Microwell hybridization assay for detection of PCR products from Mycobacterium tuberculosis complex and the recombinant Mycobacterium smegmatis strain 1008 used as an internal control

Abstract

A microwell hybridization assay was developed for the detection of the PCR products from both Mycobacterium tuberculosis complex bacteria and the recombinant Mycobacterium smegmatis strain 1008 that is used as an internal control to monitor inhibition in the PCR based on the M. tuberculosis complex-specific insertion sequence IS6110. The test is based on specific detection with digoxigenin-labeled oligonucleotide probes of biotinylated PCR products which are captured in a microtiter plate coated with streptavidin. The captured PCR products are hybridized separately with two probes, one specific for the PCR product from IS6110 from M. tuberculosis complex and the other specific for the PCR fragment from the modified IS6110 fragment from the recombinant M. smegmatis 1008. The microwell hybridization assay discriminates perfectly between the two types of amplicon. The amount of PCR product that can be detected by this assay is 10 times less than that which can be detected by agarose gel electrophoresis. The test can be performed in 2 h. It is much faster and less laborious than Southern blot hybridization. Furthermore, the interpretation of results is objective. The assay was used with 172 clinical samples in a routine microbiology laboratory, and the results were in complete agreement with those of agarose gel electrophoresis and Southern blot hybridization.

Performance of a molecular assay to detect Mycobacterium tuberculosis complex DNA in clinical specimens: multicenter study in Brazil

“…Furthermore, the Detect-TB assay is similar to an ELISA as it requires common laboratory equipment to reference laboratories from middle income countries with a high burden of TB and HIV, and it has the advantage of being a low test cost (US$ 10,00), when compared to other current molecular commercialised assays. In addition, the assay results, which are based on spectrophotometry, are independent of human interpretation, instead of visual interpretation colorimetric assays ( Kox et al 1996) .…”

Diagnostic Evaluation of Tuberculosis

“…Several techniques based on polymerase chain reaction (PCR) and isothermal amplification assay have been developed [7][8][9][10]12]. Various researchers have described the rapid detection of M. tuberculosis by PCR, and many have reported a high sensitivity in detecting M. tuberculosis in clinical samples by means of DNA amplifications [7,14]. Such techniques involve amplification of specific gene regions followed by hybridization with species specific primers, and also frequently followed by sequencing and or restriction fragment length polymorphism (RFLP) analysis [12].…”

Multicentre quality control study for detection of Mycobacterium tuberculosis in clinical samples by nucleic amplification methods

“…The sputum samples were shaken vigorously for 4 h in a plastic bottle and frozen at − 20 °C until use. Aliquots of the sputum pools were analysed by standard methods for culture and in‐house PCR to ensure that they were free from viable mycobacteria and DNA from the M. tuberculosis complex or other mycobacteria [4–6].…”