Pigeon Adenovirus (PiADV) Nucleic Acid Detection Kit

Item No：AP2308005        Specifiation：24T/48T            Storage：-20℃

Product introduction：

• Pigeon Adenovirus (PiADV) is a common and highly contagious adenovirus that infects birds such as pigeons and parrots, serving as the causative agent of acute infectious diseases, including squab adenitis.
• This virus is highly transmissible, capable of vertical transmission through egg laying, as well as horizontal transmission through direct or indirect contact.
• Currently, there are no specific antiviral drugs for PiADV, making early, rapid, and convenient detection of PiADV crucial for diagnosis, and treatment, slowing the spread of the disease, and preventing complications.
• This detection kit is designed based on the conservative sequences in the PiADV genome, utilizing primers and probes.
• Through system optimization, it enables precise fluorescence quantitative PCR testing of oral cloacal swabs, blood, and other tissue samples from birds.
• This facilitates the rapid and accurate determination of PiADV infection in the host.

Product contents：

Storage conditions：

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

 Experimental operation：

1. Sample Preparation (Sample Preparation Area)

1.1 Sample Requirements:

– Oral and cloacal swabs: Take a sterile cotton swab, insert it into the bird’s throat or cloaca, rotate three times, place it in a centrifuge tube, cut the exposed part, tightly close the cap, and label it. Samples that can be tested promptly should be stored at 2-8°C within 24 hours, and samples that cannot be tested promptly should be stored at -20°C.

– Whole blood: Use EDTA as an anticoagulant, avoid heparin anticoagulant, use fresh whole blood, or store at 2-8°C for no more than 7 days, or store at -20°C for no more than 3 months, avoiding repeated freeze-thaw cycles.

– Tissue: Take fresh muscle or organ tissue not exceeding 100mg, use 200-500μL sterile water to prepare tissue homogenate, and proceed with subsequent sample preparation.

1.2 Sample Preparation:

Refer to the SanshiBio Bio “Animal Genomic DNA Rapid Extraction Kit (for PCR analysis)” manual (Catalog: DP202), or other nucleic acid extraction kits/methods that meet relevant requirements, for nucleic acid extraction of processed samples. Place the extracted sample nucleic acid in a cooler and test as soon as possible. Store at 4°C for no more than 7 days or at -20°C for no more than 6 months.

2. Reaction System Preparation (Reaction Area)

2.1 Take out each component of the kit, completely thaw at room temperature, centrifuge for 10 seconds, and centrifuge the liquid inside the tube wall and tube cap to the bottom of the tube. Prepare the reaction solution according to the sample quantity (n+2, where n is the number of samples, it is recommended to set up 1 positive control and 1 negative control for each experiment). Specific preparation method: Take (n+2) reaction tubes containing PiADV premix solution, add 11.5µL of PiADV reaction solution to each tube, use a pipette to thoroughly mix, 23µL per well, and store in a cooler.

2.2 Then sequentially add 2μL of negative control, extracted sample nucleic acid, and PiADV positive control to the reaction solution. Close the tube cap, make a record, and the total volume of each reaction is 25μL. Mix thoroughly, centrifuge for 10 seconds, and perform amplification experiments on the PCR instrument.

3. PCR Amplification (Amplification and Product Analysis Area)

Pre-denaturation at 95°C for 2 minutes; Denaturation at 95°C for 10 seconds, annealing and extension at 60°C for 35 seconds, 40 cycles. Collect fluorescence signals at 60°C. Choose the FAM fluorescence channel (use real-time fluorescence quantitative PCR instruments such as ABI series, if necessary, contact the manufacturer or add ROX correction dye; otherwise, follow normal procedures).

4. Result Interpretation

4.1 Conditions for Experiment Validity:

Positive control FAM channel Ct value < 28 and a “S”-shaped amplification curve is observed; negative control has no Ct value or Ct value ≥ 38 and no “S”-shaped amplification curve, the experiment is valid. Otherwise, repeat the experiment; if the repeat experiment is still invalid, contact the manufacturer’s technical personnel.

4.2 Interpretation of Sample Results:

Ct value ≤ 33 and an “S”-shaped amplification curve indicate PiADV positive;

Ct value between 33 < Ct < 38 is considered suspicious, and a retest is recommended. If the FAM channel retest Ct value is < 33 after excluding aerosol contamination and there is a clear amplification curve, it is considered PiADV positive, otherwise considered negative;

No Ct value or Ct value ≥ 38 and no “S”-shaped amplification curve indicate PiADV negative.

Precautions：

• To prevent contamination, the experiment should be strictly partitioned, and physical isolation between partitions is preferred to avoid cross-contamination due to human factors. Wear lab coats and latex gloves during the experiment, use tools independently in different areas, change gloves and lab coats when needed. After PCR, do not open the lid immediately; open it after sufficient cooling to minimize aerosol contamination.
• Thaw the reagents completely before use, but avoid repeated freeze-thaw cycles. The PCR reaction tubes provided in the kit are 0.2mL; if replacement is needed, transfer after complete thawing. Strictly follow the instructions for reagent preparation and sample addition. Workstations, centrifuges, pipettes, and other instruments should be disinfected regularly with chlorine-containing disinfectants, alcohol, nucleic acid decontamination agents, or UV light.
• A negative result does not necessarily mean the host is not infected with the virus. Poor sample quality, low virus load, or the presence of strong inhibitory substances (such as alcohol, disinfectants, anticoagulants, etc.) may result in unsuccessful nucleic acid extraction (detection) and a “negative” result. Follow the result interpretation criteria, and when there are suspicious results, first exclude aerosol contamination. If there are other questions, contact the manufacturer’s technical personnel.
• This product is for one-time use only. The components in the reagent are sensitive to natural light; avoid light exposure during packaging and storage. After packaging, do not repeatedly freeze-thaw. For scientific research use only; not for clinical diagnosis or other purposes.