Manual - Fast-track Diagnostics

Manual
For 24 patients (catalog no. FTD 20-24/4)
For 48 patients (catalog no. FTD 20-48/6)
For 96 patients (catalog no. FTD 20-96/12)
FTD Stool parasites
For use with the
ABI 7500, Bio-Rad CFX96, LightCycler ® 480, RotorGene 6000
March 2009-Version 2
FTD Stool parasites 1

Table of contents
Page
1. Identification of the manufacturer 3
2. Identification of the product 3
3. Intended use 3
4. Pathogen information
Entamoeba histolytica
Cryptosporidium spp. (including parvum,
hominis, meleagridis)
Giardia lamblia
5. Contents 4
6. Precautions and warnings 5
6.1. Handling requirements 5
6.2. Laboratory procedure 5
6.3. Safe waste disposal 5
7. Storage and stability conditions 5
8. Principle of the method 5
9. Additionally required equipment 6
10. Samples 6
11. Procedure 7
11.1 Preliminary procedure 7
11.2 Main procedures 8
11.3 Programming of the thermocycler 12
12. Assay validation 13
13. Calculation of the results 13
13.1 On ABI 7500 13
13.2 On Rotor-Gene 15
14. Interpretation of results 15
15. Troubleshooting 16
FTD Stool parasites
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3
3
4
4

1. Identification of the manufacturer
Fast-track Diagnostics Luxembourg S.a.r.l
38, rue Hiehl
Z. I. Langwies
L-6131 Junglinster
Tel. +352 780290 329
Fax: +352 788894
info@fast-trackdiagnostics.com
FTD Stool parasites
2. Identification of the product
FTD Stool parasites
Category: Multiplex PCR for detection of Entamoeba histolytica, Cryptosporidium
spp. and Giardia lamblia– including internal control
Reference: FTD 20-24/4 Test for 24 patients, 6 assays, each for 4 specimens
FTD 20-48/6 Test for 48 patients, 8 assays, each for 6 specimens
FTD 20-96/12 Test for 96 patients, 8 assays, each for 12 specimens
Indication: For research use only
3. Intended use
The FTD stool parasites is an in vitro test for the detection of pathogen DNA as
an aid in the evaluation of infections by Entamoeba histolytica,
Cryptosporidium spp. (including C. parvum, C. hominis and C.
meleagridis) and Giardia lamblia (G. lamblia = G. intestinalis = G.
duodenalis).
4. Pathogen information
Entamoeba histolytica (Ent) is an anaerobic invasive
parasite infecting humans and primates. While its cysts can
survive outside the host in water, soils or foods, its active stage
can be found within the host and fresh feces. Infection can lead
to amoebic dysentery or liver abscesses accompanied by symptoms such as
diarrhea, weight loss, fatigue and abdominal pain. Its distribution is worldwide
and its morphologically indistinguishable from the apathogenic E. dispar.
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FTD Stool parasites
Giardia lamblia (Gia) is a genus of flagellated non-invasive
parasites with the most commonly found species Giardia lamblia
(synomym: Giardia intestinalis, Giardia duodenalis) infecting
mostly humans, other mammals and birds. During the active
stage of its lifecycle it remains attached to the lumen of the small intestine. The
associated disease is called giardiasis, characterized by diarrhea, hematuria,
fever and a loss of appetite. Cysts are infectious and can survive for months in
cold water.
Cryptosporidium spp. (Cry) is a pathogenic protozoan
infecting humans and other mammals but also reptilians.
Pathogenic for humans are amongst others Crypotsporidium
parvum and C. hominis which cause a short-term infection which
can become severe and non-resolving in children and immunocompromised
individuals. Symptoms of an infection include diarrhea, fever and abdominal pain.
5. Contents
Green plastic box
Label Contents FTD
20-24/4
Ent-Gia-Cry
PP MIX
IC
PC
NC
Primer/probe mixes for Entamoeba
histolytica, Cryptosporidium
spp., Giardia lamblia and IC
Internal control
(in a denaturant agent)
Positive control
(Plasmid pool)
Negative control
(in a denaturant agent)
FTD FTD
20-48/6 20-96/12
6 x 11 µl 8 x 14 µl 8 x 24 µl
6 x 11 µl 8 x 16 µl 8 x 29 µl
6 x 11 µl 8 x 11 µl 8 x 11 µl
6 x 210 µl 8 x 210 µl 8 x 210 µl
PP = primer and probe, IC = internal control, PC = positive control, NC = negative control
The box itself, the cover of the box and each vial are labeled with a lot number.
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FTD Stool parasites
6. Precautions and warnings
6.1. Handling requirements
FOR RESEARCH USE ONLY
Use of this product should be limited to personnel trained in the techniques of
PCR.
Take the normal precautions required for handling all laboratory reagents.
Do not mix reagents from different lots.
Do not use the test after its expiration date
6.2. Laboratory procedure
This product should be used in accordance with Good Laboratory Practice.
The temperature at receipt must be equal or lower than -20°C.
Pipettes should be calibrated every 6 months and equipment used with the test
should be regularly maintained in accordance with the indications of the
manufacturer.
Freezer temperatures must be supervised daily.
Do not eat, drink or smoke in the laboratory area.
Do not pipette by mouth.
All human specimens and all resulting waste should be considered potentially
infectious. Clean and disinfect all work surfaces with a disinfectant recommended
by the local authorities.
The wearing of gloves and protective laboratory coats is recommended.
Wash hands immediately after handling specimens and reagents.
6.3. Safe waste disposal
Dispose of unused reagents and waste in accordance with country, state or local
regulations.
7. Storage and stability conditions
Products should be stored in their original packaging at – 20°C until the
expiration date printed on the label.
Refreeze immediately after each set of reagents has been taken out for use and
before the rest of the reagents begin to thaw.
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FTD Stool parasites
8. Principle of the method
Entamoeba histolytica, Cryptosporidium spp. and Giardia lamblia DNA
are amplified by specific primers. Detection of product is via a dual labeled
molecular probe for each parasite of the multiplex PCR.
The assay uses murine cytomegalovirus (mCMV) as an internal control (IC),
which is introduced into the lysis buffer at the extraction stage of each sample
and the positive and negative control.
9. Additionally required equipment
The “FTD Stool parasites” is suited for use with the ABI 7500, Bio-Rad CFX96,
LightCycler ® 480, RotorGene 6000. This assay has been fully validated on an
ABI7500 with the AgPath-ID One-Step RT-PCR Kit (Ambion, cat # AM1005).
• Real time PCR master mix and enzyme. We recommend the AgPath-ID
One-Step RT-PCR Kit
• Disposable powder-free gloves
• Commercial total nucleic acid isolation kit: we recommend an automated
extractor for DNA extraction, such as the easyMAG (bioMerieux) or any of
the machines from Qiagen, but manual Qiagen columns and Invitrogen
columns are perfectly adequate if you are testing small numbers of
samples
• Pipettes (adjustable)
• Sterile pipette tips with filters
• Vortex mixer
• Desktop centrifuge
• ABI recommended 96 well PCR plates and plate sealers or Rotor-Gene 0.1
ml strip tubes and caps
• Sample rack
10. Samples
This test is for use with extracted nucleic acid from stool samples of human
origin. To reduce the risk of inhibition, we recommend the dilution of the native
specimen in S.T.A.R buffer (Roche) prior extraction. If the S.T.A.R buffer is used,
prepare the specimens according to manufacturers’ protocol.
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11. Procedure
11.1 Preliminary procedure
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Extraction of the specimens and the negative controls
1. Thaw one negative control (NC, 200 µl aliquot, white cap) and one
internal control (IC, blue cap)
! Before use, the reagents have to be thawed completely, mixed (by pipetting or short vortexing
and spinning down briefly!
2. Extract 4, 6 or 12 samples or multiples of 4, 6, 12 samples and the NC.
We recommend a starting volume for the extraction of 200 µl and an
elution volume of 50 µl.
! To increase the sensitivity of the test, you can use a higher starting volume for the extraction of
your specimens according to your extraction method i.e. a starting volume of 600 µl and an elution
volume of 50 µl.!
3. Add 2 µl internal control (IC, blue cap) directly to the lysis buffer of each
extraction
4. Do not extract positive controls
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11.2 Main Procedure
Preparation of PCR:
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1. Thaw reagents for the reaction are Ent-Gia-Cry PP, the positive control
(PC) also the enzyme and the mastermix of AgPath-ID One-Step RT-PCR
Kit
Reagents in each column are sufficient for:
FTD 20-24/4 4 patients, including controls
FTD 20-48/6 6 patients, including controls
FTD 20-96/12 12 patients, including controls
! Before use, the reagents have to be thawed completely, mixed (by pipetting or short vortexing
and spinning down briefly!
2. Pipette the required amount (see table 2 below) of the “2xRT PCR buffer”
(AgPath-ID One-Step RT-PCR Kit) to Ent-Gia-Cry PP
Take care to change the tips after each pipetting step.
3. Pipette the required amount (see table 2 below) of the “25x RT-PCR
enzyme mix” (AgPath-ID One-Step RT-PCR Kit) to Ent-Gia-Cry PP with
the “2xRT PCR buffer”.
Take care to change the tips after each pipetting step.
! See table 2 for pipetting amounts.!
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FTD
20-24/4
FTD
20-48/6
FTD
20-96/12
Number of samples 1 7
Preparation of the
PCR assay
FTD Stool parasites
PP mix 1.5 µl 10.5 µl
Enzyme 1 µl 7 µl
Mastermix 12.5 µl 87.5 µl
Total 15 µl 105 µl
Number of samples 1 9
Preparation of the
PCR assay
PP mix 1.5 µl 13.5 µl
Enzyme 1 µl 9 µl
Mastermix 12.5 µl 112.5 µl
Total 15 µl 135 µl
Number of samples 1 15.5
Preparation of the
PCR assay
PP mix 1.5 µl 23.3 µl
Enzyme 1 µl 15.5 µl
Mastermix 12.5 µl 194 µl
Total 15 µl 232.8 µl
Table 2 shows the amount of reagent that you need for one well and for
FTD 20-24/4: The PP Mix contained in one tube is sufficient for 7 reactions and includes 4
patients, 1 NC, 1 PC, 10% pipetting inacurancy.
FTD 20-48/6: The PP Mix contained in one tube is sufficient for 9 reactions and includes 6
patients, 1 NC, 1 PC, 10% pipetting inacurancy..
FTD 20-96/12: The PP Mix contained in one tube is sufficient for 15.5 reactions and includes 12
patients, 1 NC, 1 PC, 10% pipetting inacurancy.
4. Vortex the complete master mixes briefly and spin down in a centrifuge
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Preparation of the plate/tubes:
FTD Stool parasites
Preparation for the ABI7500 Preparation for the Rotor-Gene 6000
1. Take a 96 well plate Set the 0.1ml strip tubes in the
2. Pipette 15 µl of the Ent-Gia-Cry PP
with the “2xRT PCR buffer” and the
“25x RT-PCR enzyme mix” in the
wells
3. Add 10 µl of the extracted samples,
the extracted negative control and of
the positive control (which are not
extracted).
! Each run must include a negative and a
positive control.!
provided rack
Pipette 15 µl of the Ent-Gia-Cry PP
with the “2xRT PCR buffer” and the
“25x RT-PCR enzyme mix” in the
tubes
Add 10 µl of the extracted samples,
the extracted negative controls and of
the positive control (which are not
extracted).
! Each run must include a negative and a
positive control.!
5. Mix briefly by pipetting up and down. Mix briefly by pipetting up and down.
6. Close the plate with the ABI optical
adhesive film.
Close the strip tubes with strip caps.
7. Centrifuge briefly /
8. Put the plate in the ABI7500. Put the strips in the rotor. Make sure
9. Figure 1 shows an example for
location of samples and controls on a
ABI7500 plate
tubes are the correct way around.
Figure 2 shows an example for
location of samples and controls on
Rotorgene tubes
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A
B
C
D
E
F
G
H
1 2 3 4 5 6 7 8 9 10 11 12
S1 S2 S3 S4
PC NC
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Figure 1: Schematic presentation of an example for location of samples and
controls on an ABI7500 plate.
Rows A-H; columns 1-12= layout`of the ABI 7500 plate
S1; S2; S3; S4= master mixes and enzyme and samples 1-4
PC= master mixes and enzyme and positive control
NC= master mix and enzyme and negative control
1 Sample 1
2 Sample 2
3 Sample 3
4 Sample 4
5 PC
6 NC
7
8
9
10
11
12
.
.
.
Figure 2: Schematic presentation of an example for location of samples and
controls on Rotor-Gene 6000 tubes.
1-12…= layout of the Rotor
S1; S2; S3; S4= master mixes and enzyme and samples 1-4
PC= master mixes and enzyme and positive control
NC= master mix and enzyme and negative control
Yellow background = master mix and enzyme with Ent-Gia-Cry PP Mix
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11.3 Programming of the thermocycler
FTD Stool parasites
Pay particular attention to the settings for the detectors:
Detector of the ENT specific Taqman probe = VIC (excitation
wavelength/emission wavelength 530/555 nm)
Detector of GIA specific Taqman probe= Fam (excitation wavelength/
emission wavelength 483/533 nm)
Detector of CRY specific Taqman probe= Rox (excitation wavelength/
emission wavelength 615/670 nm
Detector of mCMV (internal control) specific Taqman probe = Cy5 (excitation
wavelength/emission wavelength 550/575 nm)
PCR programme:
50°C for 15 minutes hold
95°C for 10 minutes hold
40 cycles of: 95°C, 8 sec.
60°C, 34 sec.
Detailed information on programming of the thermocycler is provided in the
instruction manual of the used cycler.
NOTE:
If you use the ABI7500, it is necessary to change the setting for the passive
reference dye as this assay uses ROX for the internal control. (By default, the
ROX dye is selected).
After the step of specification the detectors and task for each well, you click
finish and the software creates the plate document.
Click on a well or click-drag to select replicate wells.
Enter the sample name and change the passive reverence in “none”.
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12. Assay validation
Set a threshold as follows:
1. All negative controls should be below the threshold. If there is a potential
contamination (appearance of a curve), results obtained are not interpretable
and the whole run (including extraction) has to be repeated.
2. All the positive controls must show a positive (i.e. exponential) amplification
trace. The positive controls must be below a ct of 33.
3. Check the “component” trace before accepting the exponential trace as real.
Contact the equipment manufacturer or Fast-track Diagnostics for advice
(info@fast-trackdiagnostics.com)
4. All internal controls must show a positive (i.e. exponential) amplification trace.
The internal control must fall within a range of CT=30 +/-3. If the internal
control falls out of this range, then the run (including extraction) must be
repeated.
13. Calculation of the results
13.1 On ABI 7500
1. In the “Experiment completed” window click on OK or open the SDS-file of
your reaction per double click
2. You are now in the “setup” folder and see your plate, Click on the “result”
folder and you can see folders for Plate, Spectra, Component, Amplification Plot,
Standard Curve, Dissociation and Report
3. Click on “Amplification Plot” and mark all
samples in the table that you want to analyze. In
the figure are the samples A1, A2 and B1, B2
marked.
4. Set the cursor in the centre of the amplification trace, RIGHT click the mouse
then highlight “Graph settings” and LEFT click the mouse.
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5. Click on the OK button. This will give a linear graph.
FTD Stool parasites
6. In the drop down menu for DETECTOR choose the desired pathogen.
7. Set the manual base line finish to 1 cycle before the
first positive trace starts to appear, hit return and then
click the analyse button.
8. Using the cursor move the threshold to a point in
the exponential phase of amplification then click on
ANALYSE.
9. Click on REPORT, which shows the Ct value and
Standard Deviation
10. Repeat from point 5 with another detector for
another pathogen.
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FTD Stool parasites
13.2 On Rotor-Gene 6000
1. Click in the tool bar on analysis and the analysis floating window is
opening
2. This window shows you the new analyses. The tab allows you to select a
analysis method
3. Select the reactions (in the right list) which you
want to analyse
4. Double click on a new analysis and the windows of “quantitation analysis”, of
“quantitation results” and “standard curve” open
5. Each time you double-click on a new analysis, the 3 windows of quantitation
open
6. Click on arrange in the tool bar and all windows will be arranged
7. To set the threshold, click on the icon then click and hold on the graph and
drag the threshold to the right. This eliminates the threshold line for low cycle
numbers
8. Repeat since point 3 for each reaction
14. Interpretation of results
The positive controls and any positive samples will show an exponential
fluorescence trace. Any specimen displaying an exponential trace is considered
as positive. For example, if a sample (e.g. with the Ent-Gia-Cry PP Mix) shows an
exponential fluorescence trace at a wavelength between 483/533 (FAM) means
that it contains Giardia lamblia DNA.
If no Giardia lamblia DNA is detectable the sample can be considered as
negative for Giardia lamblia.
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FTD Stool parasites
15. Troubleshooting
No signal with positive controls
• Incorrect programming of the temperature profile of the thermocycler
Compare the temperature profile with the SOP
• Incorrect configuration of the PCR reaction
Check your work steps by means of the pipetting scheme and repeat
the PCR, if necessary.
Check calibration of pipettes
• The storage conditions for one or more product components did not
comply with the instructions or the FTD Stool parasites test had expired.
Please, check the storage conditions and the expiration date (see the
product label) of the reagents and use a new test, if necessary.
Weak or no signal of the positive control in fluorimeter channel
• The PCR conditions do not comply with the protocol.
Check the PCR conditions and repeat the PCR with correct settings, if
necessary
• The PCR was inhibited or no sample was added
Make sure that your DNA isolation method is correct
Signals with the negative control in fluorimeter channel
• A contamination occurred during preparation of the PCR
Repeat the PCR with new reagents in replicates
Strictly pipette the positive controls at last
Make sure that work space and instruments are decontaminated at
regular intervals.
If you have any further questions or if you encounter problems, please
contact us.
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