Seegene HPV28 manual en inglés
Transcript of Seegene HPV28 manual en inglés
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AnyplexTM II
HPV28 Detection(Cat. No. HP7S00X)
AnyplexTM II PCR System for detection of human papillomavirus - 19 high-risk HPV
types(16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 69, 73, 82) and 9
low-risk HPV types(6, 11, 40, 42, 43, 44, 54, 61, 70) from cervical swab and liquid
based cytology specimens.
For use with the
1. CFX96TMReal-time PCR System(Bio-Rad)
European Union : (In Vitro Diagnostic Use)
This product can be used for IVD purposes in European union.
Other Countries : (Research Use Only)
This product should be used for RUO purposes in other countries.
Not available in the U.S.
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TABLE OF CONTENTS
NOTICES --------------------------------------------------------------------------------------------
INTENDED USE -----------------------------------------------------------------------------------
PRINCIPLES AND PROCEDURE OVERVIEW -------------------------------------------
BACKGROUND INFORMATION --------------------------------------------------------------
REAGENTS -----------------------------------------------------------------------------------------
STORAGE AND HANDLING -------------------------------------------------------------------
MATERIALS REQUIRED BUT NOT PROVIDED -----------------------------------------
PROTOCOL -----------------------------------------------------------------------------------------
REAL-TIME PCR INSTRUMENT SET UP AND RESULTS ANALYSIS ------------
RESULTS --------------------------------------------------------------------------------
TROUBLESHOOTING ---------------------------------------------------------------------------
PERFORMANCE ----------------------------------------------------------------------------------
REFERENCES -------------------------------------------------------------------------------------
EXPLANATION OF SYMBOLS ----------------------------------------------------------------
ORDERING INFORMATION --------------------------------------------------------------------
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NOTICES
This product can be used for IVD(In Vitro Diagnostics) purposes in EU as the IVD CE Mark
is reviewed by EU Directive(98/79/EC), but should be used for RUO(Research Use only)
purposes in other countries.
If this product is used with MICROLAB NIMBUS IVD and MICROLAB STARlet, maximum 5
separate runs.
This test has been validated for the following specimen types: cervical swab and
liquid based cytology specimens. This test has not been validated for any other types of
specimens.
Store DNA samples at -70
until use and keep on ice during use.
The sensitivity of an assay may decrease if samples are repeatedly frozen/thawed or stored
for a longer period of time.
Reliability of the results depends on adequate specimen collection, transport, storage and
processing procedure.
Workflow in the laboratory should proceed in a unidirectional manner.
Always wear disposable gloves in each area and change them before entering different
areas. Change gloves immediately if contaminated or treat them with DNA decontaminating
reagent.
Dedicate supplies and equipment to the separate working areas and do not move them
from one area to another.
Do not pipette by mouth.
Do not eat, drink or smoke in laboratory work areas. Wear disposable powder-free gloves,
laboratory coats and eye protections when handling specimens and reagents. Wash hands
thoroughly after handling specimens and test reagents.
Avoid contamination of reagents when removing aliquots from reagent tubes. The use of
sterile aerosol resistant disposable pipette tips is recommended.
Do not pool reagents from different lots or from different tubes of the same lot.
Do not use the product after its expiration date.
Use screw-capped tubes and prevents any potential splashing or cross-contamination of
specimens during preparations.
Please be careful not to contaminate reagents with extracted nucleic acids, PCR products,
and positive control. To prevent the contamination of reagents, the use of filter tips is
recommended.
Use separated and segregated working areas for each experiment.
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Prepare and use a different pipette set for each of the following areas: Nucleic acid
extraction, reagent mixing, nucleic acid template addition, and PCR product addition.
Only at the designated space, open the reaction tubes or strips after the amplification, to
avoid contamination with amplicon.
Store positive materials separated from kits reagents.
Laboratory safety procedures(refer to Biosafety in Microbiological and Biomedical
Laboratories & CLSI Documents) must be taken when handling specimens. Thoroughly
clean and disinfect all work surfaces with 0.5% sodium hypochlorite(in de-ionized or distilled
water).
INTENDED USE
The AnyplexTM
II HPV28 Detection is a qualitative in vitro test for the detection of human
papillomaviruses in liquid based cytology and cervical swab specimens.
The AnyplexTM
II HPV28 Detectionassay consist of two PCR reaction(A set and B set).
A setis a multiplex assay that permits the simultaneous amplification of target DNA of 14 high-
risk human papillomaviruses.
B setis a multiplex assay that permits the simultaneous amplification of target DNA of 5 high-
risk and 9 low-risk human papillomaviruses.
Category Types
A set14 high-risk HPV types
(16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68)
B set5 high-risk HPV types(26, 53, 69, 73, 82)
9 low-risk HPV types(6, 11, 40, 42, 43, 44, 54, 61, 70)
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PRINCIPLES AND PROCEDURE OVERVIEW
1. Principles
The AnyplexTM
II HPV28 Detection Assay, represents Seegenes proprietary technologies and is
based on a newly developed TOCETM
technology which makes it possible to detect multi-
pathogens in a single fluorescence channel on real-time PCR instruments.
In current melting curve analysis, temperature differences are often observed among DNAs that
have high sequence variation, resulting in issues the field of clinical diagnostic where accurate
and reproducible test results are critical. However, TOCETM
technology is designated not to be
affected by sequence variations; therefore it guaranteeing consistent Tm values.
The AnyplexTM
II HPV28 Detection can perform multiplex examination by either End point-CMTA
(End point-Catcher Melting Temperature Analysis) or Cyclic-CMTA(Cyclic-Catcher Melting
Temperature Analysis) method. Cyclic-CMTA method which represents a new class of
molecular tests can discriminate major pathogen in the co-infected samples. The AnyplexTM
II
HPV28 Detection is a multiplex real-time PCR assay that permits the simultaneous
amplification, detection and differentiation of target nucleic acids of 19 high-risk HPV types(16,
18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 69, 73, 82) and 9 low-risk HPV types(6,
11, 40, 42, 43, 44, 54, 61, 70) as well as Internal Control(IC).
In PCR, efficiency can be reduced by inhibitors that may be present in the clinical specimens.
An Internal Control(IC) is incorporated into the product as an endogenous whole process
control in order to monitor nucleic acid isolation, and to check for possible PCR inhibition. The
IC is co-amplified with the target nucleic acids within the clinical specimens. The AnyplexTM
II
HPV28 Detection uses Human house-keeping gene as an endogenous IC which can ensure
purification of DNA, verification of PCR reaction and clarification of cell adequacy from each
specimen.
The Uracil-DNA glycosylase(UDG)d-UTP system is employed in the AnyplexTM
II HPV28
Detection. The UDG-dUTP system is commonly use when performing PCR to eliminate
amplicon carry-over using UDG excises uracil residues from DNA by cleaving the N-glycosylic
bond.
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2. Procedure Overview
< AnyplexTMII HPV28 Detection procedure overview >
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BACKGROUD INFORMATION
Human Papilloma Virus(HPV) infection is linked with cervical cancer. HPV can be divided into
high-risk(HR) and low-risk(LR) groups on the basis of their association with cervical lesions.
Therefore, it is very important to know which type of HPV is infected in patients to prevent
cancer development and transmission of disease. Currently, commercially available major
products to diagnose HPV are based on probe-hybridization method to detect and/or genotype
HPV. However, main defect of the probe-hybridization based methods are high false positive
rate due to cross-reactivity between probes and various kinds of viral DNA or PCR amplicons
used for hybridization. Here we are introducing an innovative HPV detection/genotyping assay
system which amplifies only specific targets without any cross reactivity and is automated in
detection using real-time PCR method. Eventually the AnyplexTM
II HPV28 Detection only
specifically detects true HPV and accurately genotypes them. It also contains endogenous
Internal Control to check any inhibition that might occur during PCR reaction.
Cervical cancer, which progresses from the precancerous stage to invasive cancer, has 7-20
years of precancerous stage; consequently early diagnosis is possible when HPV infection is
suspected. High-risk HPV group may lead to the development of cervical cancer; especially,
HPV16 and 18 are associated with 70% of cervical cancer case. On the other hands, low-risk
HPV group including HPV6 and 11 may cause genital warts. AnyplexTM II HPV28 Detection can
identify 19 high-risk HPV types including HPV16 and 18 and also detect for 9 low-risk HPV
types such as HPV6 and 11 at the same time.
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REAGENTS
The reagents contained in one set are sufficient for 100 determinations.
AnyplexTM
II HPV28 Detection order information( HP7S00X).
AnyplexTM II HPV28 Detection
Symbols Contents Volume Description
4X HPV28 A TOM 500 LTOCE Oligo Mix(TOM):
- Amplification and detection reagents
4X HPV28 B TOM 500 LTOCE Oligo Mix(TOM):
- Amplification and detection reagents
4X Anyplex
PCR Master Mix
(with UDG)
500 L
X 2
- DNA polymerase
- Uracil-DNA glycosylase(UDG)
- Buffer containing dNTPs
HPV28 PC1 100 LPositive Control(PC) :
- Mixture of pathogen clones
HPV28 PC2 100 LPositive Control(PC) :
- Mixture of pathogen clones
HPV28 PC3 100 LPositive Control(PC) :
- Mixture of pathogen clones
RNase-free Water1,000 L
X 2
Ultrapure quality, PCR-grade
Negative Control(NC) :
- Sterilized water as Negative Control
User manual
AnyplexTM II is a trademark of Seegene Inc.
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STORAGE AND HANDLING
The components of the AnyplexTM
II HPV28 Detection should be stored at -20. All
components are stable under recommended storage conditions until the expiry date stated on
the label. Repeated thawing and freezing should be avoided, as this may reduce the sensitivity.
If the reagents are to be used only intermittently, they should be frozen in aliquots.
MATERIALS REQUIRED BUT NOT PROVIDED
Disposable powder free gloves(latex or nitrile)
Pipettes(adjustable) and Sterile pipette tips
1.5 ml microcentrifuge tube
Nucleic acid isolation kit(see Nucleic Acid Isolation)
Proteinase K(For SEEPREP12TM
, Cat. No.P4850, SIGMA)
Ice Maker
Desktop centrifuge
Vortex mixer
CFX96TMReal-time PCR system(Bio-Rad)
Optical Flat 8-Cap Strips(Cat No. TCS0803, Bio-Rad)
Low-Profile 0.2 mL 8-Tube Strips without Caps(white color, Cat. No. TLS0851, Bio-Rad)
96-Well Skirted PCR Plate, white well(Cat. No. HSP-9655, Bio-Rad)
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PROTOCOL
1. Specimen Collection, Storage, and Transport
Note: All samples have to be treated as potentially infectious materials. Only those sample
materials are permitted, which are collected, transported and stored attending strictly the
following rules and instructions :
Note: To ensure a high sample quality, the specimens should be transported as fast as possible.
The specimens have to be transported at the indicated temperature conditions.
A. Specimen Collection
Liquid based cervical cyto logy s pecimen
Follow the manufacturers instructions for collecting cervical cell specimens into ThinPrep
or SurePathTM
media.
Cervical swab sp ecimen
For the collection of cervical swab specimen, please use following materials :
Cervical swabs can be collected and transported in the following mediums :
- ENAT(COPAN)
Cervical specimen collection kit Manufacturer Cat. No.
ENAT PM 2ML L-SHAPE APPLICATOR COPAN 606CS01L*
* If you would like to purchase the above products from Seegene, Inc., please use this Cat. No.
Leave the swab in the culture transport medium. Close and label the sample container.
Stick closely to the instructions given for storage and transport.
Please follow a recommended protocol to collect columnar and squamous epithelium cells
after removal of the cervical mucus.
B. Specimen Storage
The sensitivity of an assay may decrease if samples are repeatedly frozen/thawed or stored for a
longer period of time.
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Liquid based cervical cyto logy s pecimen
Cervical cell specimens collected in ThinPrep
medium may be stored at 2 ~ 8
for up to 6weeks. Cervical cell specimens collected in SurePath
TMmedium may be restored at 2 ~ 8
for up to 2 weeks.
Note:The specimens should be extracted to nucleic acid as quickly as possible.
Cervical swab sp ecimen
If the swab specimens are not processed directly after their receipt in the laboratory, they have
to be stored at 2 ~ 8and have to be processed within seven days.
C. Specimen Transport
To ensure a high quality of sample, specimens should be transported as soon as possible at
indicated temperature.
Liquid based cytology specimen
Cervical cell specimens collected in ThinPrep medium can be transported at 2 ~ 25.
Specimens collected in SurePathTM
medium must be transported at 2 ~ 8
Cervical swab sp ecimen
Cervical swab specimens must be transported cooled.
Cervical swab specimens should be shipped to a laboratory as soon as possible after
collection, following the laboratory instructions for transports under cooling. The samples
should be transported following also the local and national instructions for the transport of
pathogen material.
2. Nucleic Acid Isolation
Various manufacturers offer nucleic acid isolation kits. Use right amount of sample according to the
protocol in use. The following isolation kits have been validated for use with this kit.
A. Pre-treatment of Liquid based cervical cytology specimen
Equilibrate samples to room temperature(19 ~ 25).
Centrifuge 1 mL of liquid based cervical cytology specimen for 15 minutes at 15,000 x g
(13,000 rpm).
The supernatant has to be discarded. Afterwards, the recommend volume(200 ~ 450 L, See
Recommended Vol. of 2-C, D) should be resuspended in 1X PBS by vortexing thoroughly
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to redissolve.
Note: Process pre-treatment step using lysis buffer in extraction kit not 1X PBS if the samples arecollected in SurePath
TMmedium and would be analyzed with NIMBUS or STARlet.
Follow the manufacturers protocol.
B.Cervical swab specimen
Equilibrate samples to room temperature(19 ~ 25).
For cervical swab specimens which contain a swab in the culture transport media specimens
should be mixed by vortexing.
The caps from specimen tubes have to be removed carefully to avoid contaminations. Any
excess mucus in the specimen should be removed at this time by collecting it on the swab.
Any residual liquid from the mucus and the swab should then be expressed by pressing the
swab against the slide of the tube. Finally the swab and the mucus should be removed and
discarded.
ENAT specimens may be processed directly out of their primary container.
C. Manual Prep Kits
Isolation Kit Manufacturer Cat. No. Recommended Vol.
QIAampDNA Mini Kit* QIAGEN 51304
Specimen: 200 L
Elution: 50 L
Ribo_spin vRD**(Viral RNA/DNA Extraction Kit)
GeneAll302-150
SG1701***Specimen: 200 L
Elution: 50 L
* Process lysis step using 180 L of ATL buffer instead of AL buffer in case of SurePathTM
media.
**Ribo_spin vRD kit is not compatible with SurePathTM
media.
*** If you would like to purchase the above products from Seegene, Inc., please use this Cat. No.
D. Automated Purification System
Note: See MICROLAB NIMBUS IVD operation manual.
Automated Purification System Manufacturer Cat. No. Recommended Vol.
MICROLABNimbus IVD Hamilton 65415-02*
STARMag 96 Tissue Seegene744300.4.
205875
Specimen: 450 L
Elution: 100 L
STARMag 48 X 8 Tissue Cartridge
Kit
Seegene744300.4.
TC384
Specimen: 450 L
Elution: 100 L
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E. Summary
QIAGEN1 GeneAll
2 SEEPREP12 NIMBUS/STARlet
Swab ENAT O O O O
LBCThinPrep O O O O
SurePathTM
O3 X O O4
1. QIAamp DNA Mini Kit
2. Robo_spin vRD (Viral RNA/DNA Extraction Kit)
3. Process lysis step using 180L of ATL buffer instead of AL buffer.
4. If DNA is extracted from SurePathTM
specimens with NIMBUS or STARlet, there is a possibility that the
sensitivity could be reduced compared to other extraction methods.
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3. Preparation for Real-time PCR
Note: The correct tubes and caps must be used(see MATERIALS REQUIRED BUT NOT
PROVIDED).
Note:Aerosol resistant filter tips and tight gloves must be used when preparing specimens. Use
an extreme care to ensure no cross-contamination.
Note: Completely thaw the reagents on ice.
Note: Briefly centrifuge the reagent tubes to remove drops from the inner cap.
5 L 4X HPV28 A TOM or B TOM
5 L 4X Anyplex PCR Master Mix (with UDG)
5 L
5 L
RNase-free Water
samples nucleic acid
20 L Total volume of PCR reaction
Note: Calculate the necessary amount of each reagent needed based on the number of
reactions(samples + controls).
Note: Use a new sterile pipette tip for each sample.
Note: For Negative Control, use 5 L of RNase-free Water instead of samples nucleic acid.
Note: For Positive Control, use 5 L of each HPV28 PC1, PC2 and PC3.
Note: Please be careful not to cross-contaminate the PCR Mastermix and samples with the
Positive Control.
Note: Do not label the cap of the reaction tubes as fluorescence is detected through the cap.
Positive Control
There are three Positive Control tubes included in the kit; HPV28 PC1, PC2 and PC3.
Each PC includes clones for 5 targets in A set(14 types of high risk and IC) and 5 targets in B
set(5 types of high risk, 9 types of low risk and IC).
Note:To run the Positive Control reaction, prepare three PCR tubes for each set, six PCR tubes
in total;
For A set, first tube with PC1, second tube with PC2 and third tube with PC3.
For B set, first tube wz`ith PC1, second tube with PC2 and third tube with PC3.
(See RESULTS)
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REAL-TIME PCR INSTRUMENT SET UP AND RESULTS ANALYSIS
1. CFX96TM
Real-time PCR System(Bio-Rad)
1.1. Real-time PCR Instrument set up
Note: The CFX96TM
Real-time PCR System(Bio-Rad) experiment setup program for the
detection of 28 types of HPV and IC can be divided into following three steps:
- Protocol Setup
- Plate Setup
- Start Run
A. Protocol Setup
1) In the main menu, click Protocol to open the Experiment Setup.
Fig. 1. Protocol Setup. Create a new protocol or load an existing protocol for the experiment.
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2) In Protocol Editor, define the thermal profile as follows:
i) Cyclic-CMTA (Melt analysis of three times)
Segment Temperature Duration No. of cycles
1 50C 4 min
2 95C 15 min
3 95C 30 sec
304 60C 1 min
5 72C 30 sec
6 GOTO 3, 29 more times7 55C 30 sec
8* Melting curve 55C ~ 85C (5 s / 0.5C)
9 95C 30 sec
1010 60C 1 min
11 72C 30 sec
12 GOTO 9, 9 more times
13 55C 30 sec
14* Melting curve 55C ~ 85C (5 s / 0.5C)
15 95C 30 sec
1016 60C 1 min
17 72C 30 sec
18 GOTO 15, 9 more times
19 55C 30 sec
20* Melting curve 55C ~ 85C (5 s / 0.5C)
Note: Plate Read on Segment 8, 14 and 20. Fluorescence is detected at Melting.
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B. Plate Setup
1) In Experiment Setup Plate, click Create New to open the Plate Editor to create a new
plate.
Fig. 6. Plate Editor. Create a new plate or load an existing plate for the experiment.
2) Click Select Fluorophores to indicate the fluorophores(FAM, HEX, Cal Red 610, Quasar
670and Quasar 705) that will be used in the experiment.
Fig. 7. Select Fluorophores(FAM, HEX, Cal Red 610, Quasar 670and Quasar 705).
3) Choose the appropriate well and then click the Sample Typefrom the drop-down menu.
- Unknown: Clinical samples
- Negative Control
- Positive Control
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4) Click the appropriate checkboxes(FAM, HEX, Cal Red 610, Quasar 670and Quasar 705)
to specify the fluorophores in the selected wells.5) Type the Sample Nameand PC(PC1, PC2, andPC3), and then press enter key.
6) In Settings of the Plate Editor main menu, choose the Plate Size and Plate Type(BR
White).
Fig. 8.Plate Setup.
7) Click OKand save a new plate set-up file.
8) Then Experiment Setupwindow will open.
Fig. 9. Experiment Setup Plate.
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B. Pre-settings for Data Analysis in CFX96
1) After the test, click the Melt curve field to confirm the Melt Peak results.
Fig. 11. Result of Melting peak.
2) Select Step number8and select Export All Data Sheets to Excelfrom Tools menu.
Note: Select Export All Data Sheets to Exceldirectly in case of End point-CMTA.
Fig. 12. Export All Data sheets to excel.
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3) Save the result to the 1folder, when a new window is opened.
Note: Save the result to the arbitrary folder in case of End point-CMTA.
Fig. 13.Export all data from spreadsheets in data analysis to designated folder.
4) Make sure the result have been saved to the folder.
Fig. 14. Exported Result files.
Note: Skip 5)~8) steps and process next analysis stage in case of End point-CMTA.
5) Return to step 2) and select Step number14. Repeat steps 3) & 4) and save data in 2
folder.
6) Return back to step 2) and select Step number 20.
7) It is necessary to regulate threshold bar before export data from step number 20. Select
only Quasar 670, the threshold bar of melt peak should be adjusted to zero.
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Fig. 15. Melt peak Threshold of Quasar 670.
8) Repeat steps 3) & 4) and save data in 3folder. Data of each step number is saved as
shown below.
Step number Designated folder
8 1
14 2
20 3
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C. Settings for Data Analysis in Seegene Viewer
1) Open the Seegene Viewer program in the screen, and click open to find the saved file in 1
folder.
Note: Open to find the saved file in arbitrary folder in case of End point-CMTA.
Fig. 16. Seegene viewer.
2) After opening the results file, click PRODUCT column and select test kit in the lists.
Fig. 17. Settings for Data Analysis in Seegene Viewer.
Note: Please check the type of the tube at the time of test kit selection (8-strip or 96 plate).
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RESULTS
1. Analyte Information
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2. Interpretation of Results
A. Cyclic-CMTA
HPV Result* IC Result
* Interpretation
+++ or ++ or + +++ or ++- HPV DNA, detected
- Target HPV type identification
+++ or ++ or + + or -
- HPV DNA, detected
- Target HPV type identification
- Additional HPV genotypes that were not detected may be
present.
- +++ or ++ - HPV DNA, not detected
- + or -
Invalid
- Weak or negative IC signal was suggestive of inadequate
specimen collection, processing or the presence of
inhibitors.
- Process another aliquot of the original specimen and
repeat the test.
Result
Cyclic-CMTA
(Cyclic Catcher Melting Temperature Analysis)
First CMTA point Second CMTA point Third CMTA point
+++ + + +
++ - + +
+ - - +
- - - -
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B. End point-CMTA
HPV Result* IC Result
* Interpretation
+ +- HPV DNA, detected
- Target HPV type identification
+ -
- HPV DNA, detected
- Target HPV type identification
- Additional HPV genotypes that were not detected may be
present.
- + - HPV DNA, not detected
- -
Invalid
- Negative IC signal was suggestive of inadequate specimen
collection, processing or the presence of inhibitors.
- Process another aliquot of the original specimen and
repeat the test.
* Internal Control or any other signals are not observed: see TROUBLESHOOTING.
Detection of the Internal Control in the Quasar 670 channel is not required for positive results. High
titer of another analyte can lead to a reduced or absent Internal Control signal.
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3. Application to Clinical Samples
A. Cyclic-CMTA
Melt Peak-1st
(First CMTA point)
Melt Peak-2nd
(Second CMTA point)
Melt Peak-3rd
(Third CMTA point)
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Melt Peak-1
st
(First CMTA point)
Melt Peak-2nd
(Second CMTA point)
Melt Peak-3rd
(Third CMTA point)
FAM HEX Cal Red 610 Quasar 670 Quasar 705 Auto interpretation
A set 66 45 58 51 59 16 33 39 52 IC 35 18 56 68 31
66,52,70
Sample + - - - - - - - ++ +++ - - - - -
B set 26 69 73 42 82 53 43 54 70 IC 61 6 44 40 11
Sample - - - - - - - - ++ +++ - - - - -
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B. End point CMTA
A set
B set
FAM HEX Cal Red 610 Quasar 670 Quasar 705 Auto interpretation
A set 66 45 58 51 59 16 33 39 52 IC 35 18 56 68 31
66,52,70
Sample + - - - - - - - + + - - - - -
B set 26 69 73 42 82 53 43 54 70 IC 61 6 44 40 11
Sample - - - - - - - - + + - - - - -
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TROUBLESHOOTING
AnyplexTMII HPV28 Detection
OBSERVATION PROBABLE CAUSES SOLUTION
Internal Control
or any other
signals are not
observed
The fluorophores for data
analysis do not comply with
the protocol
Select the correct fluorophores for data analysis.
Incorrect PCR cycle or
machine temperature
Please check the PCR conditions and repeat the
PCR under the correct setting if necessary.
Leaving reagents at room
temperature for a long time
or incorrect storage condition
Please check the storage conditions(See 9 page)
and the expiration date(see the kit label) of the
reagents and use a new kit if necessary.
Incorrect programming Repeat the detection procedure with a correct
setting.
Nucleic acid extraction failure Make sure that you use a recommended isolation
method.
Error in specimen collection If the both target and IC signal were not observed
that means specimen collected inappropriately
recollect the specimen.
Internal Control
signal is not
observed
High load of pathogen's
nucleic acid
If the target signal is observed, sample is
presumably detection for target pathogens, although
IC signal is not observed.
If you want to check the IC, dilute the specimen in
PBS(10-100x) and repeat from extraction step with
the diluted specimen.
Presence of PCR Inhibitor Dilute the specimen in PBS(10-100x), and repeat
from extraction step.
False positive or
signals
observed at the
Negative Control
Presence of cross
contamination
Decontaminate all surfaces and instruments with
sodium hypochlorite and ethanol. Use only filter tips
during the extraction procedure. Change tips among
tubes. Repeat the nucleic acid extraction with the
new set of reagents.
Cross-contamination
between PC 1, 2 and 3
Restart from extraction step
or
Restart from real-time PCR step.
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AnyplexTMII HPV28 Detection
OBSERVATION PROBABLE CAUSES SOLUTION
False negative
or no signals
observed at the
Positive Control
Error in specimen collection Recollect the specimen.
Incorrect storage of the
specimen
Recollect the specimen and repeat the whole
process. Make sure the product is stored in
recommended conditions.
Error in nucleic acid
extraction
Re-extract the nucleic acid.
Error in adding nucleic acid
to corresponding PCR tubes
Check the sample numbers for nucleic acid
containing tubes and make sure to add nucleic acidinto correct PCR tubes during detection process.
Presence of inhibitor Dilute the specimen in PBS(10-100x), and repeat
from extraction step with the diluted specimen.
The fluorophores for data
analysis do not comply with
the protocol
Select the correct fluorophores for data analysis.
Incorrect programming Repeat the PCR with corrected setting.
Incorrect PCR mixture Check whether all components are added or not(If
you use to precomposed premix, should be reduce
sensitivity). Each reagent used for homogenization
and spin down reagent tube before put the real-time
PCR.
Leaving reagents at room
temperature for a long time
or incorrect storage condition
Please check the storage condition and the
expiration date(see the kit label) of the reagents and
use a new kit if necessary.
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PERFORMANCE
1. Specificity
The high specificity of the AnyplexTM
II HPV28 Detection is ensured by the primers
specifically designed for the targets in interest and the reaction condition. AnyplexTM
II
HPV28 Detection has been tested for cross-reactivity in 85 different pathogens: result
illustrated PCR amplifications in targets only.
Organism Strain No.
Result
Acinetobacter baumannii ATCC 15150 -
Bacteroides fragilis ATCC 25285D -
Chlamydia trachomatis ATCC VR-577 -
Corynebacterium genitalium ATCC 33030 -
Enterobacter cloacae KCTC 13047 -
Enterococcus faecalis ATCC 700802D-5 -
Escherichia coli ATCC 15489 -
Fusobacterium nucleatum ATCC 25586D-5 -
Gardnerella vaginalis ATCC 14019 -
Haemophilus ducreyi ATCC 33940 -
Klebsiella pneumoniae ATCC 13883 -
Lactobacillus acidophilus ATCC 4357D-5 -
Lactobacillus crispatus ATCC 33820 -
Lactobacillus gasseri ATCC 33323 -
Lactobacillus iners ATCC 55195 -
Lactobacillus jensenii ATCC 25258 -
Mobiluncus curtisii ATCC 35241 -
Mobiluncus mulieris ATCC 35243 -
Neisseria gonorrhoeae ATCC 700825D -
Neisseria meningitidis ATCC 700532D -
Neisseria sicca ATCC 29256 -
Peptostreptococcus anaerobius ATCC 49031D-5 -
Propionibacterium acnes ATCC 6919 -
Proteus mirabilis ATCC 12453 -
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Organism Strain No.
Result
HPV6 ATCC 45150D + (HPV6)
HPV11 ATCC 45151D + (HPV11)
HPV16 ATCC 45113D + (HPV16)
HPV18 ATCC 45152D + (HPV18)
HPV26 Korean isolate + (HPV26)
HPV31 ATCC 65446 + (HPV31)
HPV33 Korean isolate + (HPV33)
HPV35 ATCC 40330 + (HPV35)
HPV39 Korean isolate + (HPV39)
HPV40 Korean isolate + (HPV40)
HPV42 Korean isolate + (HPV42)
HPV43 ATCC 40339 + (HPV43)
HPV44 Korean isolate + (HPV44)
HPV45 Korean isolate + (HPV45)
HPV51 Korean isolate + (HPV51)
HPV52 Korean isolate + (HPV52)
HPV53 Korean isolate + (HPV53)HPV54 Korean isolate + (HPV54)
HPV56 ATCC 40549 + (HPV56)
HPV58 Korean isolate + (HPV58)
HPV59 Korean isolate + (HPV59)
HPV61 Korean isolate + (HPV61)
HPV66 Korean isolate + (HPV66)
HPV68 Korean isolate + (HPV68)
HPV69 Korean isolate + (HPV69)
HPV70 Korean isolate + (HPV70)
HPV73 Korean isolate + (HPV73)
HPV82 Korean isolate + (HPV82)
SiHa Cell KCLB 30035 + (HPV16)
HeLa Cell KCLB 10002 + (HPV18)
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II HPV28 Detection
REFERENCES
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Burd EM. [Human papillomavirus and cervical cancer.] Clin Microbiol Rev. (2003) 16(1): 1-17
Castle PE. [The potential utility of HPV genotyping in screening and clinical management.] J Natl Compr Canc
Netw. (2008) 6(1): 83-95 Review
Chris JM, Peter JS, Philip EC. [Clinical utility of HPV genotyping.] Gynecol Oncol. (2006) 103: 12-17
Chun JY, Kim KJ, Hwang IT, Kim YJ, Lee DH, Lee IK, Kim JK. [Dual priming oligonucleotide system for the
multiplex detection of respiratory viruses and SNP genotyping of CYP2C19 gene.] Nucleic Acids Res. (2007)
35(6): e40
Chun JY. [High Multiplex Molecular Diagnostics.] Seegene Bulletin (2012) 1: 1-4
Giorgi Rossi P, Bisanzi S, Paganini I, Di Iasi A, Angeloni C, Scalisi A, Macis R, Pini MT, Chini F, Carozzi FM. [HPV
Prevalence Italian Working GroupPrevalence of HPV high and low risk types in cervical samples from the Italian
general population: a population based study.]BMC Infect Dis. (2010) 20(10): 214
Hwang IT. [Cyclic-CMTA: An Innovative Concept in Multiplex Quantification.] Seegene Bulletin (2012) 1: 11-15
Krane JF, Granter SR, Trask CE, Hogan CL, Lee KR. [Papanicolaou smear sensitivity for the detection of
adenocarcinoma of the cervix: a study of 49 cases.] Cancer. (2001) 93(1): 8-15
Lee DH. [TOCE: Innovative Technology for High Multiplex Real-time PCR.] Seegene Bulletin (2012) 1: 5-10
Li J,Mei J,Wang X,Hu L,Lin Y,Yang P.[Human papillomavirus type-specific prevalence in women with cervical
intraepithelial neoplasm in Western China.]J Clin Microbiol. (2012) 50(3): 1079-1081
Novaes LC, Novaes MR, Simes-Barbosa A. [Diagnosis of human papillomatosis by polymerase chain reaction in
cases of divergence between results of hybrid capture and papanicolaou cytology.] Braz J infect Dis. (2006)
10(3):169-172
Son S, Noh HT, An S. [Human papillomavirus status in cervical scrapes and biopsy specimens using the HPV
genotyping DNA microarray.]Int J Gynaecol Obstet. (2006) 93(3): 258-259
Sun ZR, Ji YH, Zhou WQ, Zhang SL, Jiang WG, Ruan Q. [Characteristics of HPV prevalence among women in
Liaoning province, China.]Int J Gynaecol Obstet. (2010) 109(2): 105-109
Wallace J, Woda BA, Pihan G. [Facile, Comprehensive High-Throughput Genotyping of Human Genital
Papillomaviruses Using Spectrally Addressable Liquid Bead Microarrays.] J Mol Diagn. (2005) 7(1): 72-80
Ursu RG, Onofriescu M, Nemescu D, Iancu LS. [HPV prevalence and type distribution in women with or without
cervical lesions in the Northeast region of Romania.]Virol J. (2011) 22(8): 558
http://www.ncbi.nlm.nih.gov/pubmed/20646310http://www.ncbi.nlm.nih.gov/pubmed/20646310http://www.ncbi.nlm.nih.gov/pubmed?term=Li%20J%5BAuthor%5D&cauthor=true&cauthor_uid=22170939http://www.ncbi.nlm.nih.gov/pubmed?term=Mei%20J%5BAuthor%5D&cauthor=true&cauthor_uid=22170939http://www.ncbi.nlm.nih.gov/pubmed?term=Wang%20X%5BAuthor%5D&cauthor=true&cauthor_uid=22170939http://www.ncbi.nlm.nih.gov/pubmed?term=Hu%20L%5BAuthor%5D&cauthor=true&cauthor_uid=22170939http://www.ncbi.nlm.nih.gov/pubmed?term=Lin%20Y%5BAuthor%5D&cauthor=true&cauthor_uid=22170939http://www.ncbi.nlm.nih.gov/pubmed?term=Yang%20P%5BAuthor%5D&cauthor=true&cauthor_uid=22170939http://www.ncbi.nlm.nih.gov/pubmed?term=Jinke%20Li%2C%20Pei%20Yanghttp://www.ncbi.nlm.nih.gov/pubmed?term=Human%20papillomavirus%20status%20in%20cervical%20scrapes%20and%20biopsy%20specimens%20using%20the%20HPV%20genotyping%20DNA%20microarrayhttp://www.ncbi.nlm.nih.gov/pubmed/20138618http://www.ncbi.nlm.nih.gov/pubmed/20138618http://www.ncbi.nlm.nih.gov/pubmed/22192090http://www.ncbi.nlm.nih.gov/pubmed/22192090http://www.ncbi.nlm.nih.gov/pubmed/22192090http://www.ncbi.nlm.nih.gov/pubmed/22192090http://www.ncbi.nlm.nih.gov/pubmed/20138618http://www.ncbi.nlm.nih.gov/pubmed/20138618http://www.ncbi.nlm.nih.gov/pubmed?term=Human%20papillomavirus%20status%20in%20cervical%20scrapes%20and%20biopsy%20specimens%20using%20the%20HPV%20genotyping%20DNA%20microarrayhttp://www.ncbi.nlm.nih.gov/pubmed?term=Jinke%20Li%2C%20Pei%20Yanghttp://www.ncbi.nlm.nih.gov/pubmed?term=Yang%20P%5BAuthor%5D&cauthor=true&cauthor_uid=22170939http://www.ncbi.nlm.nih.gov/pubmed?term=Lin%20Y%5BAuthor%5D&cauthor=true&cauthor_uid=22170939http://www.ncbi.nlm.nih.gov/pubmed?term=Hu%20L%5BAuthor%5D&cauthor=true&cauthor_uid=22170939http://www.ncbi.nlm.nih.gov/pubmed?term=Wang%20X%5BAuthor%5D&cauthor=true&cauthor_uid=22170939http://www.ncbi.nlm.nih.gov/pubmed?term=Mei%20J%5BAuthor%5D&cauthor=true&cauthor_uid=22170939http://www.ncbi.nlm.nih.gov/pubmed?term=Li%20J%5BAuthor%5D&cauthor=true&cauthor_uid=22170939http://www.ncbi.nlm.nih.gov/pubmed/20646310http://www.ncbi.nlm.nih.gov/pubmed/20646310 -
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EXPLANATION OF SYMBOLS
Explanation of symbols used in label and manual.
Symbol Explanation
In vitro diagnostic use
Research use only
Batch code
Catalogue number
Use by
Temperature limitation
Caution
Oligonucleotide Mix for amplification and detection
RNase-free Water
Positive Control
AnyplexTM
PCR Master Mix
Manufacturer
Date of manufacture
Consult instructions for use
Authorized representative in the European community
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AnyplexTM
II HPV28 Detection
ORDERING INFORMATION
Cat. No. Product Size
AnyplexTM
II HPV Series
HP7S00X AnyplexTM
II HPV28 Detection 100 rxns
Seeplex
HPV Series
HP6401Y Seeplex
HPV4A ACE Screening 50 rxns
Cervical specimen collection kit
606CS01L ENAT PM 2ML L-SHAPE APPLICATOR 50 tests
Manual extraction system
SG1701 Ribo_spin vRD(Viral RNA/DNA Extraction Kit) 50 preps
Automated extraction system
SPN1200 SEEPREP12TM
EA
SPN1004 SEEPREP12TM
Viral NA kit 96 preps
SPN1101 SEEPREP12TM Tip Set 96 tips
65415-02 MICROLAB Nimbus IVD EA
173000-075 MICROLAB STARlet EA
744300.4.205875 STARMag 96 Tissue 384T / 1box
744300.4.TC384 STARMag 48 X 8 Tissue Cartridge Kit 384T / 1box