Abstract
Molecular analyses are auxiliary tools for detecting Koch's bacilli in clinical specimens from patients with suspected tuberculosis (TB). However, there are still no efficient diagnostic tests that combine high sensitivity and specificity and yield rapid results in the detection of TB. This study evaluated single-tube nested polymerase chain reaction (STNPCR) as a molecular diagnostic test with low risk of cross contamination for detecting Mycobacterium tuberculosis in clinical samples. Mycobacterium tuberculosis deoxyribonucleic acid (DNA) was detected in blood and urine samples by STNPCR followed by agarose gel electrophoresis. In this system, reaction tubes were not opened between the two stages of PCR (simple and nested). STNPCR demonstrated good accuracy in clinical samples with no cross contamination between microtubes. Sensitivity in blood and urine, analyzed in parallel, was 35%-62% for pulmonary and 41%-72% for extrapulmonary TB. The specificity of STNPCR was 100% in most analyses, depending on the type of clinical sample (blood or urine) and clinical form of disease (pulmonary or extrapulmonary). STNPCR was effective in detecting TB, especially the extrapulmonary form for which sensitivity was higher, and had the advantage of less invasive sample collection from patients for whom a spontaneous sputum sample was unavailable. With low risk of cross contamination, the STNPCR can be used as an adjunct to conventional methods for diagnosing TB.
Highlights
Molecular analyses are auxiliary tools for detecting Koch’s bacilli in clinical specimens from patients with suspected tuberculosis (TB)
One of these is polymerase chain reaction (PCR), which has been applied to the detection of M. tuberculosis(4) (5) (7) (8) (10) (11) (12) (13) at concentrations of < 10 cells/mL in clinical samples(7) (14) . (15) Nested PCR is a variation of this technique that involves two amplification steps(7) (8) (13) (16), (17) yielding greater sensitivity and specificity(8) (16) . (17) Single-tube nested polymerase chain reaction (STNPCR) is more rapid than conventional nested PCR(8) (14) with less probability of cross-contamination and requiring a smaller amount of reagent
Various studies have reported the molecular diagnosis of TB using extrapulmonary samples, including in cases of negative acidfast bacillus (AFB) results(12) (13) . (19) The present study evaluated the performance of single-tube nested polymerase chain reaction (STNPCR) in detecting the presence of M. tuberculosis complex in blood and urine using an in-house deoxyribonucleic acid (DNA) extraction method from patients with suspected TB
Summary
Molecular analyses are auxiliary tools for detecting Koch’s bacilli in clinical specimens from patients with suspected tuberculosis (TB). This study evaluated single-tube nested polymerase chain reaction (STNPCR) as a molecular diagnostic test with low risk of cross contamination for detecting Mycobacterium tuberculosis in clinical samples. Methods: Mycobacterium tuberculosis deoxyribonucleic acid (DNA) was detected in blood and urine samples by STNPCR followed by agarose gel electrophoresis. In this system, reaction tubes were not opened between the two stages of PCR (simple and nested). One of these is polymerase chain reaction (PCR), which has been applied to the detection of M. tuberculosis(4) (5) (7) (8) (10) (11) (12) (13) at concentrations of < 10 cells/mL in clinical samples(7) (14) . In STNPCR, both amplification reactions occur consecutively and the microtubes do not need to be opened or changed for the addition of new reagents(4) (8)
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