Hepatitis B Virus(HBV) Real-Time Fluorescence PCR Detection Kit

FOR PROFESSIONAL USE ONLY

?Product Name?

Generic Name: Hepatitis B Virus(HBV) Real-Time Fluorescence PCR Detection Kit

?Packing Specifications?

50 tests/kit

?Expected Usage?

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This kit is suitable for in vitro quantitative detection of hepatitis B virus nucleic acid (HBV DNA) in human serum or plasma samples.

?Principle of Inspection?

This kit uses polymerase chain reaction (PCR) combined with Taqman technology to detect the specific nucleic acid fragment of hepatitis B virus (HBV) by real-time PCR.

?Main Ingredients?

?Storage Conditions And Validity Period?

The reagent is stored at -30 ~ -15? and store it in shade . The validity period is 12 months.

This product should be frozen and thawed no more than 5 times.

?Applicable Instrument?

Fluorescence quantitative PCR instrument: ABI 7500 fluorescence PCR instrument.; ABI Quantstudio5; Gentier 96R; SLAN-96P automatic medical PCR analysis system.

?Sample Requirements?

1. Applicable Sample

 Human serum or plasma sample

2. Sample Shipping and Storage

The samples collected above can be used for testing immediately, or stored at 2-8�C (less than 24 hours). For long-term storage, please store at -20�C.

Sample Shipping: Low temperature transportation is adopted.

?Testing Method?

1. Sample processing

The sample to be tested, HBV Quantitative Standard, Negative Control, Strong Positive Control and Critical Positive Control were processed simultaneously. Take 200?L samples and add 5?L of Internal Control to each sample, and perform nucleic acid extraction according to the instructions of nucleic acid extraction kit.

2. Reagent Preparation (Perform in the preparation area, separate from the amplification area, and operate on ice)

Note: The reagents should be completely thawed before use, vortexed before use, and centrifuged at 10,000 rpm for 5 s to ensure that the reagents are completely centrifuged to the bottom of the tube, otherwise there will be differences in the loading volume.

If there are N samples to be tested, prepare the amplification reaction solution according to the following table. After the preparation is completed, it is fully shaken and mixed, and centrifuged at 10,000 rpm for 5 s. Dispense 20?L of each tube into 0.2 mL PCR tubes, and label them well. The amplification reaction solution should be prepared and used immediately 

3. Adding Samples((Perform in the sample processing area, operate on ice)

According to the order of adding Negative Control, Sample to be tested, Critical Positive Control and Strong Positive Control, take 5?L respectively and add them to the eight-strip tube that has been subpackaged with the amplification reaction solution. The total reaction system is 25?L. Close the tube cap tightly, centrifuge briefly and place it in the real-time PCR instrument, and record the order of sample placement.

4. Amplification Reaction (In the amplification area)

FAM channel was selected for detection of hepatitis B virus (HBV) nucleic acid, and CY5 channel was selected for detection of internal standard. Set PCR amplification parameters according to the following amplification program table. Click the sample parameter interface, and set negative control (NTC), positive control and unknown sample (UNK) in the corresponding sequence of samples. For HBV Quantitative Standard, select "Standard" in "Sample Type", and enter the concentration in "Standard Concentration".

5. Quality Control

The above requirements must be met in the same experiment, otherwise, the experiment is invalid and needs to be re-run.

6. Interpretation of Results

Automatically save the results at the end of the reaction, adjust the Start value, End value and Threshold value of Baseline according to the image after analysis (you can adjust it according to the actual situation, the Start value can be set at 3~15, the End value can be set to 5~20, and the amplification curve of the negative control is straight or lower than the threshold line), click Analysis to automatically obtain the analysis results, watch the results on the Report interface, and record the unknown sample value (C).

Test result judgment

6.1 If the FAM channel has no typical amplification curve or Ct value > 40, CY5 channel has a typical amplification curve, and the Ct value is < 35, the judgment is negative.

6.2 If the FAM channel does not have a typical amplification curve or Ct value > 40, and the CY5 channel has no typical amplification curve or Ct value > 35, resampling is required.

6.3 If the FAM channel has a typical amplification curve or Ct value ? 40, it is judged as follows:

If the sample C< 100, the HBV DNA concentration of the sample < 100 IU/mL;

If the sample is 100<C<5.00E+008, the HBV DNA concentration of the sample = C IU/mL;

If the sample C> 5.00E+008, the HBV DNA concentration of the sample is >5x10⁸IU/mL. If accurate quantification is required, samples can be diluted to the linear range with negative controls before testing.

?Explanation of Test Results?

1. Laboratory environment contamination, reagent contamination, and sample cross-contamination will result in false positive results. If the negative control test is positive, further experiments are required to confirm the type of contamination. If the reagent is contaminated, a new kit needs to be opened and the experiment is repeated; if the sample is cross-contaminated, the experiment should be repeated; if the laboratory environment is contaminated, the laboratory should be thoroughly cleaned or the laboratory should be replaced before the experiment is performed.

2. Improper transportation, storage, or preparation of reagents can lead to reduced detection performance and false negative results. If the positive control test is negative, a new kit should be opened before the experiment.

?Limitations of The Detection Method?

1. The test results of this kit are for clinical reference only. The clinical diagnosis and treatment of patients should be comprehensively considered in combination with their symptoms or signs, medical history, other laboratory tests and treatment response.

2. False negatives may occur when the virus content of the sample is below the minimum detection limit. A negative result only means that the extracted nucleic acid concentration is lower than the detection limit of the kit, but it cannot completely rule out the possibility of viral infection.

?Product Performance Index?

1. Accuracy: The test results of the positive reference products of the enterprise are all positive, and the positive rate is 15/15.

2. Minimum detection limit: The minimum detection limit of the kit is 30IU/mL.

3. Coverage of different genotypes: This kit can detect positive for common HBV B, C and D samples at the lowest detection limit concentration.

4. Clinical specificity: 10 clinical HBV DNA negative samples were tested, and the results were all negative.

5. There is no cross-reaction with the following pathogens: hepatitis A virus, hepatitis C virus, adenovirus, human papillomavirus, influenza A virus, influenza B virus, varicella zoster virus, human immunodeficiency virus, hepatitis E virus, hepatitis D virus, Epstein-Barr virus, human cytomegalovirus, Staphylococcus aureus, and Candida albicans.

?Precautions?

Please read the full text of this manual carefully before starting the test.

1. Please operate strictly in accordance with the operation steps, reagent preparation and sampling and other steps, please operate on ice in strict accordance with the instructions.

2. Reagent formulation is recommended in the reagent preparation area, and sample handling requires operation in a biosafety cabinet. Clinical laboratories should be strictly in accordance with the "Management Measures for Clinical Gene Amplification Testing Laboratories in Medical Institutions" and other management norms related to molecular biology laboratories and clinical gene amplification laboratories.

3. The components in the reaction solution are sensitive to light and should be stored away from light. Repeated freeze-thaw may reduce kit sensitivity, so store the reaction solution in an appropriate volume according to the frequency of detection.

4. After the reaction is over, the amplification tube should be placed in a sealed bag and discarded, cleaned on the same day, and do not open the lid.

5. Do not mix reagents with different batch sizes, please use the kits within the validity period.

6. This product is for in vitro diagnostics only.