Detection of Mycobacterium tuberculosis in clinical samples by using polymerase chain reaction and a nonradioactive detection system

Abstract

A test based on the polymerase chain reaction (PCR) was developed for the detection of the Mycobacterium tuberculosis complex in clinical samples. In this test, a 245-bp sequence of the insertion element IS986 was amplified and detected by agarose gel electrophoresis in the presence of ethidium bromide and by Southern blot and dot blot hybridization by using a 188-bp digoxigenin-labeled probe. We tested clinical specimens from 227 patients suspected of having tuberculosis. These included 102 cerebrospinal fluid, 48 sputum, 18 pleural fluid, 5 bronchoalveolar lavage, 18 blood, 7 pus, 8 bone marrow, and 6 urine samples and 15 tissue biopsy specimens. We also tested sputum samples from 75 patients with diseases other than tuberculosis. Sputum samples were first decontaminated, and all samples were treated with proteinase K-detergent solution to extract the DNA. Part of each sample was spiked with M. tuberculosis to provide a semiquantitative assay and to control for the loss of mycobacteria or interference with the PCR which may cause false-negative results. One femtogram of M. tuberculosis DNA could be detected. PCR was positive for all 32 culture-positive (for M. tuberculosis) and Ziehl-Neelsen staining (ZN)-positive samples, 10 of 12 culture-positive and ZN-negative samples, and all 4 culture-negative and ZN-positive samples. PCR detected M. tuberculosis complex bacteria in 35 of 178 cultureand ZN-negative samples. Clinical data supported the diagnosis of tuberculosis in the majority of the 35 patients from whom those samples were obtained.

Diagnosis of tuberculosis: Available technologies, limitations, and possibilities

“…The specificity of the PCR amplification process lies in the choice of primers used. Numerous PCR-based assays for the detection and identification of individual mycobacterium species, such as M. tuberculosis, M. leprae, and M. avium, have been described (2,3,(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47). Many target sequences have been used, but the most thoroughly evaluated assays target the M. tuberculosisspecific repeated DNA element IS 6110 (40).…”

“…Globally there are about 4.6 million cases of dual HIV and tuberculosis infection. Significant mortality and morbidity rates have been reported in various parts of the world, including developed as well as developing nations (2,3). Tuberculosis has become a major concern worldwide, and without commitment and action at national and international levels, tuberculosis will claim about 30 million more lives in the next decade and there will be about 90 million new cases of active tuberculosis.…”

Individual Animals of a Cattle Herd Infected with the Same Mycobacterium bovis Genotype Shows Important Variations in Bacteriological, Histopathological and Immune Response Parameters

“…In conclusion, in a herd infected by the same strain, ante-mortem direct and immune-diagnostic parameters change, suggesting that several tests are needed for a faster control of infection at herd level. detect members of the M. tuberculosis complex and is especially useful for the direct detection of M. bovis in bovine tissue samples (Kolk et al, 1992;Kox et al, 1994;Liebana et al, 1995;Wards et al, 1995;Cornejo et al, 1998;Vitale et al, 1998;Zanini et al, 1998Zanini et al, , 2001Romero et al, 1999;Zumárraga et al, 2001Zumárraga et al, , 2005) Eradication of this disease remains an important goal in several countries. The strategy of test and slaughter has been used widely in an attempt to control dissemination of the disease and is based on the tuberculin skin test as a means for BTB diagnosis.…”

Nanoparticles: synthesis and applications

“…In the early 1990s, a diagnostic procedure based on the amplification of the insert sequence (IS) 6110 was developed and soon became prevalent. This method is displaying advantages regarding detection limit and specificity through the amplification of this signature sequence using the PCR technique (Kolk et al, 1992(Kolk et al, , 1998Kox et al, 1994;Sankar et al, 2011;Shukla et al, 2011). However, this procedure requires the time-consuming extraction of DNA from each sample, including a cell lysis step which is usually inefficient on account of the highly complex bacterial cell wall (Noordhoek et al, 1994;Ellis and Zabrowarny, 1993;Ogbaini-Emovon, 2009).…”