Single-tube nested PCR assay with in-house DNA extraction for Mycobacterium tuberculosis detection in blood and urine

Abstract

Introduction: Molecular analyses are auxiliary tools for detecting Koch's bacilli in clinical specimens from patients with suspected tuberculosis (TB). However, there are still no effi cient diagnostic tests that combine high sensitivity and specifi city 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. 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). Results: 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 specifi city 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). Conclusions: 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.

Performance of the IS6110-TaqMan® assay in the diagnosis of extrapulmonary tuberculosis from different biological samples

“…Studies have shown that, despite the low sensitivity of molecular methods on urine samples, when these samples were analyzed in combination with blood samples, the sensitivity was increased by around 8-10% 16,32 . Lima et al 16 evaluated a molecular diagnostic test for extrapulmonary TB in blood and urine samples. The sensitivity of the assay using combined samples (calculated in parallel) was 71.9%, but when using only urine samples, the sensitivity was 40.6%.…”

“…Nowadays, the most promising technique for the rapid diagnosis of TB is based on the polymerase chain reaction (PCR) method [13][14][15][16] . Real-time PCR (qPCR) has an advantage over conventional PCR due to the ability to amplify and detect deoxyribonucleic acid (DNA) simultaneously through a fluorescence system.…”

Analysis of Clinical Factors, Bacterial Genotyping, and Drug Resistance for Spinal Tuberculosis in South-Central China

“…us, identifying drug resistance in STB isolates is vitally important for thorough treatment of such cases. Delayed initial diagnosis and confirmation of STB and the increasing incidence of multidrug-resistant STB in adults drives the need to improve the etiological diagnostics of STB, taking into account the biological and molecular properties of the pathogen [11,12].…”

Rapid detection of Mycobacterium tuberculosis in children using blood and urine specimens

“…The use of polymerase chain reaction (PCR) assays has been proposed in a few studies to increase the sensitivity and specificity of the diagnosis of childhood tuberculosis 6 , 22 . Generally, they have high sensitivities and specificities using clinical samples and produce rapid results 9 , 10 , 23 - 25 . These techniques detect a specific genome DNA region in the microorganism.…”

“…Thus, it is necessary to evaluate a new system for the detection of M. tuberculosis among children, particularly because of the limitations of conventional diagnostic methods. Based on the difficulty of diagnosis of childhood TB, among patients who do not have sputum expectoration, this study proposed the use of non-invasive samples, blood and urine, to detect the DNA of M. tuberculosis by single tube PCR (STNPCR) 23 and evaluated it in comparison with other conventional tests in clinically suspected pulmonary and extrapulmonary TB. Blood and urine can be collected from outpatients regardless of the site of infection 26 .…”