ピジョンヘルペスウイルス (PiHV) 核酸検査キット

ピジョンヘルペスウイルス (PiHV) 核酸検査キット (PCR-Fluorescent Probe Method)

商品番号： AP2308002 仕様：24T/48T /96T ストレージ：-20℃

製品導入：

• ピジョンヘルペスウイルス (PiHV) Infection:
  • Viral infectious disease primarily affecting young pigeons caused by Pigeon Herpesvirus Type 1 (PiHV1).
  • Important pathogen leading to pigeon squab disease syndrome in young pigeons.
  • Clinically characterized by symptoms such as nasal and conjunctival inflammation, depression, decreased appetite, diarrhea, vomiting, and neurological symptoms.
• Importance of Detection:
  • Early, rapid, and convenient detection of PiHV is crucial for diagnosis.
  • Helps in slowing the spread of the disease and preventing complications.
• Test Kit Description:
  • Designed based on conserved sequences in the PiHV gene.
  • Optimized conditions enable accurate fluorescent quantitative PCR testing.
  • Accepts oral, and cloacal swabs, 血, and other tissue samples from birds.
  • Allows for quick determination of PiHV infection in the host.

商品内容：

保管条件：

-20℃で保存, 保存期間は少なくとも 12 月.

実験運用：

1. サンプルの準備 (サンプル準備エリア)

1.1 サンプル要件:

– 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 時間, and samples that cannot be tested promptly should be stored at -20°C.

– Whole blood: 抗凝固剤としてEDTAを使用する, avoid heparin anticoagulant, use fresh whole blood, or store at 2-8°C for no more than 7 日々, or store at -20°C for no more than 3 月, avoiding repeated freeze-thaw cycles.

– 組織: 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 サンプルの準備:

Refer to the SanshiBio “動物ゲノム DNA 迅速抽出キット (PCR分析用)” 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 月.

2. Reaction System Preparation (Reaction Area)

2.1 Take out each component of the kit, completely thaw at room temperature, 遠心分離機用 10 秒, 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: 取る (n+2) reaction tubes containing PiHV premix solution, add 11.5µL of PiHV 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 PiHV positive control to the reaction solution. Close the tube cap, make a record, and the total volume of each reaction is 25μL. 十分に混ぜ合わせてください, 遠心分離機用 10 秒, and perform amplification experiments on the PCR instrument.

3. PCR増幅 (増幅および生成物分析エリア)

Pre-denaturation at 95°C for 2 分; Denaturation at 95°C for 10 秒, annealing and extension at 60°C for 35 秒, 40 サイクル. 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. 結果の解釈

4.1 Conditions for Experiment Validity:

Positive control FAM channel Ct value < 28 そして “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 PiHV 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 PiHV positive, otherwise considered negative;

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

予防：

• 汚染を防ぐために, 実験は厳密に分割する必要があります, and physical isolation between partitions is preferred to avoid cross-contamination due to human factors. 実験中は白衣とラテックス手袋を着用してください, use tools independently in different areas, change gloves and lab coats when needed. After PCR, すぐに蓋を開けないでください; 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, 遠心分離機, ピペット, and other instruments should be disinfected regularly with chlorine-containing disinfectants, alcohol, 核酸除染剤, 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) そして “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.