1. No category

EasyScreen™ Sample
Processing Kit
(Automated Platforms)
User Guide
A method for nucleic acid extraction from microorganisms
for use with compatible EasyScreen™ IVD Kits
For in vitro Diagnostic Use
REF SP001
VER 1.0
The Whipps Consultancy Limited 272 Bath Street, Glasgow G2 4JR
Table of Contents
1. Introduction
1.Introduction ...............................................................................3
Gastroenteritis is a widespread clinical problem causing worldwide
morbidity and mortality. Gastroenteritis can be caused by a wide range
of infectious agents including viral, bacterial and protozoa. It has been
estimated that foodborne gastroenteritis alone causes 76 million episodes of
illness, 325,000 hospitalisations, and 5,000 deaths each year in the United
States alone1.
1.1 Intended Use..................................................................................... 4
2. Notice To Customers.................................................................5
2.1 Important Information ........................................................................ 5
2.2 Summary and Explanation of the Test ............................................... 6
2.3 Principle of the Procedure ................................................................. 6
2.4 Limitations ........................................................................................ 8
2.5 Warnings and Handling Precautions ................................................ 11
2.6 Specimen collection, handling, storage and stability ........................ 12
2.7 Contents of the EasyScreen™ Sample Processing Kit..................... 13
2.8 Materials and Equipment Required (Not Supplied)............................ 13
3.Methods ..................................................................................14
3.1 Reagent Preparation........................................................................ 14
3.2 Protocol........................................................................................... 14
3.3 Quality Control ................................................................................ 16
3.4 Performance Characteristics............................................................ 16
In developed countries the mortality due to gastrointestinal infections is low but
morbidity and economic consequences can be high2. Conversely in developing
countries, gastroenteritis is the second most common cause of morbidity and
mortality causing the death of about 2 million children less than 5 years of age
each year3.
Rapid, simple and accurate diagnostic methods are required to ease the
burden on testing laboratories and provide better patient diagnosis and care.
Mead PS, et al. Food-related illness and death in the United States. Emerg Infect Dis.
1999;5:607–24.
1
van Maarseveen NM, Wessels E, de Brouwer CS, Vossen A, and Claas E.
Diagnosis of viral gastroenteritis by simultaneous detection of Adenovirus group
F, Astrovirus, Rotavirus group A, Norovirus genogroups I and II and Sapovirus in
two internally controlled multiplex real-time PCR assays. J Clin. Virol. 2010:49:205-210.
2
3
World Health Organization. Children’s environmental health. Available at
http://www.who.int/ceh/en/
4.Warranty ..................................................................................24
5.Troubleshooting ......................................................................24
2
3
1.1 Intended Use
The EasyScreen™ Sample Processing Kit (Automated Platforms)
( REF SP001) is an accessory designed to rapidly isolate nucleic acids
(DNA and RNA) from stool samples via an automated purification system.
The nucleic acids are converted into 3base™ sequences prior to purification
and are compatible with our range of real-time multiplex PCR (mPCR) assays
for a wide range of gastrointestinal pathogens including bacteria, protozoan
and viral targets.
This accessory kit includes all reagents required to take a sample from
the specimen, lyse any microorganisms and convert the nucleic acids
to a 3base™ form for use with the EasyScreen™ range of real-time
PCR based IVDs. The accessory is to be used in hospital and pathology
laboratories or similar by trained personnel.
This accessory kit does not produce any results, but is a necessary
requirement for the following kits:
CDD001: EasyScreen™ C. difficile Detection Kit: A screening test for
the detection of (i) toxigenic C. difficile (targets both tcdA and tcdB), (ii) an
endogenous extraction control (EC), and (iii) an internal positive control (IPC)
for detection of inhibitors.
CDD002: EasyScreen™ C. difficile Reflex Kit: A screening test for the
detection of hypervirulent C. difficile incl. 027 & 078 via (i) tcdC gene deletion
at position 117 and (ii) binary toxin gene (iii) gyrA gene mutation (fluroquinolone
resistance) and (iv) an internal positive control (IPC) for detection of inhibitors.
EB001: EasyScreen™ Enteric Bacteria Detection Kit: A screening test for
the detection of (i) Salmonella spp., (ii) Shigella spp., (iii) Campylobacter spp.,
(iv) Yersinia enterocolitica, (v) toxigenic C. difficile, (vi) Listeria monocytogenes,
(vii) an endogenous extraction control (EC), and (viii) an internal positive control
(IPC) for detection of inhibitors.
4
EP001: EasyScreen™ Enteric Parasite Detection Kit:
A screening test for the detection of (i) Entameoba complex, (ii) Dientameoba
fragilis, (iii) Cryptosporidium, (iv) Giardia intestinalis, (v) Blastocyctis hominis,
(vi) an endogenous extraction control (EC), and (vii) an internal positive
control (IPC) for detection of inhibitors.
EV001: EasyScreen™ Enteric Virus Detection Kit:
A screening test for the detection of (i) Norovirus I/II, (ii) Astrovirus,
(iii) Rotavirus (iv) Adenovirus, (v) Sapovirus, and (vi) an internal positive control
(IPC) for detection of inhibitors and (vii) endogenous extraction control (EC).
Coming Soon:
DEC001: EasyScreen™ Diarrheagenic Escherichia coli Detection Kit:
For the detection of (i) Enterohaemorrhagic E. coli (including O104 strain) (ii)
Enterotoxigenic E. coli (iii) Enteroinvasive E. coli (iv) Enteropathogenic E. coli
(v) Enteroaggregative E. coli directly from faecal samples and
(vi) endogenous extraction control (EC).
ID001: EasyScreen™ Influenza Detection Kit
For the detection of (i) Influenza A, (ii) Influenza B, (iii) Influenza A-H3
(with extraction control)
MTB001: EasyScreen™ M. tuberculosis Detection Kit
For the rapid detection of M. tuberculosis comprising (i) the 65 kDa heat
shock protein (mycobacterial universal), (ii) IS6110, (iii) 16S rDNA
M. tuberculosis region (with an extraction control).
2. Notice To Customers
2.1 Important Information
The EasyScreen™ Sample Processing Kit is authorised as an accessory
kit for use with the EasyScreen™ range of IVD kits. To discuss licensing
of the 3base™ technology for other applications, contact Human Genetic
Signatures Pty Ltd at: [email protected]
5
2.2 Summary and Explanation of the Test
A stool specimen from a patient suspected of having gasteroenteritis
(usually liquid or soft stool) is collected and transported to the testing
laboratory. A sterile dry swab is dipped into the stool material and processed
with the EasyScreen™ Sample Processing Kit ( REF SP001), which lyses
and converts the nucleic acids to a 3base™ form. Alternatively, a cultured
sample of bacteria may be processed with the EasyScreen™ Sample
Processing Kit ( REF SP001). An aliquot of the eluate is then added to the
PCR reagents supplied with the EasyScreen™ IVD Kit of choice, as listed in
section 1.1.
All EasyScreen™ IVD Kits are designed to include an internal positive control
(IPC), to detect any specimens that may contain PCR inhibitors and an
Extraction Control (EC), which amplifies and detects the 16s rDNA sequence
of any bacteria present in the stool sample to assure that the sample has been
prepared correctly. Both of these controls also confirm the integrity of the
PCR reagents and the extraction method.
2.3 Principle of the Procedure
The EasyScreen™ Sample Processing Kits ( REF SP001 or REF SP002)
lyse any microorganisms present and convert all Cytosine bases to Uracil
(detected as Thymine after PCR amplification) to create 3base™ DNA and
RNA. The 3base™ nucleic acids have a reduced complexity compared to the
genome comprised of the native 4 bases.
This complexity reduction results in genomes that are more similar to each
other, enabling the design of primers and probes that contain less mismatches,
are more homologous, produce better amplification and importantly have less
cross-reactivity when multiple strains of the same organism are present
(see Figure 1).
6
Figure 1a. Example of the 3base™ mechanism. The example sequences below
show the increase in homology from 75% (“Before”) to 95% (“After”) via the 3base™
conversion where all C bases are detected as T bases.
Before
After
Seq 1
GAT G G C GA T A TG GT T G A C AC GAT G G T G ATA T G G T T GATA T
Seq 2
GAT G G T GACA TG GTA G A T AC GAT G G T G ATA T G G T A GATA T
Seq 3
GAT G G T GA T A TG GTGG A C AC GAT G G T G ATA T G G TG GATA T
Seq 4
GAT G G T GA T A TG GTA G A T A T GAT G G T G ATA T G G T A GATA T
Seq 5
GAT G G T GA T A TG GTGG A C AC GAT G G T G ATA T G G TG GATA T
Seq 6
GAT G G C GACA TG GT T G A T A T GAT G G T G ATA T G G T T GATA T
Seq 7
GAT G G T GA T A TG GTGG A C AC GAT G G T G ATA T G G TG GATA T
Seq 8
GAT G G T GACA TG GTA G A T AC GAT G G T G ATA T G G T A GATA T
Seq 9
GAT G G T GA T A TG GTA G A T AC GAT G G T G ATA T G G T A GATA T
Seq 10
GAT G G T GA T A TG GTGG A T AC GAT G G T G ATA T G G TG GATA T
Consensus GAT G G Y GA Y A TG GTD G A Y A Y GAT G G T G ATA T G G T D GATA T
75% homology over 20 bases
95% homology over 20 bases
48 possible primer combinations
3 possible primer combinations
The 3base™ conversion occurs during the 15 minute incubation at 95°C (See section
3.2). After the nucleic acids have been purified the eluate is ready to be added to the
EasyScreen™ PCR reaction of choice (see Section 1.1)
Figure 1b. The
regular and 3base™
DNA sequence
for 2 primers and
2 probes is shown.
The primers and
probes for the 3base™
have a more similar
melting temperature
(Tm) improving the
efficiency of multiplex
real-time PCR.
Conventional Sequence
Tm
Primer 1 G T A C A C A CCGCCCG T CGC T CC T A CC 77°C
Primer 2 G A AGGAGA AG T CG T A A C A AG
56°C
Probe 1 T GA A T A A AGAGG T GA A A T T C T AGG
59°C
Probe 2 G A AGGGCCGCGAGCCCCCGCGC
87°C
3base™ Sequence
Tm
Primer 1 G T A T A T A T T G T T T G T T G T T T T T A T T 52°C
Primer 2 G A AGGAGA AG T T G T A A T A AG
50°C
Probe 1 T GA A T A A AGAGG T GA A A T T T T AGG
59°C
Probe 2 G A AGGG T T G T GAG T T T T T G T G T
62°C
7
2.4 Limitations
a.This kit does not produce any results on its own, but is an accessory
required for use with other EasyScreen™ in vitro diagnostic assays.
b.This kit is not to be used for therapeutic purposes.
c.Results from samples not stored properly or subjected to multiple
freeze/thaw cycles should be treated with caution. See section 2.6 for
more information.
d.Different models of automated nucleic acid extraction instruments perform
differently and these differences may affect results. This kit has been
validated using a Versant Sample Preparation Kit 1.0 on a KingFisher Flex
(Thermo Scientific) system. Other systems should be validated before
use – please contact Genetic Signatures for recommended protocols for
alternate systems.
e.Potential interfering substances were assessed according to the following
list. No inhibition was observed when the substance was added at the
stated concentrations.
8
Contaminating
substance
Concentration
tested*
Active agent
Mucin
6 mg/ml
Purified mucin protein
NI^
Fecal fat
6.4 mg/ml
Stearic acid
10 mg/ml
Palmitic acid
Stearic acid
Palmitic acid
NI
Imodium
4 mg/ml
Loperamide hydrochloride
NI
Mylanta, double
strength
5%
Aluminium hydroxide 80mg/ml
Magnesium hydroxide 80 mg/ml
Simethicone 6 mg/ml
NI
Vancomycin
hydrochloride
13.8 mg/ml
Rectinol/
haemarrhoid
cream
9 mg/ml
Metranidazole
15 mg/ml
NI
Barium sulphate
7.8 mg/ml
NI
Blood
20-40% v/v
NI
Summer’s
eve™ Feminine
Lubricating Jelly
50 mg/ml
NI
KY gel
60 mg/ml
Chlorhexidine gluconate
Methyl benzoate
NI
Vagisil feminine
itching cream
60 mg/ml
Benzocaine 50 mg/g
Resorcinol 20 mg/g
NI
Vaseline white
petroleum jelly
50 mg/ml
Canesten
54 mg/ml
SP001
NI
Zinc oxid 200 mg
Cinchocaine hydrochloride 5 mg
NI
NI
200 mg Clotrimazole (1% v/v)
NI
*Mock stool sample was prepared by adding the above substances at the above
concentration, and then a swab was added and processed as per the EasyScreen™
Sample Processing Kit protocol. No inhibition was observed, with both the IPC and
EC detected in each case, when samples were amplified using the ( REF CDD001 kit).
Multiples of the above potential interfering substances were not tested. The IPC and
the EC included in each reaction are primer limited and are designed to detect if PCR
inhibition is present. ^NI - not interfering.
9
f.Incorrect test results may arise from incorrect specimen collection,
handling or storage, presence of inhibitors, technical error, sample mixup or because the number of organisms in the specimen is below the
analytical sensitivity of this test. This use of this test must be limited to
trained personnel who strictly follow the instructions provided in this
user guide.
g.The methods used here detect genetic material and are not able to
determine if the organism was viable or not. All positive results should
be treated as presumptive positive and confirmed with traditional culture
techniques if viability of the organism needs to be assessed.
h.If the Extraction Control in the downstream PCR is negative the operator
must repeat the extraction, which will lead to a delay in obtaining results.
2.5 Warnings and Handling Precautions
It is the responsibility of all users to consult the Material Safety Data Sheet
(MSDS) before using this product. The MSDS for the EasyScreen™ Sample
Processing Kit is available at www.geneticsignatures.com
• R
eagents 1 and 2 are potential irritants. Wear gloves and avoid inhaling
dust when adding Reagent 1 to the bottle containing Reagent 2.
• D
o not proceed with the sample preparation if the product has been
damaged or opened during shipment.
• This product is strictly for use by qualified laboratory and medical
technicians with appropriate training in laboratory techniques and good
laboratory practices.
• W
ear lab coats, disposable gloves and protective glasses
where appropriate.
• B
iological samples may contain infectious agents. Handle with care.
Assume all biological samples are potentially infectious and handle
according to local safety and containment regulations.
• A
ll waste produced during use of the kit must be treated as hazardous
waste. Observe local biohazard, safety and other regulations.
10
11
2.6 Specimen collection, handling, storage and stability
The EasyScreen™ Sample Processing Kit is designed to work with
stool specimens that have been collected in sterile tubes. Once collected,
the specimens should be stored between 4°C and 25°C during transport.
Specimens can be kept at room temperature (15-25°C) up to 48 hours before
testing but should be tested as soon as possible after collection. For longerterm storage, the specimens may be frozen, and tests may be performed up to
a maximum of 3 freeze/thaw cycles.
All reagents should be stored at ambient temperature (15°C-25°C).
After mixing Reagents 1 and 2, store any unused material in the dark
from 15°C-25°C for up to 4 weeks.
All reagents in this kit must be used by their expiry date printed on the outer
label and on individual reagents.
The eluted nucleic acids can be stored at -70°C for long-term storage
(up to 2 months), and tests may be performed on the sample after up to a
maximum of 3 freeze/thaw cycles.
2.7 Contents of the EasyScreen™ Sample Processing Kit
Component Name
Contents
Reagent 1
5 x 5 mL
Reagent 2
5 x 3.5 g
Elution Solution
5 x 3.5 mL
Sheathed Swabs
100
Reaction Tubes (1.5 mL)
100
Screw cap lids
100
2.8 Materials and Equipment Required (Not Supplied)
• A
n automated nucleic acid extraction system, such as: Qiagen M48,
Qiagen EZ1, Qiagen QIAsymphony, Roche MagNApure 32, Thermo
KingFisher Flex, bioMérieux easyMag
• C
ompatible reagents for use with the above platforms. Where possible
we recommend using the Versant Sample Preparation 1.0 kit
( REF 10472144).
Standard laboratory Equipment (not supplied)
•
•
•
•
•
•
•
12
imer
T
Aerosol barrier tips (10 µL – 1000 µL)
Heat block set at 95°C
Hand pipettes, up to 1000 µL
A centrifuge suitable for 1.5 mL tubes.
An incubator set at 80°C (optional).
Sterile toothpicks.
13
3. Methods
If using the EasyScreen™ Sample Processing Kit for the first time, it is highly
recommended that the detailed methodology in this User Guide be read in its
entirety before carrying out the method.
3.1 Reagent Preparation
• T ransfer the total volume of Reagent 1 to the Reagent 2 bottle and
mix by gentle inversion. Place the combined reagents at 80°C, mixing
occasionally, until all of the powder is completely dissolved. If an 80°C
incubator is not available the reagent can be dissolved by continual
shaking for 1-2 minutes. Prepare as many bottles as needed. Each
combined bottle has sufficient reagent for 20 reactions. Any left over
combined reagent can be stored at 15°C-25°C for up to 4 weeks.
3.2 Protocol
1.For an individual sample add 250 µL of combined reagent 1 and 2 into a
1.5 mL reaction tube and secure the cap.
2.Take one of the swabs provided and prepare the sample using one of the
methods below:
• F
or liquid stool specimens, dip the swab into the sample, mix well and
then transfer the swab to the reaction tube. Mix by swirling the swab
around in the buffer several times, then cap the tube. Re-sheath the
swab and discard.
14
• F
or solid or semi-solid specimens first pre-wet the swab in
combined reagent 1 and 2, then dip the swab into the specimen
several times to obtain a representative sample, as pathogens may
be non randomly distributed in the sample. Transfer the swab to the
reaction tube, mix by swirling the swab around in the buffer several
times, then cap the tube. Re-sheath the swab and discard.
• A
lternatively, if starting with cultures, it is sufficient to use a sterile
toothpick to scrape off a single colony and transfer to the Reaction
Tube. Mix well by twirling the toothpick in the reagent. Cap the tube.
3. Incubate the samples at 95°C for 15 minutes.
4.Centrifuge samples at 13000 x g for 1 min and transfer the supernatant
to a reaction tube compatible with the automated instrument of choice.
5.Transfer the reaction tube(s) to the automated instrument of choice
and follow the instrument specific instructions, eluting in 50 µL
where possible.
6.Add 10% volume of Elution Solution to the eluate (i.e. Add 5 µL Elution
Solution to 50 µL of eluate). The Elution Solution is coloured for easy
sample tracking. The converted purified nucleic acid is now ready for
PCR using any of the multiplexed kits listed in Section 1.1.
Note: Customised instructions for Qiagen M48, Qiagen EZ1, Qiagen
QIAsymphony, Roche MagNApure 32, Thermo KingFisher Flex and
bioMérieux easyMag are available for download from
www.geneticsignatures.com and it is recommended that those individual
instructions be consulted before use. The remainder of the eluate can
be stored at -70°C for long-term storage (2 months), and tests may be
performed on the eluate after up to 3 freeze thaw cycles.
15
3.3 Quality Control
3.4.1 Analytical Sensitivity
This kit is an IVD accessory and as such does not produce any measurable
results on its own. Results are generated with separate IVD kits available
from Genetic Signatures. It is recommended that a Negative Run Control be
included every time a batch of samples is processed with the EasyScreen™
Sample Processing Kit (Automated Platforms). This negative control should
be included in order to detect reagent or environmental contamination
(or carry-over) by microorganisms or amplicons from a previous PCR.
The negative control should be processed with the test samples.
Analytical sensitivity of the EasyScreen™ Sample Processing Kit
(Automated Platforms) was determined by processing a known amount
of Clostridium difficile NATtrol™ External Run Control Medium (Zeptometrix
REF NATCdi(NAP1)-ERCM) estimated to be 1.14x105 cfu/ml by the
manufacturers). The bacteria was diluted and 5 replicates were processed
with the EasyScreen™ Sample Processing Kit (Automated Platforms)
and amplified with the EasyScreen™C. difficile Detection Kit
( REF CDD001). The results are summarised below.
Additional internal controls are included in all IVD kits available from Genetic
Signatures. These include an Extraction Control (EC), which monitors reagent
failure, user error and insufficient starting material and an Internal Positive
Control (IPC), which detects any PCR inhibition that may be present in the
processed clinical specimen.
3.4 Performance Characteristics
As the EasyScreen™ Sample Processing Kit (Automated Platforms) is an
IVD accessory, it does not produce any measurable results on its own.
The following information was generated using the eluate obtained with this
kit, together with the EasyScreen™ C. difficile Detection Kit ( REF CDD001)
and is included only as an indication of performance. Additional and specific
information regarding performance characteristics is available in the User Guide
of each IVD detection kit supplied by Genetic Signatures.
Table 1 Analytical sensitivity summary
Starting
Concentration (cfu)
Amount seeded
into the PCR (cfu)
Number detected
2000
160
5/5
1000
80
5/5
500
40
5/5
250
20
5/5
125
10
5/5
0
0
0/5
The above “starting concentrations” were processed with the EasyScreen™
Sample Processing Kit (Automated Platforms) using a Versant Sample
Preparation 1.0 Reagent Kit on a KingFisher Flex robot. The sample was
eluted with 50 µL of elution solution and 4 µL was added to the PCR
components supplied with the EasyScreen™C. difficile Detection
Kit ( REF CDD001). The kit was able to detect at least 10 cfu of C.
difficile material that was prepared using the the EasyScreen™ Sample
Processing Kit (Automated Platforms).
16
17
3.4.2 Clinical Sensitivity
3.4.3 Reproducibility
Clinical sensitivity of the EasyScreen™ Sample Processing Kit (Automated
Platforms) was assessed by inoculating different starting amounts of
Zeptometrix ERCM NAP1 culture into a stool emulsion, and processed using
this kit in combination with the Versant Sample Preparation 1.0 Reagent Kit
on a KingFisher Flex extraction system. The stool emulsion was prepared by
inoculating a C. difficile negative stool sample into combined Reagents 1 and
2. The following amounts of the positive control culture were then added to
the stool emulsion 2500, 2000, 1000, 500, 250, 125, 62.5 and 31.25 cfu. Five
replicates of each control culture concentration were assessed. Four microlitres
of the eluate was then amplified with the EasyScreen™ C.difficile Detection
kit ( REF CDD001).
In order to assess the reproducibility of the EasyScreen™ Sample
Processing Kit (Automated Platforms) a panel consisting of three (3)
simulated specimen categories where each tube contained a negative stool
sample spiked with a low amount (500 cfu), medium amount (1000 cfu) and
high amount (2500 cfu) of Clostridium difficile NATtrol™ External Run Control
Medium (Zeptometrix REF NATCdi(NAP1)-ERCM) DNA. The specimens
were tested in five (5) replicates per run, on five (5) distinct days (consecutive
or not).
Results were as follows:
1
Starting
Concentration (cfu)
Amount seeded
into the PCR (cfu)
Number detected
2500
200
5/5
1000
80
5/5
500
40
5/5
250
20
5/5
125
10
5/5
62.5
5
5/5
31.25
2.5
0
Stool only
The EasyScreen™ Sample Processing Kit (Automated Platforms)
used in combination with the Versant Sample Preparation 1.0 Reagent Kit
on a KingFisher Flex robot returned a positive result for (i) the low positive
C. difficile specimen category of 100%; (ii) the medium positive C. difficile
specimen category of 100%, and (iii) the high positive C. difficile specimen
category of 100%. The negative specimen was negative 100% of the time.
The test regime above was also performed by a different operator, on a
different day, with the same result.
5/5
1
0/5
C.difficile negative stool sample was positive for 16s and IPC, and negative for tcdA/tcdB
The EasyScreen™ Sample Processing Kit (Automated Platforms) was able
to process material from 31.25 cfu (equivalent to 2.5 cfu per reaction) in 5/5
cases. This corresponds to 31.25 starting cfu on the swab being eluted into
50 µL of elution solution with 4 µL being seeded into the PCR.
18
19
3.4.4 Specific Performance Characteristics
4. Warranty
As a test of performance characteristics, a clinical testing facility processed a
total of 94 patient stool samples with the EasyScreen™ Sample Processing
Kit (Automated Platforms) in combination with the Versant Sample
Preparation 1.0 Reagent Kit on a KingFisher Flex robot. A total of 4 µL of the
eluate was examined with the EasyScreen™ C. difficile Detection Kit
( REF CDD001) and compared against toxigenic culture. All toxigenic
culture positive samples were correctly identified when processed with the
EasyScreen™ Sample Processing Kit (Automated Platforms) and amplified
with the EasyScreen™ C. difficile Detection Kit. In addition, 4 culture toxin
negative samples were called positive for presence of toxigenic C. difficile via
the EasyScreen™ C. difficile Detection Kit. These 4 samples were found to
be positive by another molecular test targeting the tcdA gene. These additional
4 samples may have contained non-viable C. difficile DNA, or it may be that the
molecular tests for C. difficile DNA are more sensitive than traditional culture
methods, in agreement with a recent study that found multiple independent
PCR positive samples that were negative using the culture toxin assay as the
gold standard (Knetsch CW, Bakker D, de Boer RF, et. al. Comparison of
real-time PCR techniques to cytotoxigenic culture methods for diagnosing
Clostridium difficile infection. J. Clin. Microbiol. 2011;49:227-31.)
The EasyScreen™ Sample Processing Kit (Automated Platforms) is
warranted to perform as described in the labelling and literature when used
in accordance with the supplied instructions. Human Genetic Signatures
Pty Ltd’s (trading as Genetic Signatures) sole obligation and the purchaser’s
exclusive remedy for breach of this warranty shall be, at the option of
Human Genetic Signatures Pty Ltd, to repair or replace the products.
Human Genetic Signatures Pty Ltd will not be liable for any incidental or
consequential damages in connection with the EasyScreen™ Sample
Processing Kit (Automated Platforms).
5. Troubleshooting
Problems
Possible Solutions
No PCR signal was observed for
any of the processed samples,
in any of the channels including
the positive control.
Confirm that the kit is within the expiry date and all
components have been stored as directed.
No PCR signal was observed
for any of the processed
samples, including the positive
control, except for the Internal
Positive Control.
The sample preparation has failed – check that the
combined Reagent 1 and Reagent 2 was no older
than 4 weeks. Make sure that all the steps in the
protocols were followed.
DNA was degraded during extraction - check that
all pipette tips etc used during the procedure were
of molecular biology quality (ie DNase free).
Sample DNA was degraded before processing
– check that the samples have been stored/
handled correctly.
20
21
Explanation of Symbols
Symbol
Meaning
The device is considered to meet the essential
requirements in Article 3 of Directive 98/79/EC on
in vitro diagnostic medical devices
Consult instructions for use
In vitro diagnostic medical device
Catalogue number
Single use, use only once
The number of reactions
25°C
15°C
Temperature limitation
Batch code
Expiration date
Name and address of the Authorised
Representative in the European Commission
Name and address of the manufacturer
22
EasyScreen™ Sample Processing Kit is covered by patents owned
by Human Genetic Signatures Pty Ltd granted in a number of countries
including AU 2005312354, CN 200580047630.3, IN 246868, ID
WO0200702024, MY 142997, MX 2007/006610, NZ 555620, SG 132883,
ZA 2007/06142 and US 7833942. Corresponding patent applications are
pending in a number of countries and regions including EP 05813335.6.
The Polymerase Chain Reaction (PCR) process is covered by U.S. Pat. Nos.
4,683,195 and 4,683,202 assigned to Hoffmann-La Roche. Patents pending
in other countries. No license under these patents to use the PCR process
is conveyed expressly or by implication to the purchaser by the purchase
of the EasyScreen™ Sample Processing Kit. Further information on
purchasing licenses to practice the PCR process can be obtained from the
director of Licensing at Applied Biosystems, 850 Lincoln Centre Drive, Foster
City, California 94404 or at Roche Molecular Systems, Inc., 1145 Atlantic
Avenue, Alameda, California 94501.
23
Human Genetic Signatures Pty Ltd
c/o Virology Research Laboratory
Level 3, Clinical Sciences Building,
Prince of Wales Hospital,
Randwick, NSW 2031, Australia
Phone: +61 2 9870 7580
Fax: +61 2 9889 4034
Email: [email protected]
www.geneticsignatures.com
VER 2.0 October 2012