Kit for pigeon sex determination

Kit for pigeon sex determination (with internal reference, PCR-fluorescent probe method)

Kit for pigeon sex determination is widely used for sex identification of different birds. PCR amplification has good effect and can accurately determine the sex of birds.This kit can amplify and detect the CHD-W conserved gene in pigeon feathers, blood and tissue samples, and then identify the pigeon's gender. By introducing endogenous internal reference, the nucleic acid extraction and amplification process can be monitored to ensure the accuracy of the test results.

Kit for pigeon sex determination product contents:

Kit for pigeon sex determination storage conditions:

Stored at -20℃, the validity period is at least 12 months.

Kit for pigeon sex determination experimental procedures:

1. Sample preparation (sample preparation area)

1.1 Sample requirements:

Feathers: Collect no less than 5 feathers (including feather bases) from the chest, abdomen or legs. Do not use feathers that fall off naturally.

Whole blood: Use EDTA as an anticoagulant. Do not use heparin for anticoagulation. It is required to use fresh whole blood, or store it at 2~8℃ for no more than 7 days, or store it at -20℃ for no more than 3 months. Try to Avoid repeated freezing and thawing.

Tissue: Take fresh muscle or organ tissue (try to avoid using samples such as skin or fascia that are difficult to process), no more than 100mg, use 200-500μL sterile water to prepare tissue homogenate, and then perform subsequent sample preparation.

1.2 Sample preparation:

Please refer to Sanshi Bio's "Animal Genomic DNA Rapid Extraction Kit (for PCR Analysis) Instructions" (Cat. No.: DP202), or other nucleic acid extraction kits/methods that meet relevant requirements, to extract nucleic acids from the processed samples. The extracted sample nucleic acid is placed in an ice box and tested as soon as possible. It should be stored at 4°C for no more than 7 days or -20°C for no more than 6 months.

2. Prepare reaction system (sample addition area)

2.1 Take out each component in the kit, thaw completely at room temperature, centrifuge for 10 seconds, and centrifuge the liquid in the tube wall and tube cap to the bottom of the tube; according to the number of samples (n+2) (i.e. n samples to be tested +1 Positive control + 1 negative control) to prepare the reaction system, the specific preparation method is as follows: respectively draw ((n+2) × 11.5µL GW reaction solution + (n+2) × 11.5µL GW master mix) into a clean centrifuge tube, mix thoroughly by pipetting, and dispense into PCR tubes, 23 μL per well, and place in an ice box for later use.

2.2 Then add 2 μL negative control, extracted sample nucleic acid and GW positive control to the reaction solution in sequence, cover the tube cap, and make records. The total volume of each reaction is 25 μL. Mix thoroughly, centrifuge for 10 seconds and perform amplification experiments on a PCR machine.

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, 35 cycles, and fluorescence signals were collected at 58°C. Select FAM (CHD-W gene) for the first fluorescence channel, and select VIC/HEX (endogenous internal reference gene) for the second channel (use an ABI series real-time fluorescence quantitative PCR instrument. If necessary, you can contact the manufacturer in advance or add ROX correction dye yourself. ; Otherwise, follow the normal procedures). 

4. Result judgment

4.1 The Ct values of the FAM channel and VIC/HEX channel of the positive control are both <28 and there is an "S" type amplification curve. The negative control has no Ct value or the Ct value is ≥35 and there is no "S" type amplification curve. The experimental results are valid; Otherwise, the experiment should be re-tested. If the re-test is still invalid, please contact the manufacturer's technical personnel.

4.2 The Ct value of the VIC/HEX channel of the sample should be ≤30 and an "S"-shaped amplification curve appears, indicating that the sample extraction and amplification are effective; if there is no Ct value in the VIC/HEX channel or the Ct value is 30 <Ct≤35, it indicates that the sample If the nucleic acid extraction is not up to standard or there are strong inhibitory interference substances (such as alcohol and other disinfectants, anticoagulant residues, etc.) that inhibit amplification, it is recommended to reprocess the sample for nucleic acid extraction and amplification;

4.3 After the sample VIC/HEX channel detection is valid, the FAM channel is determined:

If the Ct value is ≤30 and an “S”-shaped amplification curve appears, it is determined to be positive for the CHD-W gene, indicating that the sample is from a female;

If the Ct value is 30＜Ct＜32, all are judged to be suspicious, and it is recommended to conduct a retest (it is recommended to first eliminate "false positive" results caused by environmental aerosol pollution, and then re-extract nucleic acid and detect). If the aerosol pollution is eliminated, If the Ct value of the FAM channel retest is <30, and there is an obvious amplification curve, it is judged to be CHD-W gene positive, otherwise it is judged to be negative;

If there is no Ct value or Ct value ≥32 and no "S" type amplification curve, it is judged as negative for the CHD-W gene, indicating that the sample may be from a male.

Kit for pigeon sex determination Precautions:

1) In order to prevent contamination, experiments need to be strictly partitioned. It is best to physically isolate the partitions to avoid cross-contamination caused by human factors. Wear work clothes and latex gloves during the experiment, use tools independently in different areas, and need to change gloves and lab coats. After PCR is completed, do not open the lid immediately. Allow it to cool enough before opening the lid to take samples to avoid aerosol contamination to the greatest extent.

2) The reagents must be completely thawed before use, but repeated freezing and thawing should be avoided. Please strictly follow the instructions for reagent preparation and sample addition. Instruments such as operating tables, centrifuges, and pipettes should be disinfected regularly using chlorine-containing disinfectants, disinfecting alcohol, nucleic acid contamination removers, or ultraviolet lamps.

3) A negative test result does not completely mean that the nucleic acid sample is male. Unknown mutations, unqualified quality, low load, and strong interference inhibitors in the nucleic acid sample will also produce a "negative" result if the nucleic acid extraction (test) is unsuccessful. . This kit is only used for specific amplification of the CHD-W gene in pigeons. Please contact the manufacturer for other products involved.

4) This product is a disposable product. The components in the reagent are relatively sensitive to natural light. Pay attention to shading when packaging and storing. Do not freeze and thaw repeatedly. It is for scientific research only and is not for clinical diagnosis and other purposes.