Human Papillomavirus (HPV) is a type of papillomavirus A belonging to the family Lactobacillus, belonging to the DNA virus family. It can cause the proliferation of squamous epithelium in the human skin and mucosa, manifested as common warts, genital warts (genital warts), and other symptoms. With the rapid rise of the incidence rate of condyloma acuminatum among sexually transmitted diseases and the increase of cervical cancer and anal cancer, human papillomavirus infection has attracted more and more attention. There are over 130 types of human papillomavirus, and rapid and accurate identification of human papillomavirus plays an important role in the prevention and quarantine of this disease. This product is a highly sensitive PCR product developed based on the design of primers for the conserved region of human papillomavirus. It has the following characteristics:
1. Ready to use, users only need to provide DNA templates.
2. The primers have been carefully optimized and have strong specificity. They only amplify human papillomavirus and do not cross react with other pathogens.
3. Provide positive controls to distinguish false negative samples.
4. Freeze dried reagents are easy to transport and ready to use.
5.Specification: 48tests / kit-(Lyophilized in 8-well strip ) ；Storage: 2~30℃. And the kit is stable for 12 months；Compatibility:Compatible with Real-time fluorescent PCR instrument, such as ABI7500, Roche LC480, Bio-Rad CFX-96, SLAN96p, Molarray ,MA-6000 and other real-time fluorescent PCR instruments, etc
[Usage]
1. Sample processing
1.1 Sample pretreatment
Place the specialized cervical sampling brush at the cervical opening and gently rub the brush to rotate it clockwise for 4-5 turns; Slowly remove the cervical brush and place it in a sampling tube labeled with the patient's number. The sampling tube has been filled with specialized cell preservation solution. Break the brush head and insert it into the tube, then tighten the bottle cap.
1.2 DNA extraction
1) Add an equal volume of nucleic acid extraction solution to the prepared sample (50% to the solid sample) μ L nucleic acid extraction solution, treated at a constant temperature of 100 ℃ for 10 minutes, centrifuged at 13000 rpm for 5 minutes, and transferred the supernatant to a new 1.5mL EP tube, stored at -20 ℃;
2) DNA extraction can also be carried out using the DNA extraction kit (centrifugal column extraction method) produced by BT. Please strictly follow the instructions of the kit for operation.
2. Reagent preparation
Based on the total number of samples to be tested, set the required number of PCR reaction tubes as N (N=number of samples+1 negative control tube+1 positive control tube; for every 7 full samples, prepare an additional one), and prepare the following table for each test reaction system:
Reagent TB reaction solution enzyme solution
Dosage (N samples) 20 μ L 1 μ L
Divide the mixed test reaction solution into PCR reaction tubes at a rate of 21uL/tube.
3. Sampling
Take 4 samples of DNA, positive control sample, and negative control sample extracted from step 1 μ L. Add them to the corresponding reaction tubes, cover the tubes, mix well, and centrifuge briefly.
4. PCR amplification
4.2 Place the reaction tube to be tested in the reaction tank of the fluorescence quantitative PCR instrument;
4.3 Set the channel and sample information, and set the reaction system to 25 μ L;
Fluorescence channel selection: detection channel (Reporter Dye) FAM, quenching channel (Quencher Dye) NONE, ABI series instruments do not choose ROX reference fluorescence, just select None.
4.4 Recommended cycle parameter settings:
Step Cycle Number Temperature Time Collection Fluorescence Signal
1 1 cycle 95 ℃ for 10 minutes No
2 40 cycles 94 ℃ 15sec No
55 ℃ for 30 seconds
5. Result analysis and judgment
5.1 Setting of Result Analysis Conditions
Setting Baseline and Threshold: Generally, the results of automatic machine analysis are analyzed directly. When the curve shows an overall tilt, the start value (usually adjustable within the range of 3-15), stop value (usually adjustable within the range of 5-20), and Threshold value (drag the threshold line up and down to be higher than the negative control) of the baseline are adjusted based on the analyzed image, and the results are reanalyzed.
5.2 Result judgment
Positive: The Ct value of the detection channel is ≤ 35.0, and there is a clear amplification curve.
Suspicious: For samples with a detection channel Ct value>35.0 and a clear curve, it is recommended to repeat the test. If a Ct value ≤ 35.0 and a clear amplification curve also appear in the repeated test results, it is considered positive, otherwise it is considered negative.
Negative: The sample test result shows no Ct value and no obvious amplification curve.
6. Limitations of detection methods
1) The results of sample testing are related to the quality of sample collection, processing, transportation, and preservation;
2) Failure to control cross contamination during sample extraction can result in false positive results;
3) Leakage of positive control and amplification products can lead to false positive results;
4) Genetic mutations and recombination of pathogens during the epidemic process can lead to false negative results;
5) Different extraction methods have differences in extraction efficiency, which can lead to false negative results;
6) Improper transportation, storage, or inaccurate preparation of reagents may result in a decrease in reagent testing efficiency, resulting in false negatives or inaccurate quantitative testing results;
7) The test results are for reference only. If a diagnosis is necessary, please combine clinical symptoms and other testing methods.
7. Quality control standards
Negative quality control products: no obvious amplification curve or Ct value display;
Positive quality control product: The amplification curve has a significant exponential growth period, and the Ct value is ≤ 32;
The above conditions should be met simultaneously, otherwise the experiment will be deemed invalid.
【 Precautions 】
1. All operations shall be strictly carried out in accordance with the instructions;
2. All components in the reagent kit should naturally melt before use, mix well and centrifuge briefly;
3. The reaction solution should be stored away from light;
4. During the reaction, try to avoid the presence of bubbles and tightly cover the pipe cover;
5. Use disposable suction heads, disposable gloves, and work clothes for each area;
6. Sample processing, reagent preparation, and sample addition should be carried out in different areas to avoid cross contamination;
7. After the experiment is completed, treat the workbench and pipette with 10% hypochlorous acid or 75% alcohol or ultraviolet light;
8. All items in the reagent box should be treated as pollutants and disposed of in accordance with the "General Principles for Biosafety in Microbial Biomedical Laboratories".
