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. CFX96TM Real-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.

Anyplex

TM

II HPV28 Detection

TABLE OF CONTENTS
NOTICES

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

INTENDED USE

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

PRINCIPLES AND PROCEDURE OVERVIEW

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

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

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

BACKGROUND INFORMATION
REAGENTS

STORAGE AND HANDLING

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

MATERIALS REQUIRED BUT NOT PROVIDED

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

PROTOCOL -----------------------------------------------------------------------------------------

10

REAL-TIME PCR INSTRUMENT SET UP AND RESULTS ANALYSIS

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

16

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

28

RESULTS

TROUBLESHOOTING

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

34

PERFORMANCE ----------------------------------------------------------------------------------

36

REFERENCES

40

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

EXPLANATION OF SYMBOLS
ORDERING INFORMATION

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

41

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

42

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II HPV28 Detection

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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II HPV28 Detection

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 Anyplex

TM

II HPV28 Detection is a qualitative in vitro test for the detection of human

papillomaviruses in liquid based cytology and cervical swab specimens.

The Anyplex

TM

II HPV28 Detection assay consist of two PCR reaction(A set and B set).

A set is a multiplex assay that permits the simultaneous amplification of target DNA of 14 highrisk human papillomaviruses.
B set is a multiplex assay that permits the simultaneous amplification of target DNA of 5 highrisk and 9 low-risk human papillomaviruses.

Category
A set

B set

Types
14 high-risk HPV types
(16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68)
5 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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II HPV28 Detection

PRINCIPLES AND PROCEDURE OVERVIEW

1. Principles
The Anyplex

TM

II HPV28 Detection Assay, represents Seegenes proprietary technologies and is

based on a newly developed TOCE

TM

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, TOCE

TM

technology is designated not to be

affected by sequence variations; therefore it guaranteeing consistent Tm values.

The Anyplex

TM

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 Anyplex

TM

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 Anyplex

TM

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 Anyplex

TM

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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II HPV28 Detection

2. Procedure Overview

< AnyplexTM II HPV28 Detection procedure overview >

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TM

II HPV28 Detection

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 Anyplex

TM

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. Anyplex

TM

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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TM

II HPV28 Detection

REAGENTS
The reagents contained in one set are sufficient for 100 determinations.
Anyplex

TM

II HPV28 Detection order information(

HP7S00X).

AnyplexTM II HPV28 Detection

Symbols

Contents

Volume

Description

4X HPV28 A TOM

500 L

TOCE Oligo Mix(TOM):

- Amplification and detection reagents

4X HPV28 B TOM

500 L

TOCE Oligo Mix(TOM):

- Amplification and detection reagents

4X Anyplex
PCR Master Mix
(with UDG)

500 L
X2

- DNA polymerase
- Uracil-DNA glycosylase(UDG)
- Buffer containing dNTPs

HPV28 PC1

100 L

Positive Control(PC) :
- Mixture of pathogen clones

HPV28 PC2

100 L

Positive Control(PC) :
- Mixture of pathogen clones

HPV28 PC3

100 L

Positive Control(PC) :
- Mixture of pathogen clones

RNase-free Water

1,000 L
X2

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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TM

II HPV28 Detection

STORAGE AND HANDLING

The components of the Anyplex

TM

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 SEEPREP12 , Cat. No.P4850, SIGMA)

Ice Maker

Desktop centrifuge

Vortex mixer

CFX96

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)

TM

TM

Real-time PCR system(Bio-Rad)

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TM

II HPV28 Detection

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 cytology specimen

Follow the manufacturers instructions for collecting cervical cell specimens into ThinPrep
or SurePath

TM

media.

Cervical swab specimen

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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TM

II HPV28 Detection

Liquid based cervical cytology specimen

Cervical cell specimens collected in ThinPrep medium may be stored at 2 ~ 8 for up to 6

weeks. Cervical cell specimens collected in SurePath

TM

medium 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 specimen

If the swab specimens are not processed directly after their receipt in the laboratory, they have
to be stored at 2 ~ 8 and 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

Specimens collected in SurePath

TM

medium can be transported at 2 ~ 25.

medium must be transported at 2 ~ 8

Cervical swab specimen

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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TM

II HPV28 Detection

to redissolve.
Note: Process pre-treatment step using lysis buffer in extraction kit not 1X PBS if the samples are
TM

collected in SurePath

medium 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.

QIAamp DNA Mini Kit*

QIAGEN

51304

Specimen: 200 L
Elution: 50 L

Ribo_spin vRD**
(Viral RNA/DNA Extraction Kit)

GeneAll

302-150
SG1701***

Specimen: 200 L
Elution: 50 L

Recommended Vol.

* Process lysis step using 180 L of ATL buffer instead of AL buffer in case of SurePath
**Ribo_spin vRD kit is not compatible with SurePath

TM

TM

media.

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.

MICROLAB Nimbus IVD

Hamilton

65415-02*

STARMag 96 Tissue

Seegene

STARMag 48 X 8 Tissue Cartridge

Kit

12

Seegene

744300.4.
205875
744300.4.
TC384

Recommended Vol.

Specimen: 450 L
Elution: 100 L
Specimen: 450 L
Elution: 100 L

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TM

II HPV28 Detection

* If you would like to purchase the above products from Seegene, Inc., please use this Cat. No.

D-2. MICROLAB STARlet

Note: See MICROLAB STARlet operation manual.
Automated Purification System

Manufacturer

Cat. No.

MICROLAB STARlet

Hamilton

173000-075*

STARMag 96 Tissue

Seegene

STARMag 48 X 8 Tissue Cartridge

Kit

744300.4.
205875
744300.4.

Seegene

TC384

Recommended Vol.

Specimen: 450 L
Elution: 100 L
Specimen: 450 L
Elution: 100 L

* If you would like to purchase the above products from Seegene, Inc., please use this Cat. No.

D-3. SEEPREP12

TM

Automated Purification System

Manufacturer

Cat. No.

SEEPREP12

NorDiag

SPN1200*

SEEPREP12 Viral NA Kit

NorDiag

SPN1004*

Recommended Vol.

Specimen: 240 L
Elution: 60 L

* If you would like to purchase the above products from Seegene, Inc., please use this Cat. No.

Add 10 L of proteinase K(20 mg/mL) to each appropriately labeled sterile 1.5 mL sample
tube.

Transfer 240 L of specimen to the tube containing 10 L of proteinase K, mix by flicking the
tube gently.

The cartridge and assembled pump-tip are placed on the instrument.

Place 1.5 mL elution tube onto the instrument.

Press CONTINUE on the first screen to let the instrument initialize.

Press START PROTOCOL on the SEEPREP12

In the Select protocol menu, press SPN Viral NA-HT.

In the Select sample volume menu, press 250 L. And in Select elution volume, press 60

TM

main menu.

L.

Follow the onscreen instruction for loading the instrument.

After all steps are completed, close the door and start the run.

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TM

II HPV28 Detection

E. Summary

QIAGEN
Swab

GeneAll

SEEPREP12

NIMBUS/STARlet

ENAT

ThinPrep

O3

O4

LBC
SurePath

TM

1. QIAamp DNA Mini Kit

2. Robo_spin vRD (Viral RNA/DNA Extraction Kit)
3. Process lysis step using 180 L of ATL buffer instead of AL buffer.
4. If DNA is extracted from SurePath

TM

specimens with NIMBUS or STARlet, there is a possibility that the

sensitivity could be reduced compared to other extraction methods.

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TM

II HPV28 Detection

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

RNase-free Water

5 L

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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TM

II HPV28 Detection

Positive control of A set

Name
PC1
PC2
PC3

FAM

HEX

Cal Red 610

Quas ar 670

Quas ar 705

66

45

58

51

59

16

33

39

52

IC

35

18

56

68

31

+
-

+
-

+
-

+
-

+
-

+
-

+
-

+
-

+
-

+
-

Aut o int erpret at ion

Positive Control (+)
Positive Control (+)
Positive Control (+)

Positive control of B set

Name
PC1
PC2
PC3

16

FAM

HEX

Cal Red 610

Quas ar 670

Quas ar 705

26

69

73

42

82

53

43

54

70

IC

61

44

40

11

+
-

+
-

+
-

+
-

+
-

+
-

+
-

+
-

+
-

+
-

Aut o int erpret at ion

Positive Control (+)
Positive Control (+)
Positive Control (+)

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TM

II HPV28 Detection

REAL-TIME PCR INSTRUMENT SET UP AND RESULTS ANALYSIS

1. CFX96

1.1.

TM

Real-time PCR System(Bio-Rad)

Real-time PCR Instrument set up

Note:

TM

The CFX96

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)

TM

II HPV28 Detection

In Protocol Editor, define the thermal profile as follows:

i) Cyclic-CMTA (Melt analysis of three times)

Segment

Temperature

Duration

50C

4 min

95C

15 min

95C

30 sec

60C

1 min

72C

30 sec

6
7
8*

55C

30 sec

Melting curve 55C ~ 85C (5 s / 0.5C)

95C

30 sec

10

60C

1 min

11

72C

30 sec

13
14*

55C

30 sec

Melting curve 55C ~ 85C (5 s / 0.5C)

95C

30 sec

16

60C

1 min

17

72C

30 sec

19
20*

10

GOTO 9, 9 more times

15

18

30

GOTO 3, 29 more times

12

No. of cycles

10

GOTO 15, 9 more times

55C

30 sec

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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TM

II HPV28 Detection

ii) End point-CMTA (Melt analysis of one time)

Segment

Temperature

Duration

50C

4 min

95C

15 min

95C

30 sec

60C

1 min

72C

30 sec

6
7
8*

No. of cycles

50

GOTO 3, 49 more times

55C

30 sec

Melting curve 55C ~ 85C (5 s / 0.5C)

Note: Plate Read on Segment 8. Fluorescence is detected at Melting.

Fig. 2. Protocol Editor (Cyclic-CMTA)

Fig. 3. Protocol Editor (End point-CMTA)

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3)

Click on Sample Volume to directly edit the 20 L.

4)

Click OK; the Experiment Setup window will be open.

TM

II HPV28 Detection

Fig. 4. Experiment Setup Protocol (Cyclic-CMTA)

Fig. 5. Experiment Setup Protocol (End point-CMTA)

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TM

II HPV28 Detection

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

670 and Quasar 705) that will be used in the experiment.

Fig. 7. Select Fluorophores(FAM, HEX, Cal Red 610, Quasar 670 and Quasar 705).
3)

Choose the appropriate well and then click the Sample Type from the drop-down menu.
- Unknown: Clinical samples
- Negative Control
- Positive Control

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4)

TM

II HPV28 Detection

Click the appropriate checkboxes(FAM, HEX, Cal Red 610, Quasar 670 and Quasar 705)

to specify the fluorophores in the selected wells.

5)

Type the Sample Name and PC(PC1, PC2, and PC3), 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 OK and save a new plate set-up file.

8)

Then Experiment Setup window will open.

Fig. 9. Experiment Setup Plate.

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C.

Start Run

1)

In Experiment Setup Start Run, click Close Lid to close the lid.

TM

II HPV28 Detection

Fig. 10. Close Lid.

2)

Click Start Run.

3)

Store the run file either in my documents or in the designated folder. Fill in the file name,

click the SAVE, and then machine will start.

1.2. Data Analysis

A. Create specified folder for saving DATA

A-1. Cyclic-CMTA

1)

To save data for each melt point from result file, create three folders.

2)

Folder name for save the first melt point data is 1, second folder name is 2, and third

folder name is 3.

A-2. End point-CMTA

1)

To save data for melt point from result file, create one folder.

2)

Folder name can be set by the user as desired.

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TM

II HPV28 Detection

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 number 8 and select Export All Data Sheets to Excel from Tools menu.

Note: Select Export All Data Sheets to Excel directly in case of End point-CMTA.

Fig. 12. Export All Data sheets to excel.

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3)

TM

II HPV28 Detection

Save the result to the 1 folder, 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 number 14. 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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TM

II HPV28 Detection

Fig. 15. Melt peak Threshold of Quasar 670.

8)

Repeat steps 3) & 4) and save data in 3 folder. Data of each step number is saved as

shown below.

26

Step number

Designated folder

14

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II HPV28 Detection

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).

27

03/2013 V1.04

Anyplex

3)

TM

II HPV28 Detection

Check the result for each well.

Fig. 18. CFX96 test result on Seegene Viewer.

28

03/2013 V1.04

Anyplex

TM

II HPV28 Detection

RESULTS
1. Analyte Information

29

03/2013 V1.04

Anyplex

TM

II HPV28 Detection

2. Interpretation of Results

A. Cyclic-CMTA

HPV Result

+++ or ++ or +

IC Result

+++ or ++

Interpretation
- HPV DNA, detected
- Target HPV type identification
- HPV DNA, detected

+++ or ++ or +

- Target HPV type identification

+ or -

- Additional HPV genotypes that were not detected may be

present.

+++ or ++

- HPV DNA, not detected

Invalid
- Weak or negative IC signal was suggestive of inadequate

+ or -

specimen collection, processing or the presence of

inhibitors.
- Process another aliquot of the original specimen and
repeat the test.

Cyclic-CMTA
Result

30

(Cyclic Catcher Melting Temperature Analysis)

First CMTA point

Second CMTA point

Third CMTA point

+++

++

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Anyplex

TM

II HPV28 Detection

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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03/2013 V1.04

Anyplex

3.

TM

II HPV28 Detection

Application to Clinical Samples

A. Cyclic-CMTA

<A set>
st

Melt Peak-1 (First CMTA point)

nd

Melt Peak-2

(Second CMTA point)

rd

Melt Peak-3 (Third CMTA point)

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03/2013 V1.04

Anyplex

TM

II HPV28 Detection

<B set>
st

Melt Peak-1 (First CMTA point)

nd

Melt Peak-2

(Second CMTA point)

rd

Melt Peak-3 (Third CMTA point)

FAM

HEX

Cal Red 610

Quasar 670

Quasar 705

A set

66

45

58

51

59

16

33

39

52

IC

35

18

56

68

31

Sample

++

+++

B set

26

69

73

42

82

53

43

54

70

IC

61

44

40

11

Sample

++

+++

Auto interpretation

66,52,70

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03/2013 V1.04

Anyplex

TM

II HPV28 Detection

B. End point-CMTA

A set

B set

FAM

HEX

Cal Red 610

Quasar 670

Quasar 705

A set

66

45

58

51

59

16

33

39

52

IC

35

18

56

68

31

Sample

B set

26

69

73

42

82

53

43

54

70

IC

61

44

40

11

Sample

Auto interpretation

66,52,70

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03/2013 V1.04

Anyplex

TM

II HPV28 Detection

TROUBLESHOOTING
AnyplexTM II HPV28 Detection
OBSERVATION

PROBABLE CAUSES

SOLUTION

Internal Control

The fluorophores for data

Select the correct fluorophores for data analysis.

or any other

analysis do not comply with

signals are not

the protocol

observed

Incorrect PCR cycle or

Please check the PCR conditions and repeat the

machine temperature

PCR under the correct setting if necessary.

Leaving reagents at room

Please check the storage conditions(See 9 page)

temperature for a long time

and the expiration date(see the kit label) of the

or incorrect storage condition

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

High load of pathogen's

If

signal

nucleic acid

presumably detection for target pathogens, although

is

not

observed

the

target

signal

is

observed,

sample

is

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

Presence of cross

Decontaminate all surfaces and instruments with

signals

contamination

sodium hypochlorite and ethanol. Use only filter tips

observed at the

during the extraction procedure. Change tips among

Negative Control

tubes. Repeat the nucleic acid extraction with the

new set of reagents.

35

Cross-contamination

Restart from extraction step

between PC 1, 2 and 3

or
Restart from real-time PCR step.

03/2013 V1.04

Anyplex

TM

II HPV28 Detection

AnyplexTM II HPV28 Detection

OBSERVATION

PROBABLE CAUSES

SOLUTION

False negative

Error in specimen collection

Recollect the specimen.

or no signals

Incorrect storage of the

Recollect the specimen and repeat the whole

observed at the

specimen

process. Make sure the product is stored in

Positive Control

recommended conditions.
Error in nucleic acid

Re-extract the nucleic acid.

extraction
Error in adding nucleic acid

Check

the

sample

numbers

for

nucleic

acid

to corresponding PCR tubes

containing tubes and make sure to add nucleic acid

into 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

Select the correct fluorophores for data analysis.

analysis do not comply with

the protocol
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.

36

Leaving reagents at room

Please

check

the

storage

condition

and

temperature for a long time

expiration date(see the kit label) of the reagents and

or incorrect storage condition

use a new kit if necessary.

03/2013 V1.04

the

Anyplex

TM

II HPV28 Detection

PERFORMANCE
1. Specificity
The high specificity of the Anyplex

TM

II HPV28 Detection is ensured by the primers

specifically designed for the targets in interest and the reaction condition. Anyplex

TM

II

HPV28 Detection has been tested for cross-reactivity in 85 different pathogens: result
illustrated PCR amplifications in targets only.

Organism

37

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

03/2013 V1.04

Anyplex

Organism

38

Strain No.

TM

II HPV28 Detection

Result

Proteus vulgaris

ATCC 6059

Pseudomonas aeruginosa

ATCC 15522

Pseudomonas fluorescens

KCTC 49642

Serratia marcescens

ATCC 27137D-5

Staphylococcus aureus subsp.aureus

ATCC 29213

Streptococcus agalactiae

ATCC BAA-611D

Streptococcus mitis

ATCC 49456D-5

Streptococcus pyogenes

ATCC 700294D-5

Trichomonas vaginalis

ATCC 30001D

Ureaplasma urealyticum

ATCC 33695

Candida albicans

ATCC 14053

Cytomegalovirus

ATCC VR-807

Epstein-Barr virus

ATCC VR-602

Herpes simplex virus 1

ATCC VR-260

Herpes simplex virus 2

ATCC VR-734

Human Adenovirus 1

ATCC VR-1

Human Adenovirus 3

ATCC VR-3

Human Adenovirus 8

ATCC VR-1368

Human Adenovirus 18

ATCC VR-1095

Human Adenovirus 23

ATCC VR-1101

Human Adenovirus 40

ATCC VR-931

HPV1

ATCC 45021

HPV2

ATCC 45022

HPV34

Korean isolate

HPV62

Korean isolate

HPV71

Korean isolate

HPV72

Korean isolate

HPV81

Korean isolate

HPV83

Korean isolate

HPV84

Korean isolate

HPV102

Korean isolate

03/2013 V1.04

Anyplex

Organism

39

Strain No.

TM

II HPV28 Detection

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)

03/2013 V1.04

Anyplex

TM

II HPV28 Detection

2. Sensitivity
In order to determine the sensitivity of Anyplex
6

TM

II HPV28 Detection, a standard serial dilution

has been set up from 10 to 10 copy/reaction plasmid DNA and analyzed with the Anyplex

TM

II

HPV28 Detection. Detection limit for sensitivity is 50 copies/reaction.

3. Reproducibility
Reproducibility tests were carried out at 3 different points of time in the course of 10 days by 3
different experimenters. The same results were obtained in every test, confirming the
reproducibility of the product.

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Anyplex

TM

II HPV28 Detection

REFERENCES
1

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 Group Prevalence 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

9
10

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

11

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

12

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

13

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

14

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

15

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

41

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Anyplex

TM

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

Anyplex

TM

PCR Master Mix

Manufacturer

Date of manufacture

Consult instructions for use

Authorized representative in the European community

42

03/2013 V1.04

Anyplex

TM

II HPV28 Detection

ORDERING INFORMATION
Cat. No.

Product

Anyplex

TM

Size

II HPV Series

HP7S00X

Anyplex

TM

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

SEEPREP12

TM

SPN1004

SEEPREP12

TM

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

SPN1200

43

EA
Viral NA kit

96 preps

03/2013 V1.04