One of the most advanced commercial assays, Cepheid's Expert MTB/RIF, [5i-n] takes advantage of real-time PCR (rtPCR) and MB probes. [6] MB probes, first introduced by Tyagi and Kramer, [6a] are stem-loop folded oligonucleotides with fluorophore and quencher dyes attached at opposite ends (Fig. 1). Hybridization of an MB probe to a complementary DNA or RNA switches the probe to an elongated form, thus separating the fluorophore from the quencher. The resultant fluorescence increase can be quantitatively measured, which is the basis for the widespread application of MB probes in real-time detection of nucleic acids. [6] In the Expert MTB/RIF assay, five MB probes were designed to span the highly variable 81-nt core of the bacterial ** The authors are grateful to Yulia V. Gerasimova for helpful discussion and corrections and to A. Trevor Longino for careful reading of the manuscript. Support from NIHGRI (R21 HG004060) and NSF CCF (1117205) is greatly appreciated.Correspondence to: Dmitry M. Kolpashchikov.

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“…Microscopy has a low sensitivity for the diagnosis of EPTB, and reliance on culture-based detection methods can lead to substantial delays in diagnosis (8,9). Rapid TB NATs have not been optimized for nonpulmonary samples and have had variable performance in detecting TB in tissue or fluid samples (9)(10)(11)(12). Thus, there is a real need for improved methods to test extrapulmonary samples.…”

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Detection of Mycobacterium tuberculosis in Blood by Use of the Xpert MTB/RIF Assay

2013

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We have developed a novel blood lysis-centrifugation approach for highly sensitive Mycobacterium tuberculosis detection in large volumes of blood with the Xpert MTB/RIF assay. One through 20 ml of blood was spiked with 0.25 to 10 CFU/ml of the M. tuberculosis surrogate M. bovis BCG. Multiple replicates of each sample were processed by a new lysis-centrifugation method and tested with the Xpert MTB/RIF assay. The assay was very sensitive with increased blood volumes. In the 20-ml samples, BCG was detected in blood spiked with 10, 5, 1, and 0.25 CFU/ml 100, 100, 83, and 57% of the time, respectively, compared to 100, 66, 18, and 18%, of the time, respectively, in 1-ml blood samples. Assay sensitivity was influenced by the type of anticoagulant used, with acid-citrate-dextrose solution B (ACD-B) providing the best results. A limit of detection of 10 CFU/ml was established with BCG spiked into ACD-B-treated blood, and 92, 36, and 33% of the samples with 5, 1, and 0.5 CFU/ml, respectively, were assay positive. The lysis buffer was stable both at room temperature and at 4°C for 2 months. The assay was tested with blood stored for 8 days without a change in sensitivity as measured by cycle threshold. This new assay format extends the capability of the Xpert MTB/RIF test, enabling up to 20 ml of blood to be tested rapidly for the presence of M. tuberculosis. This approach may be a useful method to detect extrapulmonary tuberculosis and the risk of death in immunocompromised patients.

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Performance of the Roche LightCycler Real-Time PCR Assay for Diagnosing Extrapulmonary Tuberculosis

Cited by 12 publications

References 24 publications

A Differential Fluorescent Receptor for Nucleic Acid Analysis

A Differential Fluorescent Receptor for Nucleic Acid Analysis

Molecular Logic Gates for DNA Analysis: Detection of Rifampin Resistance in M. tuberculosis DNA

Detection of Mycobacterium tuberculosis in Blood by Use of the Xpert MTB/RIF Assay

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