Abstract：
Parvovirus B19 (B19V) is pathogenic to humans and causes various human diseases. However, no antiviral agents or vaccines currently exist for the treatment or prevention of B19V infection. Therefore, developing sensitive and specific methods for B19V infection diagnosis is essential for accurate diagnoses. Previously, a Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-Cas12a (cpf1)-based electrochemical biosensor (E-CRISPR) with a picomole sensitivity for B19V detection was established. Herein, we set up a novel nucleic acid detection system based on Pyrococcus furiosus Argonaute (PfAgo)-mediated nucleic acid detection, targeting the nonstructural protein 1 (NS1) region of the B19V viral genome (abbreviated B19-NS1 PAND). Benefiting from independent protospacer adjacent motif (PAM) sequences, PfAgo can recognize their target with guide DNA (gDNA) that is easy to design and synthesize at a low cost. In contrast to E-CRISPR, without preamplification with Polymerase Chain Reaction (PCR), the Minimum Detectable Concentration (MDC) of three guide- or single guide-mediated B19-NS1 PAND was about 4 nM, approximately 6-fold more than E-CRISPR. However, when introducing an amplification step, the MDC can be dramatically decreased to the aM level (54 aM). In addition, the diagnostic results from clinical samples with B19-NS1 PAND revealed 100% consistency with PCR assays and subsequent Sanger sequencing tests, which may assist in molecular testing for clinical diagnosis and epidemiological investigations of B19V.
摘要：
细小病毒B19（B19V）对人类具有致病性，并引起各种人类疾病。然而,目前没有用于治疗或预防B19V感染的抗病毒药物或疫苗。因此,开发敏感和特异性的B19V感染诊断方法对于准确诊断至关重要。以前,建立了用于B19V检测的具有皮摩尔灵敏度的基于聚类的定期间隔回文重复(CRISPR)-Cas12a(cpf1)的电化学生物传感器(E-CRISPR)。在这里，我们建立了一种新的核酸检测系统，该系统是基于PfAgo介导的核酸检测，靶向B19V病毒基因组的非结构蛋白1（NS1）区域（缩写为B19-NS1PAND）。受益于独立的原型间隔区相邻基序（PAM）序列，PfAgo可以用易于以低成本设计和合成的向导DNA（gDNA）识别其靶标。与E-CRISPR相比，无需使用聚合酶链反应（PCR）进行预扩增，三个引导或单个引导介导的B19-NS1PAND的最小可检测浓度（MDC）约为4nM，大约是E-CRISPR的6倍。然而,当引入扩增步骤时，MDC可以显著降低到aM水平(54aM)。此外,使用B19-NS1PAND的临床样本的诊断结果与PCR测定和随后的Sanger测序测试具有100％的一致性，这可能有助于B19V的临床诊断和流行病学调查的分子检测。
