Review of diagnostic molecular techniques

Review of diagnostic Review of diagnostic molecular techniquesmolecular techniques

Y. YANG

Current Clinical Applications in IDCurrent Clinical Applications in ID

• Detection of viral and bacterial infectious disease (majority of the current market)

• Quantitative viral load monitoring of HIV and HCV (Treatment monitoring)

• Determine antimicrobial susceptibilities• Top 5 Companies providing infectious disease

diagnostics include Bayer (Siemens), BD, Digene, GenProbe and Roche Diagnostics.

• Competitive intensity in this space is escalating.

AdvantageAdvantage

• Rapid TAT

• High sensitivity and specificity

HPAHPA• A specific DNA probe, labeled with an acridinium ester

detector molecule.

• This DNA probe hybridizes with either the target rRNA in non-amplified tests, or the amplicon produced in the Transcription-Mediated Amplification (TMA) reaction.

• In a positive sample, the bound probe is protected from alkaline hydrolysis and, upon addition of peroxides, emits detectable light (chemiluminescent signal).

• The solution phase selection/detection step does not require washing. This makes the HPA format as easy to perform as the simplest immunoassays, with the superior specificity of DNA probes.

HPAHPA

(culture identification)

• Less contamination• Highly reproducible • Used to be less sensitive; However, detection limit 3rd generation bDNA assay for HIV-1 RNA is 50 copies/ml.• HIV RNA, HBV DNA, and HCV RNA quantitation (Bayer)

• Solid phase hybridization and chemiluminescence

• Digene HPV (cervical scrapings, PAP), CMV (blood and body fluids) detection, CT/GC combo testing

• Less sensitive, quanlification assay

Hybrid Capture Assay

Digene HPV TestDigene HPV Test

The Digene HPV Test is the only FDA approved HPV test for:

• Primary screening, in conjunction with a Pap, of women age 30 years and older; and

• Triage of women of any age with ASC–US Pap results.

• Collectively detects the 13 clinically–relevant high–risk HPV types.

Clavel et.al, Brit J Cancer 2001;89:1616–1623

Amplification of the targetAmplification of the target

• PCR (RT, nested, multiplex, Real Time; Roche [AMPLICOR])

• PCR (GeneOhm Sciences-IDI)• Nucleic acid sequence-based amplification

(NASBA; bioMerieux [NucliSens])• Transcription mediated amplification (TMA; Gen-

Probe [APTIMA, MTD, Procleix])• Strand displacement amplification (SDA; BDDS

[ProbeTec])

Nested PCRNested PCR

real-timereal-time real-time

real-timereal-time

integrated systemamplifies & detects

constant monitoringfluorescent probes

rapid cycling times

quantitative

low contamination

riskassay design

PCRPCR

fast turn-around sealed system

real-timereal-time

real-timereal-timeTaqManTaqMan

real-time

Amplicon

FRET

Amplicon

Emission

EXTENSION

ANNEALING

Excitation

5’-3’ exonuclease

Reporter

Quencher

• Uses a fluorogenic probe, with reporter & quencher dyes

• Taq DNA polymerase has 5’-3’ exonuclease activity (60)

• Microtitre plate format, sealed system

• Processes 96 samples in 2½ hours

• Real-time - amplification and detection

• Quantitative results

ABI 7700


TaqManTaqManhardwarehardware

ABI Biosystems

Amplicon

molecular beaconsmolecular beacons

A

B

C

FRET

real-timereal-timereal-time

real-timereal-time

Reporter

Non-fluorescent Quencher

Excitation

ANNEALING

FRET (Fluorescence Resonance Energy Transfer) FRET (Fluorescence Resonance Energy Transfer)

using adjacent hybridization probesusing adjacent hybridization probes

FRET (Fluorescence Resonance Energy Transfer) FRET (Fluorescence Resonance Energy Transfer)

using adjacent hybridization probesusing adjacent hybridization probes

FITC

Red 640

P Phosphate

FRET Emission

P

Excitation

Amplicon


LightCyclerLightCyclerFRETFRET

• Detection of Infectious Disease agents

• Target Characterization

• Determining Microbial Load (quantitation)

• Detection of Infectious Disease agents

• Target Characterization

• Determining Microbial Load (quantitation)


LightCyclerLightCyclerApplications

Characterization of HSV by melting curve Characterization of HSV by melting curve

DNA pol

HSV

HSV-1

HSV-2

mismatch

Hybridization probes (to HSV-1)

no mismatch

Amplicon


LightCyclerLightCyclerApplication

Primers commonto HSV 1 & 2

HSV 2 HSV 1

Melting Curve Analysis

HSV 1

HSV 2

55oC

HSV 1

HSV 2

73oC

HSV 1

HSV 2

67oC


real-timereal-timePCR quantitationPCR quantitation

Microbial load testingMicrobial load testing

• For commensal organisms determine a “normal” microbial load. Elevated level determines infection.

• Detect active infection by increasing load

• Detect anti-viral drug resistance (CMV, HSV)

0.5

1.5

2.5

0 10 20 30 40 50 60 70

Cycles

F2/F

1

NEG

10

100

1000

10000

100000

1000000



Threshold Cycle

Microbial Load Testing

0

10

20

30

40

50

1 2 3 4 5 6

Concentration log 10

Th

resh

old

Cycle

0.5

1.5

2.5

0 10 20 30 40 50 60 70

Cycles

F2/F

1

Test Sample

Threshold

Threshold Cycle

Threshold Cycle = 35Load = 103.8 copies/ml

PRACTICAL APPLICATIONPRACTICAL APPLICATION



Monitoring CMV disease in transplant patients, particularly Bone Marrow Transplant recipients.

1. Early detection of disease progression to apply appropriate drug therapy

2. Detect ganciclovir drug resistance

0

1000

2000

3000

4000

5000

6000

7000

8000

Sampling Time (Wks)

40

30

20

10

0

AntigenemiaPositive cells

per 200,000 cells

AntigenemiaPositive cells

per 200,000 cells

g

eno

me

cop

ies

q-PCR

1 2 3 4 5 6 7 8 9 10 11

Ganciclovir

BMT PATIENT 1BMT PATIENT 11 2 3 4 5 6 7 8 9 10 11

ROCHE PCR

“in house” PCR

Antigenemia


real-time PCRreal-time PCRViral LoadViral Load

ADVANTAGES OF REAL-TIME PCRADVANTAGES OF REAL-TIME PCR

Rapid cycling times (1 hour)

High sample throughput (~200 samples/day)

Low contamination risk (sealed reactions)

Very sensitive (3pg or 1 genome eq of DNA)

Broad dynamic range (10 - 1010 copies)

Reproducible (CV < 2.0 %)

Allows for quantitation of results

Software driven operation

No more expensive than “in house” PCR ($15/test)

real-timereal-timereal-time PCRreal-time PCR

Summary Summary

real-timereal-timereal-time PCRreal-time PCR

SummarySummary

DISADVANTAGES OF REAL-TIME PCRDISADVANTAGES OF REAL-TIME PCR

Current technology has limited capacity for multiplexing. Simultaneous detection of 2 targets is the limit.

Development of protocols needs high level of technical skill and/or support. (Requires R&D capacity and capital)

High capital equipment costs ($ 50,000 -160,000).

Transcription mediated amplification (TMA)

TMATMA• TMA is isothermal. A water bath or heat block is used

instead of a thermal cycler.• TMA produces RNA amplicon rather than DNA amplicon.

Since RNA is more labile in the laboratory environment than DNA, this helps reduce the possibility of carry-over contamination.

• TMA produces 100-1000 copies per cycle in contrast to PCR and LCR that produce only two copies per cycle. This results in a 10 billion fold increase of copies within about 15-30 minutes.

• Direct detection of Mycobaterium tuberculosis (rRNA), detection of CT/NG, WNV (Gen-probe)

Amplification of the probeAmplification of the probe

• Ligase chain reaction (LCR; Abbott)

• Cleavase-invader technology (Third Wave Technologies)

• Cycling probe technology (ID Biomedical Corp)

Amplification of the probesAmplification of the probes

Like PCR, LCR requires a thermal cycler to drive the reaction and each cycle results in a doubling of the target nucleic acid molecule.The LCR reaction is also completed in about 90 minutes

HIV Viral Load Response HIV Viral Load Response to Antiretroviral Therapyto Antiretroviral Therapy

• Duration of response not predicted by baseline HIV RNA

• Following viral load throughout therapy is important

• If never < 1000 copies/ml, rapid rebound toward baseline

• Determine of resistant virus may be developing

Significance of viral load Significance of viral load reductionreduction

Significance of viral load Significance of viral load reductionreduction

Kempf, et al. In: Program and abstracts of the International Workshop on HIV Drug Resistance,

Treatment Strategies and Eradication; 25–28 June 1997; St Petersburg, Florida, USA

Duration of maximal response

Pla

sma

HIV

-1 R

NA

viral load = replication = mutations = resistance =

durability

AMPLICOR HIV-1 MONITORAMPLICOR HIV-1 MONITOR

• First HIV viral load test approved by the FDA

• Sensitivity: UltraSensitive 50 c/mL

• Upper limit for quantitation:

– Standard: 750,000 c/mL

– UltraSensitive: 100,000 c/mL

Automation for improved reliability and ease-of-use

BLOOD COLLECTION AND BLOOD COLLECTION AND STABILITYSTABILITY• Collect Blood in EDTA PPT tubes

– Heparinized tubes not acceptable

– Separate plasma within 3 hours of draw

• Store plasma frozen or at 2-8°C

Test should be performed within 3 days. If delay of more than 3 days is anticipated, then plasma should be separated to a different tube.

A Continuing Epidemic: HIV




(OD * Dilution Factor) (OD * Dilution Factor) HIVHIV

(OD * Dilution Factor) (OD * Dilution Factor) QSQS

HIV Copy / ml = Input QS CopiesX X 4

BROAD DYNAMIC RANGE BROAD DYNAMIC RANGE with Two Sample Processing Methodswith Two Sample Processing Methods

• Test will include both methods for a broad linear range:– Ultrasensitive: 50 to 75,000 HIV RNA copies/mL– Standard: 400 to 750,000 HIV RNA copies/mL

• Suggested algorithm:– Request Standard method for:

• Patients with no prior viral burden measurements• Patients not on antiviral therapy• Baseline measurements: before initiating or

changing therapy– Request Ultrasensitive method for:

• Patients with prior results < 2000- 5,000 c/mL• After initiation or changing antiviral therapy

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Review of diagnostic molecular techniques