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Thursday, August 4, 2011

Hybridization and automated Molecular Methods for Laboratory assessment of Mycobacterium tuberculosis


lRapid detection and identification of Mycobacterium tuberculosis (M. tuberculosis) complex  in respiratory samples are extremely important for optimal diagnosis and effective treatment, as well as for prevention and control of tuberculosis transmission
lDiagnostic means of tuberculosis (TB), have considerably improved. Available standardized nucleic acid-based amplification techniques were shown to yield reliable results within 5 to 7 hours of sample processing.
lIn spite of their theoretical ability to detect even a single mycobacterial cell, nucleic acid amplification tests (NAATs) are not sufficiently reliable to replace conventional diagnostic methods for pulmonary tuberculosis (TB)
l Both inherent test characteristics and errors in testing procedures may account for their inaccuracy
lOn the other hand, false positive results arise most often from contamination of negative samples with either organisms or target DNA from samples containing large numbers of mycobacteria or from amplicons contaminating the laboratory room
lTo overcome these problems, automated commercial systems were developed which were made more robust by the use of standardized procedures and reagents for sample processing, amplification, and detection.
lOn the other hand, false positive results arise most often from contamination of negative samples with either organisms or target DNA from samples containing large numbers of mycobacteria or from amplicons contaminating the laboratory room
lTo overcome these problems, automated commercial systems were developed which were made more robust by the use of standardized procedures and reagents for sample processing, amplification, and detection.
lThese procedures, which allow different steps of the process to take place in a single sealed tube, were intended to reduce the risk of contamination.
lAt the same time, the use of larger sample volumes or the introduction of internal amplification controls to detect inhibitors was adopted to cut down the rate of false negative results
lThe US Centers for Disease Control (CDC) recommend that commercial NAATs be used with microscopy to improve diagnostic certainty (pending culture results and/or patient’s response to treatment) and that clinicians should rely on their clinical judgment in the interpretation of results.
lAccording to the CDC, the diagnosis of pulmonary TB can be presumed in smear positive (acid-fast bacilli (AFB)+) patients with a positive NAAT result and in smear negative (AFB–) patients with two subsequent positive NAAT results.
lHere we described  our experience in Egypt for diagnosis of Mycobacterium tuberculosis with molecular techniques
lWe use  fully automated PCR system that use seminested PCR  (Acu-Gene M. tuberculosis quantitative test ) for determination of Mycobacterium tuberculosis determine positive clinical samples with low level of bacilli doses.
lAlso we asses the presence of rifampicin resistant tuberculosis strains by cultural method by BACTEC 460 system and   proportional agar method compared to determination  the type of  mutation pattern of rpoB gene in those strains by line probe  assay  and DNA sequence analysis
 Acu-Gene M. tuberculosis quantitative test (Amplisen­sor assay for quantitative TB)
lDNA Amplisensor assay for M. tuberculosis is based on the principle of amplisensor  assay to determine the initial target ­dosage of M. tuberculosis through PCR amplification.
lThe test involves a two-stage amplification scheme: (1) an initial asymmetric amplification to overproduce one strand of the target sequence and (2) a subsequent heminested amplification to monitor AmpliSensor (a fluorogenic primer duplex) as it is converted into amplification product.
lThe assay quantifies the amount of the target sequence by correlating the initial target dosage to a “transition” cycle number, which marks the beginning of signifi­cant AmpliSensor conversion. To prevent false negative, the assay calls for a polymerase activity check all the re­agents for sample preparation and complete reaction mixes for amplification and detection.
l All the procedures of assay set up and reagent preparation should be per­formed in an UV irradiated clean hood.
 Line probe assay
lTest principle: It is based on amplification of the rifampicin resistance region of gene encoding for the subunit of RNA polymerase (rpoB). Amplified biotinylated DNA material was hybridized with specific oligonucleotide probes immobilized as parallel lines on membrane based strips.
l After hybridization, streptavidin labeled with alkaline phosphatase was added and bound to any biotinylated hybrid previously formed. Incubation with BCIP/NBT chromogen resulted in purple/brown precipitate.
lThe presence of MTB complex in the sample was monitored by MTB complex-specific probe. The reactivity of an amplified fragment with one or more of the 5 wild type probes (Sl to S5) was prevented by the presence of a mutation within the probe region. Four additional probe; R2, R4a, R4b and R5 were expected to hybridize to mutant sequences of the most commonly observed mutations
 
 
 

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