Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA, USA.
Department of Statistics, College of Liberal Arts and Sciences, Iowa State University, Ames, IA, USA.
J Vet Diagn Invest. 2023 Sep;35(5):521-527. doi: 10.1177/10406387231182102. Epub 2023 Jun 20.
Based on publications reporting improvements in real-time PCR (rtPCR) performance, we compared protocols based on heat treatment or dilution followed by direct rtPCR to standard extraction and amplification methods for the detection of porcine reproductive and respiratory syndrome virus (PRRSV), influenza A virus (IAV), porcine epidemic diarrhea virus (PEDV), or (MHP) in swine oral fluids (OFs). In part A, we subjected aliquots of positive OF samples to 1 of 4 protocols: protocol 1: heat (95°C × 30 min) followed by direct rtPCR; protocol 2: heat and cool (25°C × 20 min) followed by direct rtPCR; protocol 3: heat, cool, extraction, and rtPCR; protocol 4 (control): extraction and then rtPCR. In part B, positive OF samples were split into 3, diluted (D1 = 1:2 with Tris-borate-EDTA (TBE); D2 = 1:2 with negative OF; D3 = not diluted), and then tested by rtPCR using the best-performing protocol from part A (protocol 4). In part A, with occasional exceptions, heat treatment resulted in marked reduction in the detection of target and internal sample control (ISC) nucleic acids. In part B, sample dilution with TBE or OF produced no improvement in the detection of targets and ISCs. Thus, standard extraction and amplification methods provided superior detection of PRRSV, IAV, PEDV, and MHP nucleic acids in OFs.
基于报道实时 PCR（rtPCR）性能改进的文献，我们比较了基于热处理或稀释后直接进行 rtPCR 的方案与标准提取和扩增方法，用于检测猪繁殖与呼吸综合征病毒（PRRSV）、甲型流感病毒（IAV）、猪流行性腹泻病毒（PEDV）或细小病毒（MHP）在猪口腔液（OFs）中的存在。在 A 部分中，我们将阳性 OF 样本的等分试样进行以下 4 种方案之一的处理：方案 1：加热（95°C×30 分钟）后直接进行 rtPCR；方案 2：加热和冷却（25°C×20 分钟）后直接进行 rtPCR；方案 3：加热、冷却、提取和 rtPCR；方案 4（对照）：提取后再进行 rtPCR。在 B 部分中，阳性 OF 样本被分成 3 份，稀释（D1=用 Tris-硼酸-EDTA（TBE）稀释 1:2；D2=用阴性 OF 稀释 1:2；D3=未稀释），然后使用 A 部分中表现最佳的方案（方案 4）通过 rtPCR 进行测试。在 A 部分中，除了偶尔的例外情况外，热处理导致靶标和内部样本对照（ISC）核酸的检测显著减少。在 B 部分中，用 TBE 或 OF 稀释样本并没有改善靶标和 ISC 的检测。因此，标准提取和扩增方法在 OFs 中提供了对 PRRSV、IAV、PEDV 和 MHP 核酸的更好检测。
