macro micro-test HWTS-CC008A 14 High-Risk HPV with 16-18 Genotyping Test Instruction Manual

macro micro-test HWTS-CC008A 14 High-Risk HPV with 16-18 Genotyping Test

Instruction for Use

(V1.0)

[REF] HWTS-CC008A
[Product Name] 14 High-Risk HPV with 16/18 Genotyping Test Kit（Fluorescence PCR）
[Packaging Size] 50 tests/kit
[Research Use Only]

The kit is used for qualitative fluorescence-based PCR detection of nucleic acid fragments specific to 14 human papillomavirus (HPV) types (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68) in cervical exfoliated cells in women, as well as for HPV 16/18 genotyping to detect HPV infection. Cervical cancer is one of the most common malignant tumors in the female reproductive tract. It has been shown that persistent HPV infection and multiple infections are one of major causes of cervical cancer. [Test principle]

The kit uses multiplex fluorescence-based qPCR. It contains highly specific primers and probes designed according to target sequences in the HPV L1 gene. The specific probes are labeled with four distinct fluorophores, FAM (HPV 18), VIC (16), ROX (HPV31,33,35,39,45,51,52,56,58,59,66 and 68) and CY5 (Internal Control) at the 5′-end, and a quencher (BHQ1 or BHQ2) at the 3’-end. During PCR amplification, the specific primers and probes separately bind to their own target sequences. When the Taq DNA polymerase encounters the probes binding to their target sequences, it exhibits the exonuclease activity at the 5′-end to isolate the fluorophores (reporter dyes) from the quencher, thereby allowing the fluorescence-monitoring system to receive fluorescence signals, i.e., when a DNA strand is amplified, a fluorescent molecule will be formed to achieve complete synchronization of PCR product formation and fluorescence signal accumulation, thereby qualitatively detecting nucleic acid fragments specific to 14 HPV types (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 ,68) in cervical exfoliated cells.

Main Components

Note: The components of the kit in different batches are not interchangeable. Reagents required but not provided: Macro & Micro-Test Viral DNA/RNA Kit(HWTS-3001, HWTS-3004-32, HWTS-3004-48)from Jiangsu Macro & Micro- Test Med-Tech Co., Ltd., TIANamp Virus DNA/RNA Kit (DP315) manufactured by Tiangen Biotech (Beijing) Co., Ltd., QIAamp DNA Mini Kit (Cat. no.51304) manufactured by QIAGEN. Consumables required but not provided: 1.5 mL RNase- and DNase-free centrifuge tubes, RNase- and DNase-free tips, fluorescence- based qPCR tubes, benchtop centrifuge, and benchtop vortex mixer. [Storage Conditions and Shelf-life] The kit should be stored below -18°C and protected from light, the shelf life is 12 months. After opening the container, please use it up within 3 months. It shall not be subjected to more than 4 freezing- thawing cycles.

Applicable Instruments

SLAN ® -96P Real-Time PCR Systems, Applied Biosystems 7500 Real-Time PCR Systems, QuantStudio™5 Real-TimePCR Systems, LightCycler® 480 Real-Time PCR system, LineGene 9600 Plus Real-Time PCR Detection System(FQD-96A), MA-6000 Real-Time Quantitative Thermal Cycler. [Specimen Requirements]

1. Specimen collection Before sampling, use a cotton swab to gently wipe the excessive secretions from the cervix. Discard the used swab. Allowa new cotton swab infiltrated with cell preservation solution or a brush for sampling cervical exfoliated cells to adhere closely to the cervical mucosa, and rotate it clockwise for 3-5 times to obtain cervical exfoliated cells. Slowly take out the cotton swab or brush and put it in 1 mL sterile saline. After fully rinsing it, squeeze the cotton swab or brush against the tube wall to dry it and then discard it. Tighten the tube cap and mark the name (or number) of the specimen on the tube.
2. Storage Specimens to be tested should not be stored for more than 48h at 2-8°C or for more than six months below -18°C; they can be stored for a long time at -70°C. Avoid repeated thawing and freezing.
3. Transportation
   It is recommended to use liquid nitrogen and dry ice for ultra-low temperature transportation.

Test Procedure (Please read this operating procedure carefully before use)

1. Preparation of reagents (reagent preparation area)

2. Take out each component from the kit, place it at room temperature for recovery to room temperature, then shake to mix it well, and centrifuge it briefly for later use.

3. Take out the reaction buffer for HPV nucleic acid detection from the kit, dissolve it at room temperature, and shake to mix it well. Calculate the number (n) of samples to be prepared (n=Number of specimens + HPV blank control +HPV positive control). Add 25 μL of the reaction buffer for HPV nucleic acid detection to a fluorescence-based qPCR tube, press the tube cap tightly, and quickly transfer it to the specimen processing area. Immediately store the remaining reaction buffer below -18°C.

4. Specimen processing (specimen processing area)
   Extract genomic DNA from specimens of cervical exfoliated cells in humans. DNA extraction should guarantee that the quality and amount of DNA meet the test requirements, and extracted DNA samples should be stored below-18°C. It is recommended to extract DNA in strict accordance with the package insert using TIANamp Virus DNA/RNAKit (DP315) manufactured by Tiangen Biotech (Beijing) Co., Ltd., or QIAamp DNA Mini Kit (Cat. no.51304) manufactured by Qiagen. The HPV positive control and HPV blank control are extracted in parallel according to the recommended extraction reagent instructions.

5. Sample addition/loading
   Add 5μL of DNA extracted in step 2, 5μL of the HPV positive control and 5μL of the HPV blank control to the reaction buffer for HPV nucleic acid detection aliquoted in step 1, press the tube cap tightly and centrifuge the tube briefly. Put the PCR tube into the fluorescence PCR instrument, and record the loading sequence.

6. PCR amplification (testing area)
   Channel setting:
   Reference Dye: Select None (only applicable to ABI series instruments);
   Setting of PCR conditions is shown in the following table, and the reaction volume is set to 30 μL. For specific settings for detection channels, refer to the user manual for each instrument.

7. Result analysis

8. Baseline and threshold setting for Applied Biosystems 7500 Real-Time PCR Systems Baseline setting: Take the fluorescence signal in 1-6 cycles as a baseline. Threshold setting: The threshold for each channel should be set separately. When setting the threshold line for a certain channel, first select blank control, remove the checked automatic threshold line, change the option “□√ Auto” to “□ Auto”, and then manually adjust the threshold line to the point where the threshold line just exceeds the peak of the normal blank control’s amplification curve (irregular noise line) in the channel.

9. Baseline and threshold setting for SLAN ® -96P Real-Time PCR Systems Baseline setting: Take the fluorescence signal in 1-6 cycles as a baseline. Threshold setting: The threshold for each channel should be set separately. When setting the threshold line for a certain channel, first set the “baseline optimization” in the “basic parameters” to manual optimization, and then manually adjust the threshold line to the point where the threshold line just exceeds the peak of the normal blank control’s amplification curve (irregular noise line) in the channel.

10. Baseline and threshold setting for QuantStudio
    ® 5 Real-Time PCR System
    Baseline setting: Select the default baseline Threshold setting: Use the threshold set automatically by software. When the threshold needs to be set manually, click
    “Show Plot Setting”, select the gene to be viewed, and tick the option before “Show: Threshold”. The corresponding
    threshold line will appear on the amplification curve. Input the threshold or drag the threshold line with the mouse. After setting, click “Analyze” to analyze the result.

11. LightCycler® 480 Real-Time PCR system
    Baseline setting: Select the default baseline Threshold setting: Adjust the Threshold value according to the analyzed image (the user can adjust it according to the
    actual situation: set the Threshold Value in the Log spectrum window, so that the threshold line is in the exponential phase of the amplification curve, and the amplification curve of the negative control is flattened or located below the threshold line), click Analysis to automatically obtain the analysis results, and read the test results in the “Report” window.

12. LineGene 9600 Plus Real-Time PCR Detection System（FQD-96A)
    Baseline setting: Select the automatic baseline by default Threshold setting: Use the threshold set automatically by the software. When it is required to manually set the threshold, manually drag the threshold line to just exceed the peak of the normal blank control’s amplification curve (irregular noise line) in each channel. The threshold in each channel should be set separately: first select the channel to be set, and then drag the threshold line.

13. MA-6000 Real-Time Quantitative Thermal Cycler Baseline setting: Select the automatic baseline by default
    Threshold setting: Use the threshold set automatically by the software. When it is required to manually set the threshold, first keep the box in front of “Auto Threshold” unchecked, then manually drag the threshold line to just exceed the peak of the normal blank control’s amplification curve (irregular noise line) in each channel. The threshold should be set separately: first select the channel to be set, and then drag the threshold line.

Quality control

HPV blank control: There is no Ct value at FAM, ROX and VIC/HEX channels, Ct value＞25 or no Ct value at CY5channel. 6.2 HPV positive control: There are obvious amplification curves at FAM, ROX, CY5 and VIC/HEX channels with a Ct value ≤ 25.

Note: The above requirements must be met at the same time in the same test, otherwise, such test is invalid and needs to be repeated. [Positive judgment value or reference interval] The cycle threshold (Ct) value for the kit is analyzed using the ROC curve analysis. It is determined that the positive judgment value for the kit is a Ct value ≤ 28; and Ct value ≤ 25 in the Internal Control. [Interpretation of test results]

1.  If FAM of the reaction buffer for HPV nucleic acid detection has a Ct value ≤ 28, then it is determined that the specimen tests positive for HPV18.

2. If VIC/HEX of the reaction buffer for HPV nucleic acid detection has a Ct value ≤ 28, then it is determined that the specimen tests positive for HPV 16.

3. If ROX of the reaction buffer for HPV nucleic acid detection has a Ct value ≤ 28, then it is determined that the specimen tests positive for HPV 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68. The types and corresponding channels are detailed in the table below.
   If the specimen tests positive for FAM, VIC/HEX and ROX at the same time, then, a positive HPVtest will be reported.

4. If the Ct value in the channels FAM, VIC/HEX and ROX of the reaction buffer for HPV nucleic acid detection is not value or the Ct value＞28, and the Ct value of Internal Control is ≤ 25, then a negative result will be recorded.

5. If the Ct value in the channels FAM, VIC/HEX and ROX of the reaction buffer for HPV nucleic acid detectionis＞28 or there is no Ct value at those channels, and the Ct value of the Internal Control is＞25, then re-sample and test

Limitations of the test method

1. The test results obtained by the kit are for reference only.
2. Any of unreasonable processes of specimen collection, transportation and handling/processing may result in inaccurate test results.
3. Contamination of amplification products and cross-contamination between specimens subjected to nucleic acid extraction are prone to cause false positive results. Therefore, laboratories should be equipped with equipment in strict accordance with the relevant regulations, and operators should strictly follow the package insert. 4. A negative test does not mean that the patient is non-HPV infected.. A negative test may be caused by: ① unreasonable processes of specimen collection, transportation and handling/processing, or low pathogen titers in specimens;
   ② variations in target sequences of pathogens; ③ other interference factors that have not been verified, such as oral administration of antibacterial.

Product performance indicators

1. The appearance of the kit is intact and the components are complete; the liquid components are clear, translucent, and free of insoluble; the packaging label should be clear and easy to identify.
2. HPV positive control: There are obvious amplification curves at the FAM, ROX, CY5 and VIC/HEX channels and the Ct value is ≤25; HPV blank control: There is no Ct value at FAM, ROX, and VIC/HEX channels, and there is no Ct value or Ct value＞25 at CY5 channel.
3. Both the positive and negative reference tested by the kit meet the requirements.
4. Limit of detection (LOD): The LOD of the kit is 100 copies/mL.
5. When the kit is used to detect non-specific specimens that may cross-react with it, all results are negative, including
   Ureaplasma urealyticum, Chlamydia trachomatis, Candida albicans, Neisseria gonorrhoeae, Trichomonas vaginalis, and mildew, Gardnerella and other HPV types not tested by the kit.

Precautions

1. Research use only. Please read the package insert carefully before testing.

2. The results obtained by the kit will be affected by factors including the source and quality of the specimen itself, specimen collection, specimen transportation conditions and specimen pretreatment, as well as restricted by the quality of extracted DNA, working status of fluorescence-based qPCR instruments, operating environments and the limitations of current molecular biology technology, which may result in false-positive or false-negative test results. The user needs to understand the potential errors and accuracy limitations that can arise during testing.

3. The kit should be transported and stored at a low temperature. Before use, all reagents in the kit should be completely thawed and shaken evenly, and then centrifuged briefly. Avoid unnecessary repeated thawing and freezing.

4. All reagents in the kit have been specially prepared for the detection of the above pathogens. Replacement of any reagent in the kit may affect its effect. The components of the kit in different batches cannot be mixed with each other.

5. When carrying out a test, perform different operations separately in different areas:
   Area 1: Pre-PCR area – to prepare the reagents needed for amplification;
   Area 2: Specimen processing area – to handle specimens to be tested and controls (reference standards);
   Area 3: Testing area – to perform a PCR assay. Testing should be performed in strict accordance with relevant laboratory SOPs for gene amplification testing issued by
   administrative departments. Items in each area are for exclusive use and shall not be cross-used to avoid contamination; please clean the workbench after testing. The lysates from samples stored below -18°C or -70°C should be thawed at room temperature before loading, and used after brief centrifugation.

6. During testing, avoid contamination of the reagents by exogenous nucleic acids, and note to add the DNA sample followed by the positive control. It is recommended to use separate, dedicated pipettes and pipette tips when preparing reagents and adding templates.

7. The reaction tube containing the reaction buffer should be capped and put into an airtight bag before transfer to the specimen processing area.

8. When aliquoting the reaction buffer, avoid air bubbles as far as possible. Before loading, check whether each reaction tube is tightly capped to prevent the leakage of fluorescent substances, which may contaminate the instrument.

9. When a sample is added, it should be completely dropped into the reaction buffer without adhering to the tube wall, followed by immediate capping.

10. After amplification is completed, take out the reaction tube immediately, seal it in a dedicated plastic bag, and discard it in a designated place.

11. All centrifuge tubes and tips used during testing must be free of RNase and DNase. All centrifuge tubes and tips used during testing must be processed in a harmless manner. All pipette tips used during testing should be directly put into a tank containing 84 disinfectants for disposal of waste, and sterilized them together with other waste items before discarding them.

12. The workbench and various experimental supplies should be regularly disinfected with 75% alcohol or ultraviolet light.

13. All chemicals are potentially dangerous. During operation, please wear appropriate laboratory overalls and take protective measures including disposable gloves. All used kits should be disposed of properly.

References

1. Guo Yumei, Sun Jie, Chen Yadan, et al. HPV testing and its significance in the diagnosis of cervical cancer [J]. Chinese Journal of Laboratory Diagnosis, 2009,13: 1306-1308.
2. Li Hua, Gao Guolan. Advance in research on HPV and cervical cancer [J]. The Practical Journal of Cancer, 2007, 22: 420-422.
3. He Guishan. Advance in research on HPV detection methods [J]. Gansu Medical Journal, 2009, 28: 182-183.
4. Ning Tang, Shihai Huang, Brian Erickson, et al. High-risk HPV detection and concurrent HPV 16 and 18 typing with Abbott realtime high-risk HPV test [J]. Journal of Clinical Virology, 2009, 45: S25-S28.

Index of Symbols

Basic Information
Jiangsu Macro & Micro-Test Med-Tech Co., Ltd. Manufacturer Address: No. 888, Zhujiang Road, Juegang Street, Rudong County, (Life and Health Industrial Park of
Rudong High-tech Zone), 226499 Nantong City, Jiangsu Province, PEOPLE’S REPUBLIC OF CHINA
Tel：+86-513-80562880
Website：http://www.hongweitest.com

14 High-Risk HPV with 16/18 Genotyping Test Kit (Fluorescence PCR) Instructions

References

• Antigen Test, Rapid Tests, Antigen Test Rapid - Macro & Micro-Test

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