Molecular Testing and Clinical Diagnosis

Amplified nucleic acid testing

Part III

• Describe and evaluate types of target sequences (DNA, mRNA, tRNA, rRNA) (C3)

• Describe and compare amplification processes including (C3)– Basic steps of an amplification process – Principles of methods available

PCR, LCR, SDA, NASBA, TMA– List and describe the function PCR components in the

reaction mix (C2)• Describe the variations of PCR process (C2)

– LCR– Reverse Transcription-PCR– Real time PCR

Objectives: At the end of this lesson the student will:

• Explain the application of PCR to STR testing (C2)– Paternity testing– Forensic testing– RFLP mapping

• Describe the significance of the following PCR considerations (C2, A2)– Contamination– Quality control– Lab space allocation

Objectives: At the end of this lesson the student will:

Strand Displacement Amplification- BDPobeTec ET system• One hour assay

• fluorescence detection

• automated and semi-automated systems

• pre-dispensed reagent devices

Strand Displacement Process

• step 1: primer hybridization

• step 2: primer extensions by DNA polymerase leads to strand displacement

• step 3: extended probe binds complimentary strand

• step 4: probe is extended creating BsoBI site

• step 5: BsoBI cleaves dsDNA

Detection linked with amplification.Target must be amplified and double stranded to enable the restriction enzyme to function. Fluorescence only occurs when there is cleavage.

SDA lends itself to automation since it is isothermal.

Transcription-Mediated Amplification

RNA transcription amplification system using two enzymes: RNA polymerase and reverse transcriptase

Isothermal amplification of nucleic acid target producing RNA product amplification

Rapid kinetics results in excess of ten billion-fold amplification within 15-30 minutes

Combined with Hybridization Protection Assay detection in a single tube format

Transcription-Mediated Amplification Components

Primers: Oligonucleotides that hybridize to target and initiate the reaction

Nucleotides Enzymes drive the reaction:

– T7 RNA polymerase• transcribes RNA from DNA

– Reverse transcriptase (MLV):• synthesizes DNA from RNA or DNA• RNAse H activity: degrades RNA after it has

been copied into DNA

Transcription-Mediated Amplification

TMA: Gen-Probe Second GenerationAPTIMATM Assays

Target Capture sample processing partially purifies target nucleic acid

Transcription-Mediated Amplification-- amplified target

Dual Kinetic Assay (DKA) technology simultaneously detects two organisms

.....GAUCGAUCCCCCCUAGCGGUGCAUCUAGCAUCUA....

•••••••••••••••••••••••••••••••••••••

one micron

magnetic particle

––TTTTTTTTTTTTTT

––TTTTTTTTTTTTTT

––TTTTTTTTTTTTTT

––TTTTTTTTTTTTTT

––TTTTTTTTTTTTTT

––TTTTTTTTTTTTTT

––TTTTTTTTTTTTTT

AAAAAAAAAAAAAAAAAAGGATCGCCACGTAGATCGGCCTC

Mag

net

N

S

Bead Bead OligoOligo

Capture Capture Oligo "Tail"Oligo "Tail"

Target Target SequenceSequence

Capture Capture SequenceSequenceThese are washed away:These are washed away:

non-specific non-specific DNA/RNADNA/RNA

ProteinProteinCell Cell debrisdebris

PlasmaPlasma

••••••••••••••••••••••••••

Gen-Probe Proprietary Target Capture Technology

Detection by Dual Kinetic Assay (DKA) Technology

Hybridization Protection Assay (HPA) Technology

Two modified acridinium

ester labels with different

light-off kinetics on

different DNA probes

– “Flasher” fast

– “Glower” slow– Simultaneous detection of different organisms

Dual Kinetic Assay (DKA)

0

20,000

40,000

60,000

80,000

100,000

120,000

.04 .20 .36 .52 .68 .84 1.00 1.16 1.32 1.48 1.64 1.80 1.96

CT + GC

CT

GC

RL

U

Time in Seconds

1E0

1E2

1E4

1E6

1E8

1E10

1E12

1E14

Am

plic

on c

opie

s

0 5 10 15 20 25 30 35 40 45 50 55 60

Time (min.)

TMA Amplicon ProductionStarting with 1000 copies of Target

TMA Amplicon Production

Gen-Probe Instrumentation Systems

Fully automated, APTIMATM amplification assays for TIGRISTM

Target Capture system

VIDAS* dual platform: Amplified assays and immunoassays

*from bioMérieux

Challenges with Current Nucleic Acid Amplification Tests

Carry-over contamination can cause false positives

Verification of positive results is difficult Inhibition can cause false negatives Compared with current microbiology tests:

– Increased labor– Higher cost– Low throughput

Methods for Control of Carryover Contamination in automated TMA

Assays Unidirectional workflow Single-tube format Oil as a barrier to the environment HPA format eliminates wash steps and

potential aerosols Treatment of RNA amplicon with

detection reagents Bleach destroys nucleic acids

Comparison of TMA with PCR and LCR Amplification Methods

TMA (Gen-Probe ) PCR (Roche ) LCR ( Abbott )

Thermal

Conditions Isothermal reaction Thermal cycling Thermal cycling

AmplifiedProduct RNA DNA DNA

Wash step

Homogeneousassay no washsteps Wash step required

Wash steprequired

Detection System Chemiluminescence Absorbance Fluorescence

Specialequipmentneeded Luminometer

Thermal cycler,microtiter plate

reader/washerThermal cycler,LCx instrument

RNA polymeraseReverse transcription DNA polymerase

LigaseDNA polymeraseEnzymes

Step OneStep One

SampleSampleProcessingProcessing

Extract RNAExtract RNA

~ 90 minutes~ 90 minutes

(Hybridized target (Hybridized target

captured on to captured on to microparticles)microparticles)

Gen-Probe HIV-1/HCV dual-assay protocol for blood supply

Step TwoStep Two

TMATMA Add Add

Amplification Amplification

Reagent, Oil Reagent, Oil ReagentReagent

10 minutes 10 minutes 41.5°C41.5°C

Add Reverse Add Reverse Transcriptase, Transcriptase,

RNA PolymeraseRNA Polymerase

60 minutes 60 minutes 41.5°C41.5°C

Step ThreeStep Three

HPAHPA Add Probe Add Probe

ReagentReagent(Hybridizes to (Hybridizes to

amplicon)amplicon)

15 minutes 60°C15 minutes 60°C

Add Selection Add Selection ReagentReagent

10 minutes 60°C10 minutes 60°C

Read in Read in LuminometerLuminometer

Pooling Scheme

128Donations

16 16

16 16

16 16

16 16

128Pool

11

11

11

1

Resolution Testing

Identification of SingleDonation

16 16

16 16

16 16

16 16

ReactivePool

11

11

11

11 1

Test Primary Pools

128

HCV Panel 6211 – Virologic/Serologic Profile

DaysDays

S/C

OS

/CO

HC

V P

CR

Qua

ntita

tion

HC

V P

CR

Qua

ntita

tion

46 Days46 Days

PCR

100

1,000

10,000

100,000

1,000,000

10,000,000

0 20 40 60 80 100 120 140 160 180 200

0

1

2

3

4

5

6

7

8

9

10

Antibody

E991685 7-14-99 25

Summary: Amplification Methods• Much like a culture technique, they increase

likelihood of detection and identification• Enzymes are used to increase target

sequence for detection• May be automated or semi-automated more

easily if isothermal

Summary Amplification Methods

• Increased sensitivity– amplification– detection systems

• Specificity – primers– probe/detection systems