Abstract：
BACKGROUND: Currently, millions of people suffer from undiagnosed chronic hepatitis B (CHB) infection each year, which leads to high mortality rates attributed to cirrhosis and hepatocellular carcinoma. Previously reported assays, such as PCR-based assays, have limitations in terms of convenient for CHB screening in high-burden regions and resource-limited settings. Recently, diagnosis based on CRISPR/Cas, which has been considered as a potential method of point-of-care test (POCT) in resource-limited settings, offers a significant advantage in terms of high sensitivity and specificity. Therefore, there is an urgent need for the hepatitis B virus (HBV) detection utilizing CRISPR/Cas system.
RESULTS: We have proposed a one-pot of one-step method for CRISPR/Cas12b assisted loop-mediated isothermal amplification (LAMP) to facilitate the quick, sensitive, and precise quantification of HBV DNA. This method is designed for point-of-care testing following genomic extraction or sample heat treatment. We have optimized several critical factors, such as the reaction buffer, AapCas12b-gRNA concentration, reporter and its concentration, reaction temperature, and chemical additives, to significantly enhance the performance of the one-pot assay for HBV. Importantly, it exhibited no cross-reactivity between HBV and blood-borne pathogens. Moreover, the assay is capable of quantifying HBV DNA within 1 h with a limit of detection (LOD) of 25 copies per milliliter. Additionally, when tested on 236 clinical samples, the assay demonstrated a sensitivity of 99.00 % (198/200) and a specificity of 100.00 % (36/36) at the 99 % confidence level compared to real-time quantitative PCR.
CONCLUSIONS: The utilization of convenient and reliable point-of-care diagnostic methods is crucial for reducing the burden of CHB globally. The assay we developed was helpful to improve the ability of HBV diagnosis for practical clinical translation, especially in high-burden regions and resource-limited settings. It has great advantages for rapid screening of CHB as well as evaluation of therapeutic efficacy as a companion diagnostic method.
RESULTS: We have proposed a one-pot of one-step method for CRISPR/Cas12b assisted loop-mediated isothermal amplification (LAMP) to facilitate the quick, sensitive, and precise quantification of HBV DNA. This method is designed for point-of-care testing following genomic extraction or sample heat treatment. We have optimized several critical factors, such as the reaction buffer, AapCas12b-gRNA concentration, reporter and its concentration, reaction temperature, and chemical additives, to significantly enhance the performance of the one-pot assay for HBV. Importantly, it exhibited no cross-reactivity between HBV and blood-borne pathogens. Moreover, the assay is capable of quantifying HBV DNA within 1 h with a limit of detection (LOD) of 25 copies per milliliter. Additionally, when tested on 236 clinical samples, the assay demonstrated a sensitivity of 99.00 % (198/200) and a specificity of 100.00 % (36/36) at the 99 % confidence level compared to real-time quantitative PCR.
CONCLUSIONS: The utilization of convenient and reliable point-of-care diagnostic methods is crucial for reducing the burden of CHB globally. The assay we developed was helpful to improve the ability of HBV diagnosis for practical clinical translation, especially in high-burden regions and resource-limited settings. It has great advantages for rapid screening of CHB as well as evaluation of therapeutic efficacy as a companion diagnostic method.
摘要：
背景：目前，每年有数百万人患有未诊断的慢性乙型肝炎（CHB）感染，导致高死亡率归因于肝硬化和肝细胞癌。以前报道的测定，例如基于PCR的检测，在高负担地区和资源有限的环境中方便CHB筛查方面有局限性。最近,基于CRISPR/Cas的诊断，在资源有限的环境中，它被认为是一种潜在的即时测试(POCT)方法，在高灵敏度和特异性方面具有显著优势。因此,迫切需要利用CRISPR/Cas系统进行乙型肝炎病毒（HBV）检测。
结果：我们提出了一种用于CRISPR/Cas12b辅助环介导等温扩增（LAMP）的一步法，以促进快速，敏感,和HBVDNA的精确定量。该方法设计用于基因组提取或样品热处理后的即时测试。我们优化了几个关键因素，如反应缓冲液，AapCas12b-gRNA浓度，记者及其浓度，反应温度,和化学添加剂，显着增强一锅法检测HBV的性能。重要的是,它表现出HBV和血源性病原体之间没有交叉反应性。此外，该测定法能够在1小时内定量HBVDNA，检测限（LOD）为每毫升25个拷贝。此外,当在236个临床样本上测试时，与实时定量PCR相比,在99%置信水平下,该检测的灵敏度为99.00%(198/200),特异性为100.00%(36/36).
结论：使用方便可靠的即时诊断方法对于降低全球CHB的负担至关重要。我们开发的检测方法有助于提高HBV诊断的实际临床翻译能力，特别是在高负担地区和资源有限的环境中。它对于CHB的快速筛查以及作为伴随诊断方法的治疗效果评估具有很大的优势。
结果：我们提出了一种用于CRISPR/Cas12b辅助环介导等温扩增（LAMP）的一步法，以促进快速，敏感,和HBVDNA的精确定量。该方法设计用于基因组提取或样品热处理后的即时测试。我们优化了几个关键因素，如反应缓冲液，AapCas12b-gRNA浓度，记者及其浓度，反应温度,和化学添加剂，显着增强一锅法检测HBV的性能。重要的是,它表现出HBV和血源性病原体之间没有交叉反应性。此外，该测定法能够在1小时内定量HBVDNA，检测限（LOD）为每毫升25个拷贝。此外,当在236个临床样本上测试时，与实时定量PCR相比,在99%置信水平下,该检测的灵敏度为99.00%(198/200),特异性为100.00%(36/36).
结论：使用方便可靠的即时诊断方法对于降低全球CHB的负担至关重要。我们开发的检测方法有助于提高HBV诊断的实际临床翻译能力，特别是在高负担地区和资源有限的环境中。它对于CHB的快速筛查以及作为伴随诊断方法的治疗效果评估具有很大的优势。
