© Cepheid – Proprietary and Confidential

What is the principle behind the assay?

Presented By : BIMAN CHANDRA DEY Territory Business Executive - Diagnostics

Principle

1h52 min

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.

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

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

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.

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

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

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

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…

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

MTB Result Ct (Threshold Cycle)

High < 16

Medium 16 -22

Low 22 - 28

Very low > 28

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

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

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

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

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

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

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