Use of Real-Time PCR and Fluorimetry for Rapid Detection of Rifampin and Isoniazid Resistance-Associated Mutations  in Mycobacterium tuberculosis

Torres, Marı́a José; Criado, Antonio; Palomares, J.C.; Aznar, Javier

doi:10.1128/jcm.38.9.3194-3199.2000

Cited by 111 publications

(28 citation statements)

References 22 publications

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“…Real-time PCR offers the potential to detect gene mutations responsible for drug resistance within hours from patient specimens compared with the average of 2 weeks required for traditional susceptibility test methods. The rpoB and katG genes are the most common Mycobacterium tuberculosis targets utilized in real-time PCR methods and well-known mutations in these genes correlate with resistance to rifampin and isoniazid, respectively (106,110,135,353,379,495,496,505). The significance of other gene targets such as kasA, ahpC-oxyR, and inhA for the prediction of isoniazid resistance is still somewhat controversial (378).…”

Section: Mycobacteriamentioning

confidence: 99%

Real-Time PCR in Clinical Microbiology: Applications for Routine Laboratory Testing

et al. 2006

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SUMMARY Real-time PCR has revolutionized the way clinical microbiology laboratories diagnose many human microbial infections. This testing method combines PCR chemistry with fluorescent probe detection of amplified product in the same reaction vessel. In general, both PCR and amplified product detection are completed in an hour or less, which is considerably faster than conventional PCR detection methods. Real-time PCR assays provide sensitivity and specificity equivalent to that of conventional PCR combined with Southern blot analysis, and since amplification and detection steps are performed in the same closed vessel, the risk of releasing amplified nucleic acids into the environment is negligible. The combination of excellent sensitivity and specificity, low contamination risk, and speed has made real-time PCR technology an appealing alternative to culture- or immunoassay-based testing methods for diagnosing many infectious diseases. This review focuses on the application of real-time PCR in the clinical microbiology laboratory.

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Section: Mycobacteriamentioning

confidence: 99%

Real-Time PCR in Clinical Microbiology: Applications for Routine Laboratory Testing

et al. 2006

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“…However, specific bacterial species are more frequently the focus for real-time PCR assays, especially when long culture times can be replaced by rapid and specific gene detection. Leptospira genospecies, Mycobacterium and Propionibacterium spp., Chlamydia spp., Legionella pneumophila and Listeria monocytogenes have all been detected and in some cases quantitated with the use of real-time PCR assays [41,[234][235][236][237][238][239][240][241][242][243][244][245][246].…”

Section: Bacteriamentioning

confidence: 99%

Real-time PCR in the microbiology laboratory

Mackay

2004

Clinical Microbiology and Infection

651

338

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Use of PCR in the field of molecular diagnostics has increased to the point where it is now accepted as the standard method for detecting nucleic acids from a number of sample and microbial types. However, conventional PCR was already an essential tool in the research laboratory. Real-time PCR has catalysed wider acceptance of PCR because it is more rapid, sensitive and reproducible, while the risk of carryover contamination is minimised. There is an increasing number of chemistries which are used to detect PCR products as they accumulate within a closed reaction vessel during real-time PCR. These include the non-specific DNA-binding fluorophores and the specific, fluorophore-labelled oligonucleotide probes, some of which will be discussed in detail. It is not only the technology that has changed with the introduction of real-time PCR. Accompanying changes have occurred in the traditional terminology of PCR, and these changes will be highlighted as they occur. Factors that have restricted the development of multiplex real-time PCR, as well as the role of real-time PCR in the quantitation and genotyping of the microbial causes of infectious disease, will also be discussed. Because the amplification hardware and the fluorogenic detection chemistries have evolved rapidly, this review aims to update the scientist on the current state of the art. Additionally, the advantages, limitations and general background of real-time PCR technology will be reviewed in the context of the microbiology laboratory.

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“…PCR has long been the preferred method for diagnosis of bacteria that are diYcult to cultivate, and many real-time PCR assays have already been developed to replace standard PCR methods for bacteria such as Listeria monocytogenes [121], Legionella pneumophila [122], Mycobacterium spp. [123][124][125][126], Propionibacterium spp. [127], Borrelia burgdorferi [128], and Leptospira genospecies [129].…”

Section: Bacterial Agentsmentioning

confidence: 99%