The overall goal of this procedure is to simultaneously detect within two hours mycobacterium, tuberculosis and rifampicin resistance in expectorated sputum, or concentrated sputum sediments. Begin with a simple, highly effective sample decontamination protocol. Then transfer each processed sample to a single use disposable expert, M-T-B-R-I-F cartridge that holds PCR reagents and hosts the PCR process.
Load the cartridges onto a gene expert DX system and start the ultra sensitive HEMI nested PCR for the simultaneous detection of mycobacterium tuberculosis and rifampicin resistance. Ultimately, read the test results and report them to the physician. The expert MTPR assay is intended for use with specimens from patients for whom there is clinical suspicion of pulmonary tuberculosis and who fulfill the criteria outlined in the compan text.
Do note that it should not be used for monitoring the effects of drug therapy.Crystal. A technician from my laboratory will now demonstrate the protocol As per standard two of the international standards for tuberculosis care. Collect two specimens for bacteriological examination from each patient suspected of having pulmonary tb.
Allocate one gene expert DX system module for each test specimen labeling each cartridge by specimen id. Now for each sample assay, transfer one milliliter of sputum to a 15 milliliter conical screw capped tube with another sterile transfer pipette. Add two milliliters of M-T-B-R-I-F sample reagent.
Replace the lid vigorously. Shake the tube 10 to 20 times. Then stand the tube upright for five minutes at room temperature after vigorously shaking the tube.
Again, equilibrate at room temperature for 10 minutes. Verify that all sample specimens are ED with no visible clumps of sputum First label cartridges with specimen ID of each sputum sediment sample to each sediment sample, add 1.5 milliliters of M-T-B-R-I-F sample reagent, and to transfer 0.5 milliliters to a 15 milliliter conical screw capped tube. Replace the lid vortex for 30 seconds and set the tube upright for five minutes after a second.
Mixing by vortex, stand the tube upright for 10 minutes. Inspect the liquified samples to verify absence of clumps of sputum. Match the label on the tubes with the corresponding cartridges.
Do not open the cartridge until you are ready to perform testing and use the cartridge within 30 minutes of opening the cartridge lid. Using the sterile transfer pipette, aspirate the liquified specimen until the meniscus is above the minimum mark. Load the sample slowly into the cartridge.
Close the cartridge lid. Start the test within 30 minutes of adding the sample to the cartridge. First switch on the computer and then the instrument on the desktop.
Double click the gene expert DX icon. Log on to the system software. Select create test to open the scan cartridge barcode dialogue box.
Then type in the sample ID and scan the barcode on the cartridge. When the create test window appears, click start test and enter the password in the dialogue box. Now open the instrument module door and load the cartridge.
Close the door to start the test. When the green light turns off, the test is complete. Once the system releases the door lock, open the module door and remove the cartridge.
Finally, dispose of used cartridges in the appropriate waste containers. Each test cartridge includes reagents for QPCR of Mycobacterium tuberculosis complex rifampicin resistance, and the appropriate experimental controls. The probe check control verifies reagent rehydration PCR tube filling in the cartridge probe integrity and dye stability.
The sample processing curve serves as a positive control for adequate processing of the target bacteria and also indicates the presence of any PCR inhibitors. The primers are designed to differentiate between the conserved wild type sequence and mutations in the core region of the RPOB gene associated with RIF resistance. The CT values are inversely proportional to the concentration of target M-T-B-D-N-A template in the test sample.
The test result dialogue box concludes MTB target DNA is detected and RIF resistance not detected in this patient sample. The MTB target DNA is detected in low levels with indication of rifampicin resistance. Both controls SPC and PCC meet the assigned acceptance criteria.
This assay result is deemed invalid as the SPC is negative and PCR products are absent. Either the sample was not properly processed or PCR was inhibited. Repeat the assay.
As you have seen on the video testing, is standardized and requires only moderate laboratory infrastructure and training. Therefore, I believe that the expert M-T-B-R-F assay has the potential to bring standardized, sensitive and very specific diagnostic testing for both tuberculosis and rifampicin resistance to universal access point of care settings.
