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
real-timereal-time real-time
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
real-timereal-time real-time
LightCyclerLightCyclerFRETFRET
• Detection of Infectious Disease agents
• Target Characterization
• Determining Microbial Load (quantitation)
• Detection of Infectious Disease agents
• Target Characterization
• Determining Microbial Load (quantitation)
real-timereal-time real-time
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
real-timereal-time real-time
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-timereal-time
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
real-timereal-timereal-time
real-timereal-timePCR quantitationPCR quantitation
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
real-timereal-timereal-time
real-timereal-timePCR quantitationPCR quantitation
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-timereal-time real-time
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
A Continuing Epidemic: HIV
A Continuing Epidemic: HIV
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