十二烷基硫酸钠、月桂酰肌氨酸钠和十二烷基苯磺酸钠提取法检测 HBV DNA 的对比研究。
A comparative study of extraction free detection of HBV DNA using sodium dodecyl sulfate, N-lauroylsarcosine sodium salt, and sodium dodecyl benzene sulfonate.
机构信息
Department of Epidemiology, Infectious Disease Control and Prevention, Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3, Kasumi, Minami, Hiroshima, 734-8551, Japan.
Project Research Center for Epidemiology and Prevention of Viral Hepatitis and Hepatocellular Carcinoma, Hiroshima University, Hiroshima, Japan.
出版信息
Sci Rep. 2024 Oct 26;14(1):25442. doi: 10.1038/s41598-024-75944-7.
This study aimed to develop an extraction-free method for quantitative and qualitative detection of HBV DNA compared to traditional nucleic acid extraction. Paired serum and dried blood spot (DBS) samples from 67 HBsAg-positive and 67 HBsAg-negative individuals were included. Two samples with known HBV DNA titers (~ 10 copies/mL) were examined by extraction-free detection using three surfactants (0.2 to 1% of Sodium dodecyl sulfate:SDS, N-Lauroylsarcosine sodium salt:NL, Sodium dodecyl benzene sulfonate:SDBS), two stabilizing agents (0.1 or 0.01% 2-Mercaptoethanol:2ME and 3.5 or 7% Bovine Serum Albumin:BSA) and two Taq polymerases (Fast Advanced and Prime Direct Probe). HBV DNA in all 67 HBsAg-positive and 67 HBsAg-negative serum and DBS samples was directly quantified by Rt-PCR using 0.4% SDS or 0.4% NL with Fast Advance or Prime Direct Probe Taq. Extraction-free amplification was also performed. Detection limits were varied by different surfactants and Taq. SDS combined with Fast Advanced Taq showed lower detection limits, while SDS with Prime Direct Probe Taq outperformed NL or SDBS-based detection. Adding 2ME to SDS improved detection limit with Prime Direct Probe Taq but not significantly compared to SDS alone. BSA did not significantly enhance detection limits but provided insights into sample stability. The senitivity and specificity of 0.4% SDS and NL in combination with either Fast advanced or Prime Direct Probe Taq polymerase in serum samples were > 90% and 100% resepctively, while it was > 80% and 100% respectively in DBS samples. Extraction-free HBV DNA amplification provided 100% identity with original genomes. Our study suggests that SDS, NL or SDBS-based extraction-free HBV DNA detection strategies using Prime Direct Probe Taq have potential to simplify and accelerate HBV DNA detection with high sensitivity and specificity in both serum and DBS samples, with implications for resource-limited settings and clinical applications. Utilizing surfactants with 2ME is optional, and further research and validation are necessary to broaden its application in real-world diagnostics.
本研究旨在开发一种无需提取的方法，与传统核酸提取方法相比，可对 HBV DNA 进行定量和定性检测。纳入了 67 例 HBsAg 阳性和 67 例 HBsAg 阴性个体的配对血清和干血斑 (DBS) 样本。使用三种表面活性剂（0.2%至 1%十二烷基硫酸钠：SDS、N-月桂酰肌氨酸钠：NL、十二烷基苯磺酸钠：SDBS）、两种稳定剂（0.1%或 0.01%2-巯基乙醇：2ME 和 3.5%或 7%牛血清白蛋白：BSA）和两种 Taq 聚合酶（Fast Advanced 和 Prime Direct Probe）对两种已知 HBV DNA 滴度（~10 拷贝/mL）的样本进行了无提取检测。使用 0.4% SDS 或 0.4% NL 与 Fast Advance 或 Prime Direct Probe Taq 通过 Rt-PCR 直接对所有 67 例 HBsAg 阳性和 67 例 HBsAg 阴性血清和 DBS 样本中的 HBV DNA 进行定量。还进行了无提取扩增。不同的表面活性剂和 Taq 导致检测限有所不同。SDS 与 Fast Advance Taq 联合使用显示出较低的检测限，而 SDS 与 Prime Direct Probe Taq 联合使用则优于 NL 或 SDBS 检测。向 SDS 中添加 2ME 可提高 Prime Direct Probe Taq 的检测限，但与单独使用 SDS 相比并不显著。BSA 并未显著提高检测限，但提供了有关样品稳定性的信息。在血清样本中，SDS 和 NL 分别与 Fast Advanced 或 Prime Direct Probe Taq 聚合酶联合使用时，检测下限的灵敏度和特异性均>90%和 100%，而在 DBS 样本中，其灵敏度和特异性分别为>80%和 100%。无提取 HBV DNA 扩增与原始基因组完全一致。本研究表明，基于 SDS、NL 或 SDBS 的无提取 HBV DNA 检测策略，使用 Prime Direct Probe Taq，具有在血清和 DBS 样本中以高灵敏度和特异性简化和加速 HBV DNA 检测的潜力，对资源有限的环境和临床应用具有重要意义。使用含有 2ME 的表面活性剂是可选的，需要进一步的研究和验证来拓宽其在实际诊断中的应用。
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