Kit for pigeon sex determination

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

Kit for pigeon sex determination allows to determine the sex of pigeons by analyzing bird DNA starting from feather samples by polymerase chain reaction (PCR) using a prepared PCR mixture.This kit can amplify and detect the CHD-W conserved gene in pigeons' feathers, blood and tissues, and then identify the gender of pigeons. The nucleic acid extraction and amplification process can be monitored by introducing endogenous internal references to ensure the accuracy of the detection results.

Kit for pigeon sex determination product content:

Kit for pigeon sex determination storage conditions:

Store at -20℃ for a validity period of at least 12 months.

Kit for pigeon sex determination experimental operation:

1. Sample preparation (sample preparation area)

1.1 Sample Requirements:

Feathers: Collect no less than 5 feathers on the chest, abdomen or legs (including the feather root part). Do not use naturally falling feathers.

Whole blood: Use EDTA as an anticoagulant, do not use heparin to anticoagulant. Fresh whole blood is required, or stored at 2~8℃ for no more than 7 days, or stored at -20℃ for no more than 3 months. Try to avoid repeated freeze-thawing.

Tissue: Use fresh muscle or organ tissue (not easily treated samples such as skin or fascia as possible) to not exceed 100 mg, use 200-500 μL of sterile water to prepare the tissue homogenate, and then perform subsequent sample preparation.

1.2 Sample preparation:

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

2. Prepare the reaction system (sample loading area)

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

2.2 Then add 2 μL of negative control, extracted sample nucleic acid and GW positive control to the reaction solution, cover the tube cover, and keep records. The total volume of each reaction is 25 μL. Mix thoroughly, centrifuge for 10 seconds before performing amplification experiment on a PCR instrument.

3. PCR amplification (amplification and product analysis area)

Predenaturation at 95℃ for 3 minutes; denaturation at 95℃ for 10 seconds, annealing at 58℃ for 30 seconds, 35 cycles, and fluorescence signals were collected at 58℃. FAM (CHD-W gene) is selected for the first channel of fluorescence, and VIC/HEX (endogenous internal reference gene) is selected for the second channel (using the ABI series real-time fluorescence quantitative PCR device, if you have any requirements, you can contact the manufacturer in advance or add ROX correction dye yourself; otherwise, refer to the normal procedure). 

4. Result judgment

4.1 The Ct values of the positive control FAM channel and VIC/HEX channel are both <28 and the "S"-type amplification curve appears. The negative control has no Ct value or the Ct value ≥35 and there is no "S"-type amplification curve. The experimental results are valid; otherwise, the experiment should be carried out again. If the retest experiment is still invalid, please contact the manufacturer's technician.

4.2 The Ct value of the sample VIC/HEX channel should be ≤30 and a "S"-type amplification curve appears, indicating that the sample extraction and amplification are effective; if there is no Ct value or the Ct value of 30＜Ct≤35, it indicates that the sample nucleic acid extraction does not meet the standards or there are strong inhibitory interference substances (such as disinfectants such as alcohol, anticoagulants, etc.) to 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, then the FAM channel is determined:

The Ct value is ≤30 and a "S"-type amplification curve appears, which is determined to be positive for the CHD-W gene, indicating that the sample is from females;

If the Ct value is 30＜Ct＜32, it is determined that it is suspicious. Retesting is recommended (it is recommended to first exclude the "false positive" results caused by environmental aerosol pollution, and then re-extract and test). If the FAM channel re-testing Ct value is <30 after aerosol pollution is eliminated and there is a significant amplification curve, it is determined that the CHD-W gene is positive, otherwise it is determined to be negative;

There was no Ct value or Ct value ≥32 and no "S"-type amplification curve, and it was determined that the CHD-W gene was negative, indicating that the sample may be derived from males.

Kit for pigeon sex determination notes:

1) In order to prevent pollution, the experiment requires strict partitioning operations, and it is best to physically isolate the partitions to avoid cross-contamination caused by human factors. During the experiment, work clothes and latex gloves were worn, tools were used independently in different areas, and gloves and experimental clothes were needed to be replaced. After the PCR is over, do not open the cover immediately. After cooling thoroughly, open the cover to take samples to avoid aerosol contamination to the greatest extent.

2) The reagent should be completely thawed before use, but repeated freeze-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 regularly disinfected with chlorine-containing disinfectants, disinfected alcohol, nucleic acid contamination removers or ultraviolet lamps.

3) The test result is negative, which does not completely mean that it is male. If the nucleic acid sample has an unknown mutation, unqualified quality, too low load, and strong interference inhibitors, it will also produce a "negative" result. This kit is only used for specific amplification of the CHD-W gene in pigeons. Please consult the manufacturer for other products involved.

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