Transcript PACE 2C
PACE 2C
Genetic probe screening for C.
Trachomatis & N. Gonorrhoeae in
Endocervical and Male urethral
specimens
Heidi Philips and Michael S. Joseph
Outline
Biological background on Chlamydia
Trachomatis and Neisseria
Gonorrhoeae
Flow chart
Biology of the PACE 2C
Assay Applications
Assay Components
Interpretation of the assay
Why test for this disease?
Gonorrhea in the US, nearly 392,200
cases
Public health measures requires
surveillance by monitoring and screening
as a preventative and allocation of public
funds.
Background: Neisseria Gonorrhoeae
Gonorrhea is a very common sexually transmitted disease
(STD).
The disease is caused by the gonococcus bacteria that can
multiply and grow in moist, warm areas of the body.
In women, gonorrhea can infect the cervix (opening to the
uterus, or womb), uterus, and fallopian tube.
It can also infect the urethra (urine canal) in men and
women, as well as the mouth, throat, and rectum.
Gonorrhea can be treated and cured by antibiotics.
Background: Chlamydia Trachomatis
Chlamydia is caused by the bacterium Chlamydia
trachomatis.
It persists at body sites that are inaccessible to
phagocytes, T-cells, and B-cells.
Endemic trachoma leads to blindness, whereas inclusion
conjunctivitis is associated with the sexually transmitted
form and does not lead to blindness.
The surface of chlamydia does not contain proteins that
are distinctive enough to induce a full immune response.
The organism is also extremely temperature sensitive and
must be refrigerated at 4 C as soon as a sample is
obtained.
What is PACE 2C Assay?
Probe Activated Chemiluminescent
Enhancement 2C
Direct Genetic probe
DNA-RNA hybridization
With Chemiluminescent probe
Acridinium ester
Direct assay with qualitative results
Steps in PACE
One swab, one tube, two
test convince.
1. Sample Preparation
2. Hybridization
-incubation
3. Separation
-incubation
-magnetic separation
4. Detection
Flow Chart
Obtain Sample
Reagent Preparation
- All reagents aside from the
probe reagent must reach
room temperature prior to
use.
Sample Preparation
- Sample must reach
room temperature prior
to use.
- Samples must be obtained using the
PACE Specimen Collection Kits.
Reconstitute Primer
-The solid phase must be
reconstituted in the selection
reagent prior to use.
-Temperature dependent
Hybridization
- Incubation
Separation
- Forceful wash
- Incubation
- Magnetic separation unit
Detection
- Chemiluminescent probes
- Leader Illuminometer
What makes it attractive
PACE 2C is a qualitative assay for both C.
Trachomatis and N. Gonorrheae.
Detects rRNA of the bacteria
Initial screening
High specificity of DNA Probe
Enhanced by targeting rRNA, which is highly
abundant w/o amplification
Free from false negative, due to amplification inhibition.
Hybridization protocol with addition of
Chemiluminescent label detection.
What makes it attractive cont.
Cost-effective, do not need amplification
Increased efficiency and lower costs by
testing CT and GC from single
specimen
Simple workflow
high tolerance for various specimen
quality
Samples
Endocervical for
female and male
urethral swab
specimens
collected with the
Gen-Probe PACE
Specimen
Collection Kits
Samples cont.
The kit consists of
single tube assay
There is no transfer
sample from collection
container to assay
tube.
Avoiding
transportation of
sample
But once in the
specimen collection
kit,it can last for 7
days.
Additional Products & Reagents
GEN-PROBE® PACE Specimen Collection
Kits
GEN-PROBE® Detection Reagent Kit
GEN-PROBE® Leader® Luminometer
GEN-PROBE® Magnetic Separation Unit
Vortex Mixer
Covered water bath (60°±1° C)
Micropipettes (100 µL)
Pipettes capable of delivering 1-25 mL
Absorbent paper
Economics of the PACE 2C
Luminometer $29,550
Separation Unit $450
PACE 2A kit $1000
100 test per kit
Specimen Collection Kit $500
50 tubes per kit
Sample Limitations
ONLY sampled from the endocervical area
and the male urethra.
Performance with other samples has not been
tested.
Only to be used with swabs transported and
stored in the GEN-PROBE transport medium.
Transport and store sample at 2º-25º until use
if before 7 days.
If longer storage is needed prepare sample
for freezing at -20º to -70ºC.
Detection Scheme
Specificity
rapid DNA probe test which utilizes the nucleic
acid hybridization to screen for the presence of
Chlamydia trachomatis and/or Neisseria
gonorrhoeae
System uses single-stranded DNA
probes with chemiluminescent labels
complementary to the ribosomal RNA of
the target organism.
Target Capture
Magnetic particles (1m in
diameter), with synthetic
oligonucleotides/ antibody
specific/ charge specific
for target nucleic acid are
bound to the surface
The beads are used to
capture target nucleic acid
target nucleic acid is the
DNA/rRNA probe
Detection
It confers specificity because the relationship
between the DNA/rRNA hybrid molecule and
the magnetic bead
It is selective because a minimal amount of
non amplified rRNA is need to have
quantifiable amount.
Detection: specificity cont.
Specificity- complementary base pairing
- Precise hybridization conditions
- Lyophilized probe
The use of single-strand DNA probes with
chemiluminescent labels that are
complementary to the ribosomal RNA of the
target organism allows the DNA probes to
combine with the rRNA of the target organism
to form stable DNA:RNA hybrids.
Wash/ Separation
Washing/ Separation – Labeled DNA:RNA
hybrids are separated and removed from the
non-hybridized probes.
Incubation with separation suspension buffer
Magnetic separation with stringent/ forceful
washes
Visualization
The luminometer measures the amount of
chemiluminescent emission from reactive patient
samples (PACE assays)
light emitted from these reactions is detected by a
photomultiplier tube
It is converted into a digital signal
The final measurable signal is directly proportional to
the number of photons released by the respective
reaction.
Visualization: chemiluminescence
Acridinium ester made pre-attached to the probe.
The probe binds to the target rRNA of the
bacterium.
Selection reagent is added which will hydrolyze
acridinium ester of the single stranded probe.
However, the probes that have hybridized
DNA/rRNA probe, the acridinium ester is
protected in the double helix of the DNA RNA
probe.
Detection reagent is added and no light is emitted
from the ss probes, because the acradidium ester
has been hydrolyzed.
In the hybridized probe, the acradidium ester is
protected and can emit light, and the light can be
quantified in the luminometer as Relative Light
Unites.
Sensitivity
Relatively large number of organisms in a typical
sample collected in swab. No amplification is need
Targeting of rRNA
Background Reduction
-Separation- Forceful washing: Angle wash to front
sides of tube with the appearance of foam head to
ensure adequate wash procedure.
Controls
The Negative Reference and Positive Controls
provided, control for the PACE 2C assay ONLY.
Negative Reference control- Non-infectious nucleic
acid in a buffered solution containing <5% detergent.
Provides a measure of the assay background and is
used to calculate the run cut-off.
Chlamydia trachomatis Positive Control- Noninfectious C. trachomatis nucleic acid in a buffered
solution containing <5% detergent
Neisseria gonorrhoeae Positive Control- Noninfectious N. gonorrhoeae nucleic acid in a buffered
solution containing <5% detergent
Results calculation:
Result is calculated based on the difference the
Response Light Units of the specimen and the
mean of the negative reference.
Mean of the Negative Reference =
(65 RLU + 71 RLU + 80 RLU)/3 = 72 RLU
Assigned Cut-off = 300 RLU
Calculated Cut-off = 300 RLU + 72 RLU = 372
RLU Negative
Specimen Response = 900 RLU Positive
The limit RLU = 600 to 3200
Interpretation
A negative test does not exclude the
possibility that the numbers of C.
trachomatis and/or N. gonorrhoeae
organisms may be below the level of
detection of the assay.
Second test is required
Lack of forceful washing may result in false
positive
Detection Cont.
Sample
Number of Replicates
Mean Response (RLU)
Standard Deviation (RLU)
Coefficient of Variance
C. trachomatis
Positive Control
69
1837
329
5.6%
N. gonorrhoeae
Positive Control
69
2165
425
5.1%
Critical Parameters
Hybridization and separation temperature
- Temperature Dependent
- Uniform equilibration of reaction tubes needed
Background Reduction
-Separation- Forceful washing: Angle wash to front
sides of tube with the appearance of foam head to
ensure adequate wash procedure.
Conclusion
Results form the PACE 2C system should be used in
conjunction with other data. This is a screening test
only.
Detection is based on luminometer RLU unites
Positive 600-3200 RLU
Negative 372 or less
Reference
1.
http://www.gen-probe.com/prod_serv/std_pace.asp
2.
3.
http://www.4woman.gov/faq/stdgonor.htm
Iwen, P.C., Walker, P.A., Warren, K.L., Kelly, D.M., Hinrichs, S.H., and
Linder, J. Evaluation of Nucleic Acid-Based Test (PACE 2C) for
Simultaneous Detection of Chlamydia trachomatis and Neisseria
gonorrhoeae in Endocervical Specimens. 1995, Journal of Clinical
Microbiology, Vol. 33, No.10; 2587-2591