Parrot Sex Nucleic Acid Test Kit (PCR-fluorescence probe method) – 100T

Pigeon order sex nucleic acid test kit (with internal reference, PCR-fluorescence probe method)

Product introduction：

This reagent kit can amplify and detect the CHD-W conserved gene in samples such as feathers, blood, and tissues of parrots, thereby determining their gender. By introducing an endogenous internal reference, the nucleic acid extraction and amplification processes can be monitored to ensure the accuracy of the test results.

Product contents：

Reagent componentsAN2304001-01（24T）AN2304001-02（48T）CW reaction solution23µL/well×8wells/strip×3strips23µL/well×8wells/strip×6stipsCW Positive control100μL100μLNegative control100μL100μLStorage conditions：

Store at -20℃, the shelf life is at least 12 months

Experimental operation：

1. Sample preparation (Sample preparation area)

1.1 Sample requirements：

Feathers: Collect at least 5 feathers from the chest, abdomen, or legs (including the feather shaft portion), and do not use naturally shed feathers.

Blood: Use EDTA as an anticoagulant, do not use heparin as an anticoagulant. Fresh whole blood should be used, or it can be stored at 2~8℃ for up to 7 days, or stored at -20℃ for up to 3 months. Avoid repeated freezing and thawing as much as possible.

Tissue: Take fresh muscle or organ tissue (avoid using samples with skin, tendons, or fascia that are difficult to process) of no more than 100mg. Prepare tissue homogenate using 200-500μL of sterile water, and then proceed with subsequent sample preparation.

1.2 Sample preparation：

Please refer to the Three Lions Biotech “Animal Genomic DNA Rapid Extraction Kit (for PCR analysis)” manual (Part Number: DP202), or other nucleic acid extraction kits/methods that meet the relevant requirements, to extract the processed samples. Place the extracted sample nucleic acid in an icebox and try to proceed with the testing promptly. It can be stored at 4°C for up to 7 days or at -20°C for up to 6 months.

2 . Preparation of reaction system (Reaction area for adding samples).

2.1 Take out n+2 reaction tubes containing CW positive control, CW negative control, and the reaction solution needed for the experiment (n represents the number of samples to be tested, plus 1 positive control, and 1 negative control). Thaw the reagents completely at room temperature, centrifuge for 10 seconds, and spin the liquid inside the tube walls and caps to the bottom of the tube. Place them in an icebox for later use.

2.2 Then, add 2μL of negative control, extracted sample nucleic acid, and CW positive control to the reaction solution in sequence. Close the tube caps, make proper records, and ensure that the total volume of each reaction is 25μL. Thoroughly mix the contents, centrifuge for 10 seconds, and then proceed with the amplification experiment on the PCR instrument.

3. PCR amplification (Amplification and product analysis area).

Pre-denaturation at 95°C for 3 minutes; Denaturation at 95°C for 10 seconds, Annealing and extension at 58°C for 30 seconds, for 35 cycles, collecting fluorescence signal at 58°C. The fluorescence channel 1 should be set to FAM (CHD-W gene), and channel 2 should be set to VIC/HEX (endogenous reference gene) (when using the ABI series real-time fluorescence quantitative PCR instrument, if necessary, contact the manufacturer in advance or add ROX calibration dye by yourself; otherwise, follow the normal procedure).

4. Result determination

4.1 If the Ct values of the positive control in both the FAM channel and the VIC/HEX channel are <28 and show an “S”-shaped amplification curve, and the negative control has no Ct value or a Ct value ≥35 and no “S”-shaped amplification curve, the experimental result is valid. Otherwise, the experiment should be repeated, and if the repeat experiment is still invalid, please contact the manufacturer’s technical personnel.

4.2 The Ct value of the sample in the VIC/HEX channel should be ≤30 and show an “S”-shaped amplification curve, indicating that the sample extraction and amplification are effective. If there is no Ct value or the Ct value is 30 < Ct ≤ 35 in the VIC/HEX channel, it indicates that the sample nucleic acid extraction is not up to standard or there is strong inhibition interference (such as residues of alcohol or disinfectants, anticoagulants, etc.) that inhibits amplification. It is recommended to reprocess the sample for nucleic acid extraction and amplification.

4.3 After the detection in the VIC/HEX channel is valid, the determination in the FAM channel is performed as follows:

• Ct value ≤ 30 and an “S”-shaped amplification curve is observed, and the Ct difference between the two channels is less than 5, it is determined as positive for the CHD-W gene, indicating that the sample is from a female.
• Ct value ≤ 30 and an “S”-shaped amplification curve is observed, but the Ct difference between the two channels is ≥ 5, or Ct value is 30 < Ct < 32, all are considered as suspicious, and a retest is recommended (it is suggested to first exclude “false-positive” results caused by environmental aerosol contamination, and then proceed with nucleic acid extraction and testing). If the retest in the FAM channel after excluding aerosol contamination shows a Ct value < 30 and a clear amplification curve, and the Ct difference between the two channels is less than 5, it is determined as positive for the CHD-W gene; otherwise, it is determined as negative.
• No Ct value or Ct value ≥ 32 and no “S”-shaped amplification curve, it is determined as negative for the CHD-W gene, indicating that the sample may come from a male.

Precautions：

1）To prevent contamination, the experiment should be conducted strictly with partitioned operations. It is preferable to have physical isolation between partitions to avoid cross-contamination caused by human factors. Wear lab coats and latex gloves during the experiment, and use separate tools in different areas. Change gloves and lab coats as needed. After the PCR, do not open the lids immediately. Wait for sufficient cooling before opening for sampling to minimize aerosol contamination.

2）Thaw the reagents completely before use, but avoid repeated freezing and thawing. Follow the instructions strictly for reagent preparation, sample addition, and other steps. The workbench, centrifuge, pipettes, and other instruments should be regularly disinfected using chlorine-containing disinfectants, ethanol, nucleic acid contamination removal agents, or ultraviolet lamps.

3）A negative result does not necessarily mean a male. Unknown mutations, poor sample quality, low concentration, or the presence of strong inhibitory substances in the nucleic acid extraction (testing) can also lead to “negative” results. This kit is only used for the specific amplification of the CHD-W gene in parrots. For other products, please consult the manufacturer.

4）This product is for single use only. Do not repeatedly freeze and thaw. It is for scientific research purposes only and should not be used for clinical diagnosis or other purposes.

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