The Second Clinical Medical College, Guizhou University of Traditional Chinese Medicine, Guiyang, Guizhou, 550003, People's Republic of China.
Department of Scientific Research, The Second Affiliated Hospital, Guizhou University of Traditional Chinese Medicine, Guiyang, Guizhou, 550003, People's Republic of China.
BMC Microbiol. 2024 Nov 6;24(1):455. doi: 10.1186/s12866-024-03610-z.
Viral hepatitis caused by hepatitis B virus (HBV) and hepatitis C virus (HCV) infections remain a major global public health challenge, particularly in low- and middle-income countries. It is crucial to utilize a pointof-care (POC) testing platform that is sensitive, specific, rapid, and user-friendly for screening and diagnosis of the two infections. Here, a novel molecular diagnostic assay, integrating multiplex loop-mediated isothermal amplification with a gold nanoparticle-based lateral flow biosensor (mLAMP-AuNPs-LFB) was developed and applied for one-step, visual, rapid, sensitive, and specific identification of HBV and HCV.
The AuNPs-based LFB was devised and constructed for the simultaneous detection of HBV and HCV. The HBV-LAMP and HCV-LAMP primers were designed against the S and 5'-untranslated region (5'-UTR) genes from the major HBV genotypes (B, C, D, B/C recombinant, and C/D recombinant) and HCV subtypes (1b, 2a, 3a, 3b, and 6a) in China, respectively. Our assay conditions, both multiplex-LAMP amplification temperature and time were optimized. The sensitivity and specificity of our assay were tested, and the feasibility of our assay was verified through clinical samples.
The AuNPs-based LFB used here was successfully manufactured according to our devise manual. The two unique independent primer pairs were successfully designed based on the S and 5'-UTR genes, respectively. The optimal mLAMP-AuNPs-LFB detection process, involving rapid nucleic acid isolation (10 min), mLAMP (63 °C for 35 min), and visual AuNPs-LFB interpretation (less than 2 min), could be completed within 50 min. The HBV&HCV-mLAMP-AuNPs-LFB assay can detect the target genes (HBV-S and HCV-5'-UTR) with as low as 20 copies of plasmid template per test, and the specificity was 100% for the experimental pathogens.
The preliminary results manifested that our mLAMP-AuNPs-LFB assay is a valuable tool and has tremendous potential as a POC testing approach for HBV and HCV identification, especially in undeveloped regions.
乙型肝炎病毒（HBV）和丙型肝炎病毒（HCV）感染引起的病毒性肝炎仍然是一个重大的全球公共卫生挑战，特别是在低收入和中等收入国家。利用一种敏感、特异、快速、用户友好的即时检测（POC）平台对这两种感染进行筛查和诊断至关重要。在这里，我们开发了一种新型分子诊断检测方法，将多重环介导等温扩增与基于金纳米粒子的侧向流动生物传感器（mLAMP-AuNPs-LFB）相结合，用于一步、可视化、快速、敏感和特异性检测 HBV 和 HCV。
设计并构建了基于 AuNPs 的 LFB 用于同时检测 HBV 和 HCV。HBV-LAMP 和 HCV-LAMP 引物分别针对中国主要 HBV 基因型（B、C、D、B/C 重组和 C/D 重组）和 HCV 亚型（1b、2a、3a、3b 和 6a）的 S 和 5'-非翻译区（5'-UTR）基因设计。优化了多重-LAMP 扩增温度和时间等实验条件。对本研究检测方法的灵敏度和特异性进行了测试，并通过临床样本验证了其可行性。
根据我们的设计手册，成功制造了基于 AuNPs 的 LFB。成功设计了基于 S 和 5'-UTR 基因的两个独特的独立引物对。最佳的 mLAMP-AuNPs-LFB 检测过程包括快速核酸提取（10 分钟）、mLAMP（63°C 35 分钟）和可视化 AuNPs-LFB 解释（不到 2 分钟），整个过程可在 50 分钟内完成。HBV&HCV-mLAMP-AuNPs-LFB 检测方法可检测到每个测试低至 20 个拷贝质粒模板的靶基因（HBV-S 和 HCV-5'-UTR），且对实验病原体的特异性为 100%。
初步结果表明，我们的 mLAMP-AuNPs-LFB 检测方法是一种有价值的工具，具有作为 HBV 和 HCV 鉴定的 POCT 方法的巨大潜力，特别是在欠发达地区。
