Amplification and Detection of MTB and MTB-Rif

Procedure

Equipment and reagents

• Truelab Uno/ Duo/ Quattro Real Time Quantitative micro–PCR analyzer
• Truenat MTB micro-PCR kit
• Truenat MTB Rif micro-PCR kit
• Truepet fixed volume (6 μl) precision micropipette

Amplification and detection of Mycobacterium tuberculosis (MTB)

1. Wear gloves, laboratory coats, mask.
2. Clean working surfaces with 1% Sodium hypochlorite, followed by 70% alcohol.
3. Clean instruments with a paper towel dipped in 70% alcohol.
4. Switch on the Truelab device by pressing the red button for two seconds.
5. Select test bay.
6. Select test profile: “MTB”, enter Details (Referred By, Patient ID, Gender, Patient Name, Age).
7. Select the sample type.
8. Press “Start Reaction". For Truelab Uno Dx, Press the eject button to open the chip tray. For Truelab Duo/Quattro, the chip tray opens automatically on tapping the “Start Reaction” .
9. Take out “Truenat MTB chip” from the chip sleeve (check desiccant colour. If blue -chip can be used; discard chip if desiccant is pink/ white which indicates moisture is absorbed).
10. Place the chip on the tray by aligning the registration holes with tray pins without touching the white reaction well.  (the white reaction well should face upward).
11. Open the microtube containing the freeze-dried PCR reagent (white colour) and place it on the microtube stand.
12. Pipette 6 μl DNA from the Extraction Chamber (After DNA extraction and purification step)  into the microtube.
13. Wait 30-60 seconds for the DNA elute and PCR reagent to mix (clear solution obtained).
14. Use the same tip to pipette 6 μl treated DNA from the microtube and load on the white reaction well of the chip.
15. Discard the microtube and microtip (1% sodium hypochlorite).
16. Start the test run.
17. The test completes in 35 minutes, press ‘’RESULT’’ to view the result screen.
18. Possible results:
    • MTB Detected/ Not Detected/ Errors/ Invalid
19. If MTB detected, test the same DNA eluate for Rif resistance using MTB-Rif chip. Select the MTB-Rif assay.
20. If the result is Invalid/ Error, repeat amplification using the same extracted DNA and a new “Truenat MTB chip”. If Invalid again, repeat the test with a fresh sample.
21. Tap “Open/ Close Tray” button to eject the chip tray.
22. Lift the chip and discard (1% sodium hypochlorite).

Amplification and Detection of MTB Rif

1. DNA from the MTB positive elutes is tested for Rif-resistance using the MTB-Rif chip.
2. Select the test profile “MTB Rif”.
3. Select the sample type.
4. Press “Start Reaction". For Truelab Uno Dx, Press the eject button to open the chip tray. For Truelab Duo/Quattro, the chip tray opens automatically on tapping the “Start Reaction”.
5. Take out “Truenat MTB Rif Dx chip” from the chip sleeve (check desiccant colour. If blue -chip can be used; discard chip if desiccant is pink/ white which indicates moisture is absorbed).
6. Follow steps 10-16 as described in Amplification and detection of MTB (above).
7. The test completes in 55 minutes, press ‘’RESULT’’ to view the result screen.
8. Possible results:
   • MTB Rif resistance detected/ MTB Rif resistance not detected/ Error/ Indeterminate.
9. If result is Indeterminate/ Error, repeat the amplification using the same extracted DNA and new “Truenat MTB Rif Dx chip”. If Indeterminate/ Error again, repeat the test with a fresh sample.
10. Tap “Open/ Close Tray” button to eject the chip tray.
11. Lift the chip and discard (1% sodium hypochlorite).

The algorithm for Truenat MTB Assay (Figure) describes interpretation of test results obtained for Truenat MTB and Truenat MTB Rif Dx assays.

Figure: Truenat MTB Assay Results Interpretation: Algorithm; Source: Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance

# Test is Valid as Internal Positive Control amplified

Resources

• Practical Guide to Implementation of TrueNAT Tests for the Detection of TB and Rifampicin Resistance.
• Truenat MTB Pack Insert.
• Truenat MTB Rif Pack Insert.

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