Comparison of amplified Q beta replicase and PCR assays for detection of Mycobacterium tuberculosis.

Journal of Clinical Microbiology
Q AnD M Olive

Abstract

Because of the long time required to isolate Mycobacterium tuberculosis in culture, there is an acute need for simple rapid methods for direct detection of M. tuberculosis from human sputum specimens. We have developed and characterized quantitative manual Q beta replicase and PCR assays for M. tuberculosis. The Q beta replicase assay was based on reversible target capture of M. tuberculosis 23S rRNA followed by amplification of a replicatable detector probe with Q beta replicase. For PCR assays, primers generating a 370-bp amplification product from the IS6110 insertion element were used in combination with a control plasmid containing an internal deletion in the IS6110 amplicon. Serial dilutions of M. tuberculosis were spiked into sputum and subjected to digestion and decontamination with N-acetyl-L-cysteine and NaOH. Assay conditions were optimized for hybridization and sample processing chemistries in order to maximize sample utilization. Following assay optimization, the sensitivities of the Q beta replicase and PCR assays of spiked sputum samples were 0.5 and 5.0 CFU per assay reaction, respectively. The effects of sputum matrix on each assay were examined by testing 20 patient sputum samples which had been cultured for M...Continue Reading

References

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Citations

Mar 31, 2010·PLoS Neglected Tropical Diseases·Wenjun LiDidier Raoult
Jan 1, 1996·Annual Review of Microbiology·A C Whelen, D H Persing
May 1, 1997·APMIS : Acta Pathologica, Microbiologica, Et Immunologica Scandinavica·H Soini, M K Viljanen
Aug 5, 2009·Molecular Diagnosis & Therapy·Seetha V BalasinghamTone Tønjum
Dec 29, 2017·European Journal of Clinical Microbiology & Infectious Diseases : Official Publication of the European Society of Clinical Microbiology·J S ShahR Ramasamy

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