The Xpert MTB/RIF assay uses molecular beacon probes to detect mutations in the rpoB gene associated with rifampin resistance. It contains five probes that bind to the wild-type rpoB sequence and one probe for an internal sample processing control. If one of the five probes does not bind, it indicates a mutation and rifampin resistance. The assay interprets results based on cycle threshold values and differences in values between probes to determine MTB detection level and presence of resistance. Internal controls verify reagent rehydration, tube filling, and detect inhibition to help prevent false negatives.

1. © Cepheid – Proprietary and Confidential
What is the principle
behind the assay?
Presented By :
BIMAN CHANDRA DEY
Territory Business Executive - Diagnostics

2. Principle
1h52 min

3. Intended Use
• Product Code : GXMTB/RIF-10
• Detection of Mycobacterium tuberculosis complex DNA and the detection
of rifampin-resistance
• Semi Quantitative in vitro diagnostic test
• Specimen :
– Sputum samples or concentrated sediments prepared from induced or
expectorated sputa
• Detect
– Portion of the rpoB gene. 5 probes are able to differentiate between
the conserved wild-type sequence and mutations
– SPC
• 10 cartridges per Kit.

4. PAGE | 4
Molecular
Beacon
Target
Hybrid
The Xpert MTB/RIF Molecular Beacon Assay
5-Probes bind to wild type (do not bind to
mutant sequence)
1-Probe for SPC (B. globigii)
6-fluorescent dyes detected simultaneously
Delta Ct Max 5
rpoB gene

5. Common rpoB mutations
● Rifampin binds to the beta
subunit of the RNA
polymerase, preventing
transcription.
● Mutations in the rpoB gene
prevent the binding of
rifampin.
● As documented by WHO,
RIF resistance is most
commonly seen in multi-drug
resistant (MDR-TB) strains
and has a reported frequency
of greater than 95% in such
isolates

6. Controls
Cepheid designed specific molecular methods to include internal controls that enable the
system to detect specific failure modes : Two elements of the molecular design were
created to address all the key failure modes that could result in a false negative result :
- Probe Check Control  Positive Control
- Specimen Processing Controls  Internal Control, and much more.
- DNA Négative Control (NC)  No need! No wet interface between instrument and cartridge , 1
isolated cartridge for 1 sample only.
ATCC strains can be used as External Positive & Negative Controls:
- External Positive Controls : QC organisms such as ATCC 27290 as BCG Copenhagen
- External Negative Controls : ATCC SmT Mc2 2500 as Mycobacterium avium
Or ATCC 35790 or 35771 as Mycobacterium intracellular
External controls should be used in accordance with local, state accrediting organizations, as applicable.

7. Probe Check Control
What is it?
• Just prior to starting thermal cycling, multiple fluorescence
readings at different temperatures are made.
• The results are automatically compared to pre-established
factory settings in the software.
What does it do?
Probe check control verifies
• Bead re-hydration
• PCR tube filling
• Probe integrity
• Dye or quencher instability

8. Sample Processing Control (SPC)
• Intact organisms, DNA
• Detection probe: A fluorescently-labeled hybridization probe with different color
reporter dye than that of the target probe(s) or the internal control (IC); present in
primer-probe bead
• Primers: Can be the same as those for the target or IC or can be unique; present in
primer-probe bead
What does it do?
•Co-processed with target organism through entire sample prep process
•Co-amplified with targets
-The SPC assesses the effectiveness of the sample processing steps, including and up to
reaction tube filling
-Detects degradation of enzyme(s) or other components of system
-Detects sample inhibition

9. Specimen Processing Control (SPC)
• The SPC assesses the effectiveness of the sample
preparation steps, including reaction tube filling
• The SPC controls for:
– Missing primer/probe or enzyme beads
– Incomplete reagent reconstitution
– Incomplete reaction tube fill
– Enzyme degradation
– Specimen processing
– Sample inhibition

13. Simple Sample Processing – Direct Sputum
1. Add 2:1 Sample
Buffer to sample
2. Shake then stand 10 minutes
3. Shake then stand
further 5 minutes
4. Transfer 2ml
to cartridge
Begin Test…

14. Simple Sample Processing – Processed Deposit
2. Add 1.5ml Sample
Reagent to 0.5ml
deposit
3. Shake then stand
10 minutes
1. Inoculate media
prepare smear from
deposit
5. Transfer the
2ml to
cartridge
Begin Test…
4. Shake then stand
further 5 minutes

15. MTB Result Ct (Threshold Cycle)
High < 16
Medium 16 -22
Low 22 - 28
Very low > 28

18. All 5 Probes are Positive
Delta Ct=Highest Ct- Lowest Ct is 1.4 ≤ 4
(15.9 -14.5 )
Hence RIF Resi NOT DETECTD
Lowest Ct is 14.5 which is < 16
Hence HIGH

19. Only 4 Probes are Positive
One Probe is Negative
Hence RIF Resistance DETECTD
Lowest Ct is 21.9 which is between 16 - 22
Hence MEDIUM

20. All 5 Probes are Positive
Delta Ct =Highest Ct- Lowest Ct is which is 2.2 ≤ 4 (25.4 -23.2)
Hence RIF Resi NOT DETECTD
Lowest Ct is 23.2 which is between 22 - 28
Hence LOW

21. All 5 Probes are Positive
Delta Ct =Highest Ct- Lowest Ct is 3 which is ≤ 4 (32.4 -29.4)
Hence RIF Resi NOT DETECTD
Lowest Ct is 29.4 which is above 28
Hence VERY LOW

22. All 5 Probes are Positive
Delta Ct =Highest Ct- Lowest Ct is 3 which is ≤ 4 (32.4 -29.4)
Hence RIF Resi NOT DETECTD
Lowest Ct is 29.4 which is above 28
Hence VERY LOW

23. All Probes are NEGATIVE
Only SPC is Positive
Hence MTB is Not Detected

24. All Probes are NEGATIVE
Also SPC has Failed
Reason- PCR Inhibitor in sample