Review of diagnostic

molecular techniques

Y. YANG
 Current 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.
 Advantage
• Rapid TAT
• High sensitivity and specificity
 HPA
• 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.
HPA
(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)
 Hybrid Capture Assay

• Solid phase hybridization

and chemiluminescence
• Digene HPV (cervical
scrapings, PAP), CMV
(blood and body fluids)
detection, CT/GC combo
testing
• Less sensitive,
quanlification assay
 Digene 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 Clavel et.al, Brit J Cancer 2001;89:1616–1623
clinically–relevant high–
risk HPV types.
 Amplification 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 PCR
 real-time real-time
real-time
real-time
PCR

amplifies & detects integrated system

fluorescent probes constant monitoring
fast turn-around rapid cycling times
sealed system low contamination
quantitative risk
assay design
 real-time
real-time
Reporter real-time real-time
TaqMan
Quencher

Excitation
FRET
Emission
• Uses a fluorogenic
probe, with reporter &
Amplicon
quencher dyes
ANNEALING

• Taq DNA polymerase

has 5’-3’ exonuclease
activity (60°)
EXTENSION

Amplicon

5’-3’ exonuclease
 real-time real-time
TaqManreal-time
hardware
ABI Biosystems
ABI 7700

• Microtitre plate format, sealed system

• Processes 96 samples in 2½ hours
• Real-time - amplification and detection
• Quantitative results
 real-time
real-time
real-time
real-time
molecular beacons
A
Excitation
B Reporter
C
Non-fluorescent
FRET Quencher

ANNEALING

Amplicon
 real-time real-time
LightCycler real-time
FRET
FRET
FRET (Fluorescence
(Fluorescence Resonance
Resonance Energy
Energy Transfer)
Transfer)
using
using adjacent
adjacent hybridization
hybridization probes
probes

FITC
Red 640
P Phosphate
Excitation Emission
FRET

Amplicon
 real-time real-time
LightCycler real-time
Applications

• Detection of Infectious Disease agents

• Target Characterization

• Determining Microbial Load (quantitation)

 real-time real-time
LightCycler
real-time
Characterization of HSV by melting curve Application

HSV
DNA pol
Primers common
to HSV 1 & 2

Hybridization probes (to HSV-1)

HSV-1
no mismatch
Amplicon

HSV-2

mismatch
 Melting Curve Analysis

HSV 1
55 C
o

HSV 2

HSV 2 HSV 1 HSV 1

67oC

HSV 2

73oC
HSV 1

HSV 2
 real-time
real-time
real-time real-time
PCR quantitation

Microbial 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)

 real-time
real-time
real-time real-time
PCR quantitation
Microbial Load Testing
2.5

N EG
Threshold
Cycle 10
100
F2/F1

1.5 1000
10000
100000
1000000

0.5
0 10 20 30 40 50 60 70
C ycle s
 50
Threshold Cycle
40
Threshold Cycle = 35
30 Load = 103.8 copies/ml

20

10

0
Concentration log 10
1 2 3 4 5 6

2 .5

Test Sample
Threshold
F2/F1

1 .5

Threshold Cycle
 real-time
real-time
real-time real-time
PCR quantitation

PRACTICAL 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

 real-time real-time
real-time PCR real-time
Viral Load
BMT PATIENT 1
1 2 3 4 5 6 7 8 9 10 11
ROCHE PCR

“in house” PCR

8000 40
Ganciclovir
7000
genome copies

30
6000 q-PCR
5000 Antigenemia
20 Positive cells
4000 per 200,000 cells

3000 10

2000
Antigenemia
1000 0
1 2 3 4 5 6 7 8 9 10 11
0 Sampling Time (Wks)
 real-time
real-time PCR
real-time
Summary

ADVANTAGES 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-time
real-time PCR
real-time
Summary

DISADVANTAGES 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)
 TMA
• 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 probe

• Ligase chain reaction (LCR; Abbott)

• Cleavase-invader technology (Third Wave
Technologies)
• Cycling probe technology (ID Biomedical
Corp)
Amplification 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
to 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
reduction
↓ viral load = ↓ replication = ↓ mutations = ↓ resistance =
Plasma HIV-1 RNA
↑ durability

Duration of maximal response

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
AMPLICOR 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
STABILITY
• 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) HIV
HIV Copy / ml =
(OD * Dilution Factor) QS
X Input QS Copies X 4
 BROAD DYNAMIC RANGE
with 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