Xpert- MTB-RIF Data _Biman
Transcript of Xpert- MTB-RIF Data _Biman
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© Cepheid – Proprietary and Confidential
What is the principle behind the assay?
Presented By : BIMAN CHANDRA DEY Territory Business Executive - Diagnostics
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Principle
1h52 min
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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.
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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
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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
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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.
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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
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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
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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
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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…
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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
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MTB Result Ct (Threshold Cycle)
High < 16
Medium 16 -22
Low 22 - 28
Very low > 28
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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
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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
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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
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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
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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
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All Probes are NEGATIVE Only SPC is Positive Hence MTB is Not Detected
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All Probes are NEGATIVE Also SPC has Failed Reason- PCR Inhibitor in sample