Visual Format for Detection of Mycobacterium tuberculosis and M. bovis in Clinical Samples Using Molecular Beacons

A real-time polymerase chain reaction (PCR) assay for the direct identification of Mycobacterium tuberculosis and M. bovis using molecular beacons was developed. The assay was modified for use in regular thermal cyclers. Molecular beacons that were specific for M. tuberculosis (Tb-B) and M. bovis (Bo-B) were designed. The fluorescence of the target PCR product-molecular beacon probe complex was detected visually using a transilluminator. The results were then compared with those of conventional multiplex PCR (CM-PCR) assays and biochemical identification. The detection limit of Tb-B and Bo-B beacons was 500 fg and 50 fg by the visual format and real-time PCR assay, respectively, compared with 5 pg by CM-PCR assay. Pulmonary and extrapulmonary samples were examined. The agreement between culture and the two assays was very good in sputum samples and fair in extrapulmonary samples. The agreement between clinical diagnoses with the two assays was moderate in extrapulmonary samples. There was very good agreement between CM-PCR and visual format assays for all samples used in the study. Concordance in the identification of isolates by the visual, CM-PCR assay, and biochemical identification was seen. Hence, the use of molecular beacon detection of M. tuberculosis and M. bovis in clinical samples is feasible by setting up two asymmetric PCRs concurrently. The assay is sensitive, specific, simple to interpret, and takes less than 3 hours to complete.For effective treatment of tuberculosis (TB), rapid and accurate diagnosis is essential. Conventional polymerase chain reaction (PCR)-based assays designed to detect Mycobacterium tuberculosis that involve electrophoresis based analysis of amplicons are relatively fast but are laborious and the potential risk of inadvertent dispersal of amplicons leading to contamination of untested samples exists. To overcome these difficulties a number of probes, including TaqMan probes, molecular beacons, and side-by-side probes, that can report the amplification of the correct amplicon in sealed tubes have been developed.^1,2,3 Several different instruments that measure increase in fluorescence of these probes while simultaneously performing amplification have also become available. Although such real-time PCR assays are more quantitative than conventional ones, the instruments used are not commonly available in resource-poor locations. Since such localities are often where tuberculosis is more prevalent, it is important to develop sealed tube assays that can be implemented on conventional PCR machines but have the simplicity and accuracy of probe-based detection.In the previously developed tests different coding and intergenic regions from the M. tuberculosis genome, such as devR, IS6110, IS986, RNA polymerase gene, and ribosomal RNA gene have been used as targets for amplification using molecular beacons or fluorescent probes.^4,5,6,7 One of the limitations of these tests is that they have exclusively focused on M. tuberculosis. However, other closely related bacteria such as M. bovis have been often associated clinically with human and bovine samples in practice.^8,9,10 Therefore an ideal test should distinguish between M. tuberculosis and M. bovis. We used the mce3 operon that has a differential organization in the M. tuberculosis and M. bovis genome as a suitable target for the assay.¹¹To develop a simple sealed tube test that can reliably detect M. tuberculosis in clinical samples using conventional PCR instrument for amplification and a common lamp for transillumination, we used molecular beacons as probes. However, the signal intensity in PCRs performed with molecular beacons is often limited because the two product strands of the amplicon bind to each other excluding the probe from the target strand. We improved the fluorescence intensity of these reactions by performing asymmetric PCR that produced more of the molecular beacon target strand than of the opposite strand. The optimizations were performed using a real-time instrument. We show that our assay is robust, specific, sensitive, and can be completed in less than 3 hours. The presence of mycobacteria in the sample can be conclusively established by visualizing the fluorescent amplicon in a blue light transilluminator by the naked eye. We also compared and correlated our endpoint visual format assay with acid-fast bacilli (AFB) smear microscopy; a gel-based conventional multiplex PCR (CM-PCR) assay, culture, and clinical diagnosis. The simplified visual format assay was found to be an accurate predictor of the presence of M. tuberculosis and M. bovis in clinical samples.