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As genomics makes progress, there arise an increasing number of ingenious innovations that should replace old systems and methodologies for elevated efficiency and sensitivity. In the field of infection, we witness one of the recent examples.Tuberculosis claims more than 3 million death in a year world-wide and in some underdeveloped countries, it is a more serious disease than AIDS. In addition to its pandemic nature, multi-drug resistant causative agent, Mycobacterium tuberculosis, has been emerging to lead to high mortality rates. Antibiotic susceptibility tests are used to guide therapy, but conventional testing may take 2 to 8 weeks to let many infected patients die. Further, infected areas are not always equipped with adequate facilities.
Approximately 96% of rifampin-resistant strains have the mutation (H526Y) in a single 81-bp core region of the rpoB gene (Telenti A et al. 1993: uid=93196283) and susceptible strains are always wild type in this region of the gene. Several methodologies are targeted at this region to detect drug resistance of M. tuberculosis isolates but they require considerable time, expertise and/or high cost. Piatek AS et al (1998: uid=98216568) now report molecular beacon sequence analysis for detecting drug resistance in closed tubes where real-time PCR is performed and visual inspection for rpoB gene mutation is possible under UV.
Molecular beacons are a new type of reporters designed to detect short regions of DNA accurately, rapidly and simply. Molecular beacons used here are single stranded DNA (25- to 30-mer) that possesses a loop complementary to the target sequence (H526Y mutation) of rpoB gene and complementary stems attached to 5' and 3' ends. Tetramethylrhodamine (fluorescing red), fluorescein (fluorescing green), or other colors are coupled to the 5' stem, whereas a quencher, 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL) is attached to the 3' stem. Thus the molecular beacons get colored upon hybridization to the target codon 526-containing region. The color may be detected under UV as cDNA is synthesized by PCR in real time. Piatek AS et al (1998: uid=98216568) typically used the following molecular beacons (C to T mutation at the first base of codon 526 thus G to A in the probe):
H526Y mutant probe: fluoresein-5'-CCACGGCGCTTGTAGGTCACGTGG-3'-DABCYL
Wild type probe: TMrhodamine-5'-CCACGCTTGTGGGTCAACCCCCGTGG-3'-DABCYL
In a blinded study of 52 rifampin-resistant and 23 susceptible clinical isolates, this method correctly detected mutations in all of the resistant strains and in none of the susceptible strains. The assay was entirely in sealed PCR tubes and simple to perform and interpret. It is reasonably inexpensive to enable field tests for rapidly assessing M. tuberculosis drug resistance in hours, instead of days to weeks.
As a matter of fact, molecular beacons described here can detect change in any DNA sequence of moderate length with single base pair accuracy that similar molecular beacons should be used to detect common mutations for physiologic aberrations and pathogenesis.
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