Evaluation of Amplicor‐ and IS6110‐PCR for direct detection of Mycobacterium tuberculosis complex in Singapore

Abstract

SummaryOne hundred and seventy-eight samples from 168 individuals were tested for Mycobacterium tuberculosis complex (Mtc) using Amplicor PCR, IS6110-PCR (in-house), acid fast (AF)-staining and culture. Thirty-one samples were positive by culture, but 37 samples were later resolved to be truly positive for Mtc. Of these, Amplicor detected 32 (86.5%), IS6110-PCR detected 31 (83.6%), and AF-staining 21 (56.8%). None of the 141 Mtc-negative samples was positive by these tests, thus giving 100% specificity. Although the IS6110-PCR was more sensitive than Amplicor in detecting spiked Mtc DNA, it was not more sensitive than the latter in detecting Mtc in clinical samples. Reasons likely to account for the PCR false negativity were (i) sample inoculum size, (ii) nonuniform samples due to clumping effect of Mtc and (iii) the absence of target gene sequences for IS6110-PCR. Culture negativity, on the other hand, was likely to be associated with nonviable Mtc. Amplicor PCR is promising for direct detection of Mtc. The IS6110-PCR, however, may not be as suitable because of possible existence of IS6110-deleted Mtc strain in Singapore.

Molecular Analysis of Sarcoidosis Tissues forMycobacteriumSpecies DNA

“…A second possibility is that M. tuberculosis is present but the strains do not contain IS6110, since strains that possess one copy or no copies of IS6110 have been reported (31,32). In the United States, all of approximately 14,000 strains of M. tuberculosis tested have been shown to possess IS6110; some in low-copy number (33).…”

Evaluation of Sensitivity of Multiplex PCR for Detection of Mycobacterium tuberculosis and Pneumocystis jirovecii in Clinical Samples

“…The assay successfully amplified the positive controls and samples for which nonspecific bands were not observed. The specificity of IS6110 in detecting the M. tuberculosis complex (MTC) using the primer specific sets TB1/TB4 and TB2/ TB3 was confirmed by Se Thoe et al in 1997 (17). These primers did not amplify non-MTC strains (M. fortuitum, M. intracellulare, M. kansasii, M. scrofulaceum, and M. smegmatis).…”

“…P. jirovecii was detected using toluidine blue O stain and immunofluorescence assay (IFA). The results for the samples were confirmed by a PCR specific for each organism (17,19,20). Each sample was recorded as a code number and kept at Ϫ20°C in order to use in a blind study of the multiplex nested PCR.…”

“…PCR amplification was performed using primers described previously (17). The first-round amplification, using primer TB1 as a forward outer primer and TB4 as a reverse outer primer, amplified a 302-bp region of repetitive sequence ( Table 1).…”

“…The reaction mixture consisted of deionized distilled water, Go Taq Flexi buffer, 2 mM MgCl 2 , 0.2 mM each deoxynucleoside triphosphate (dNTP), 25 pmol of each primer, 1.25 U Taq DNA polymerase (Promega), and 1 l DNA template. The protocol for IS6110 amplification was modified from that used in a previous study (17) and was initiated with denaturation at 95°C for 5 min, and then the mixture was subjected to 40 cycles of 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s and had a final extension of 72°C for 5 min.…”