Seegene HPV28 manual en inglés

7/26/2019 Seegene HPV28 manual en ingls

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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 --------------------------------------------------------------------

3

4

5

7

8

9

9

10

16

28

34

36

40

41

42


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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





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.


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 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


15 95C 30 sec

1016 60C 1 min

17 72C 30 sec

18 GOTO 15, 9 more times

19 55C 30 sec


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


- 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


+ -

- HPV DNA, detected


- 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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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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II HPV28 Detection

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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II HPV28 Detection

Organism Strain No.

Result

HPV6 ATCC 45150D + (HPV6)




HPV26 Korean isolate + (HPV26)

HPV31 ATCC 65446 + (HPV31)


HPV35 ATCC 40330 + (HPV35)




HPV43 ATCC 40339 + (HPV43)





HPV53 Korean isolate + (HPV53)HPV54 Korean isolate + (HPV54)

HPV56 ATCC 40549 + (HPV56)










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

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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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II HPV28 Detection

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

Seegene HPV28 manual en inglés

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