Chapter 6 : GeneXpert Machine

Enabling Objectives:

•By the end of this chapter participants will be able to:

 Explain the working principle and the purpose of PCR machine

 Explain working principles of GeneXpert machines

 Explain the GeneXpert technology

 List down basic parts of GeneXpert machine

 Practice safety precautions needed on GeneXpert machines

 Demonstrate the daily, weekly, monthly maintenance of the GeneXpert

 Troubleshooting the problem of GeneXpert system

Outline
1.Introduction
2.PCR
3.GeneXpert technology and Parts
4.Maintenance of GeneXpert Machine
5.Troubleshooting of GeneXpert Machine
6.Summary
Introduction
Basics of Molecular Biology: The Genetic
information of the cell is contained in DNA.

 DNA is built in a double helix

 DNA encodes the genetic information (different

genes, different information)

 DNA is organized into a long chain called

chromosomes

 23 pairs of chromosomes are in the nucleus of

human cells
Genetic information of bacteria and Virus

 Genetic information in bacteria is encoded in DNA

 Most bacteria have a genome that consists of a single circular DNA molecule, located in a region called

nucleoid (not bound by a membrane)

 Extrachromosomal genetic elements such as plasmids and bacteriophages often determine resistance to

antimicrobial agents, production of virulence factors, or other functions.

 A virus is a small parasite that cannot reproduce by itself. It relies on the host cell machinery

 Viral genome can be found in various forms: RNA or DNA, single or double stranded, linear circular or even

segmented
DNA building block

DNA is made up of 4 nucleotides

• A = Adenine
• T = Thymine
• C = Cytosine
• G = Guanine

Most DNA is double stranded and

pairs in a unique way:
DNA Replication

• New DNA is made by enzymes called

DNA polymerases.
• They synthesize DNA in the 5' to 3'
direction only
• Another enzyme called primase makes
an RNA primer to prime the
Polymerase
• Once the RNA primer is in place, DNA
polymerase "extends" it, adding
nucleotides one by one to make a new
DNA strand that is complementary to
the template strand
PCR

Activity 6.1: Group Discussion

What is PCR and discuss type of

PCR?
Time: 5 min
•PCR (Polymerase Chain Reaction) is a chain reaction which generates multiple copies of a specific sequence

of DNA present in the sample.

•DNA amplification occurs by repeated thermal cycles

•The number of copies of the specific sequence doubles after each cycle

•After forty cycles, a single copy becomes around 2 trillion copies

Components of a PCR reaction

The various components required for PCR include

• a DNA sample,

• DNA primers,

• free nucleotides called ddNTPs and,

• DNA polymerase.
The phases of PCR

PCR is based on three simple steps required for any DNA synthesis reaction:

(1) denaturation of the template into single strands;

(2) annealing of primers to each original strand for new strand synthesis; and

(3) extension of the new DNA strands from the primers

Illustration showing the main steps in PCR.
Activity 6.2: Group Discussion

Discuss the FRET technology.

Time: 10 min
FRET Technology
Definition of Ct – based on threshold
 Polymerase Chain Reaction (PCR) Evolution

Homebrew PCR Batch Xpert Tests Flexi

Patient Sample(s) Patient Sample(s)
Batch
Washing Sample Preparation

Nucleic acid extraction

Purification

MasterMix

Amplification and Detection

Result Interpretation by Lab Technician Automatic Result Interpretation

1
6
 GeneXpert History of Ongoing System Innovation

2020/21:
2018: Edge **Omni
2018: Xpress

2011: Infinity-80
2009: Infinity-48
2006: GeneXpert
2000: System
SmartCycler

Information as of July 2020 **Product in development. Not for use in diagnostic

6 © Cepheid * Not all systems available in all countries US-IVD and CE-IVD. For in vitro diagnostic use. procedures. Not reviewed by any regulatory body.
 The Package

GeneXpert® System* Cartridge

• Thermal and optical module(s) • Self-Contained
• Computer System and GeneXpert Dx Software • Disposable
• Barcode scanner • Assay Definition Protocol (ADF)

Recommended Accessories Optional Accessories

• UPS • Batteries/Power generator
• Surge protector • Printer

* FDA cleared

7 © Cepheid US-IVD and CE-IVD. For in vitro diagnostic use.

 Proven Technology

I-CORE®

GeneXpert Module

2000 2006 2009 2011 2018

SmartCycler GeneXpert Infinity-80 GeneXpert

Infinity-48
System System System Edge
System
9 © Cepheid * Not all systems available in US-IVD and CE-
all countries IVD. For in vitro
 GeneXpert® Technology

• Integrated and closed system

‒ No direct contact between instrument and sample to eliminate carry-over
‒ The sample is enclosed in the cartridge
‒ Integrated ultrasonic horn for cell lysis (when applicable)
• Fluid transfer: Micro fluidics based reconstitution, and automated filling
‒ Advanced micro fluidics technologies to enable complex sample preparation
processing protocols
‒ Software driven motors for valve movement and integral hydraulic drives
20
• Multiple integrated controls to validate every step
© Cepheid US-IVD and CE-IVD. For in vitro diagnostic use.

• Automated protocol, data reduction and results interpretation

Automated Xpert® Protocol

3 Nucleic acids
are purified
Purified
Nucleic acids
mix with the
4
PCR reagents

2 5
Simultaneous
The cartridge is
loaded into the amplification
instrument and detection
occurs

1
Sample is

added to the
cartridge
Results are
ready to view 6
 GeneXpert® Module

Plunger Motor

Motherboard

I-CORE®

Module door
22 © Cephe US-IVD and CE-IVD. For in vitro diagnostic use.

Valve Drive Motor

“For complete details on the GeneXpert System, please refer to the GeneXpert System Operators Manual”
Cartridge Bay

I-CORE® Slit
Plunger Rod
PCR amplification &
detection Facilitates movement of sample
and reagents into different
chambers

Ultrasonic Horn
Valve Drive
Lyses the sample
(if applicable) Rotates the cartridge valve body
to allow access to the different
cartridge chambers
The I-CORE® Module

Building Block of Fan

Heater
the GeneXpert®
Rapid,
System precis
e
temper
ature
control
Circuitry
Passes optical
information to the Inserts into the I-
computer for analysis CORE®
and display

Optical Blocks C
a
Optical analysis, detect
r
and quantify up to
10 different DNA t
targets r
simultaneously i
d
 GeneXpert Maintenance

Preventative maintenance tasks:

• Performed on regular basis by end-user: daily, weekly, monthly,
yearly

• Ensures good performance of the system

• Helps to avoid problems and malfunction

Software based PM
Required tools/products
Daily Maintenance
Weekly Maintenance
Maintenance of I_CORE

Use the Video demonstration

Weekly Maintenance …
Monthly maintenance
Annual Instrument Maintenance

• Check the module performance with Xpert check once a year (after monthly
maintenance)

• Calibration is performed before a system is shipped.

• Xpert Check is performed annually by Cepheid Authorized Personnel

• Replacement modules are calibrated prior to shipment from the Cepheid Repair
Center
 Troubleshooting
Troubleshooting Approach

1. In case of an issue, a message will be displayed (often, with an error code)

2. Check if the error affects one particular module

3. Refer to the Operator Manual and look for the error code, and follow the recommended corrective actions

4. If the problem remains, contact your local service provider or Cepheid

Software messages?
Where to find Software messages?

Where to find Software messages?

Whole GeneXpert not detected
Module replacement

Use Video demonstration

Calibration

Use Video Demonstration