A more reliable PCR for detection of Mycobacterium tuberculosis in clinical samples

Abstract

Diagnostic techniques based on PCR have two major problems: false-positive reactions due to contamination with DNA fragments from previous PCRs (amplicons) and false-negative reactions caused by inhibitors that interfere with the PCR. We have improved our previously reported PCR based on the amplification of a fragment of the Mycobacterium tuberculosis complex-specific insertion element IS6110 with respect to both problems. False-positive reactions caused by amplicon contamination were prevented by the use of uracil-Nglycosylase and dUTP instead of dTTP. We selected a new set of primers outside the region spanned by the formerly used primers to avoid false-positive reactions caused by dTTP-containing amplicons still present in the laboratory. With this new primer set, 16 copies of the IS6110 insertion element, the equivalent of two bacteria, could be amplified 1010 times in 40 cycles, resulting in a mean efficiency of 77% per cycle. To detect the presence of inhibitors of the Taq polymerase, which may cause false-negative reactions, part of each sample was spiked with M. tuberculosis DNA. The DNA purification method using guanidinium thiocyanate and diatoms effectively removed most or all inhibitors of the PCR. However, this was not suitable for blood samples, for which we developed a proteinase K treatment followed by phenol-chloroform extraction. This method permitted detection of 20 M. tuberculosis bacteria per ml of whole blood. Various laboratory procedures were introduced to reduce failure or inhibition of PCR and avoid DNA cross contamination. We have tested 218 different clinical specimens obtained from patients suspected of having tuberculosis. The samples included sputum (n = 145), tissue biopsy samples (n = 25), cerebrospinal fluid (n = 15), blood (n = 14), pleural fluid (n = 9), feces (n = 7), fluid from fistulae (n = 2), and pus from a wound (n = 1). The results obtained by PCR were consistent with those obtained with culture, which is the "gold standard." We demonstrate that PCR is a useful technique for the rapid diagnosis of tuberculosis at various sites.

Development of improved PCR to prevent false positives and false negatives in the detection ofTetracapsula bryosalmonae, the causative agent of proliferative kidney disease

“…Sensitivity of reactions, including the detection of the 548 bp mimic for highlighting false negatives, was estimated at five target copies, two times less than reactions without mimics. The mimic was over double the size of the target and could not compete for raw materials at high numbers of target copies, an effect also demonstrated by Kox et al (1994).…”

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.…”

Identification of Mycobacterium spp. isolated from snakehead, Channa striata (Fowler), and Siamese fighting fish, Betta splendens (Regan), using polymerase chain reaction–reverse cross blot hybridization (PCR–RCBH)

“…The latter can be a significant problem as it can be re-amplified with the same set of primers, thus yielding a false-positive reaction. False positive reactions were controlled in this study by adding the enzyme uracil DNA glycosylase into the PCR mixture in order to prevent any contaminating amplicons from previous experiments (Kox et al 1994). The method of DNA preparation is crucial to reduce inhibitors, which may be present in the sample.…”

“…Deoxyribonucleic acid prepared from reference strains M. marinum (NCIMB1297), M. fortuitum (NCIMB1294) and M. chelonae (KIT4358) was diluted to 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 and 10 -11 g DNA mL -1 , with TE buffer; these weights correspond to 2 · 10 8 , 2 · 10 7 , 2 · 10 6 , 2 · 10 5 , 2 · 10 4 and 2 · 10 3 mycobacteria mL -1 , respectively (Kox et al 1994(Kox et al , 1995. Ten lL of each DNA solution was added to 40 lL PCR mixture and amplified as described above.…”