A rapid RNA extraction-free lateral flow assay for molecular point-of-care detection of SARS-CoV-2 augmented by chemical probes.
A rapid RNA extraction-free lateral flow assay for molecular point-of-care detection of SARS-CoV-2 augmented by chemical probes.
复制标题
DOI：
10.1016/j.bios.2021.113900
复制
发表时间：
2022-03-15
影响因子：
12.6
通讯作者：
Pan D
中科院分区：
文献类型：
--
作者：
Dighe K;Moitra P;Alafeef M;Gunaseelan N;Pan D
冠状病毒病2019年(新冠肺炎)大流行突显了全球医疗系统的主要缺陷，即无法快速准确地诊断疾病。目前，诊断新冠肺炎的分子方法主要是利用逆转录聚合酶链式反应(RT-PCR)从SARS冠状病毒2型(SARS-CoV-2)RNA中扩增出c DNA。尽管有报道称分子检测具有特异性，但假阴性相当常见。此外，从字面上看，所有这些测试都需要一个涉及RNA分离的步骤，这并不能使它们成为真正意义上的关注点(POC)。在这里，我们报告了一种基于侧向流动纸条的RNA提取和无扩增核酸测试(NAT)，用于POC新冠肺炎阳性病例的快速诊断。该方法使用高度特异的6-羧基荧光素(6-FAM)和生物素标记的反义寡核苷酸(ASO)作为探针，针对SARS-CoV-2的N基因序列进行靶向。此外，我们还使用了半胱胺修饰的金纳米颗粒(cyst-AuNPs)来进一步增强信号以提高灵敏度。在没有任何大型固定设备和训练有素的工作人员的情况下，在我们的案例中，从患者拭子样本采集到最终诊断结果，整个样本到答案的方法只需不到30分钟。此外，当对60个临床样本进行评估并用FDA批准的用于新冠肺炎诊断的TaqPath RT-PCR试剂盒验证时，该方法获得了近99.99%的准确性和特异性。我们预计，新建立的对SARS-CoV-2RNA进行低成本、无扩增的检测将有助于开发一种平台技术，用于对新冠肺炎和其他病原体进行快速和POC诊断。
The coronavirus disease 2019 (COVID-19) pandemic has highlighted the major shortcoming of healthcare systems globally in their inability to diagnose the disease rapidly and accurately. At present, the molecular approaches for diagnosing COVID-19 primarily use reverse transcriptase polymerase chain reaction (RT-PCR) to create and amplify cDNA from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. Although molecular tests are reported to be specific, false negatives are quite common. Furthermore, literally all these tests require a step involving RNA isolation which does not make them point-of-care (POC) in the true sense. Here, we report a lateral flow strip-based RNA extraction and amplification-free nucleic acid test (NAT) for rapid diagnosis of positive COVID-19 cases at POC. The assay uses highly specific 6-carboxyfluorescein (6-FAM) and biotin labeled antisense oligonucleotides (ASOs) as probes those are designed to target N-gene sequence of SARS-CoV-2. Additionally, we utilized cysteamine capped gold-nanoparticles (Cyst-AuNPs) to augment the signal further for enhanced sensitivity. Without any large-stationary equipment and highly trained staffers, the entire sample-to-answer approach in our case would take less than 30 min from a patient swab sample collection to final diagnostic result. Moreover, when evaluated with 60 clinical samples and verified with an FDA-approved TaqPath RT-PCR kit for COVID-19 diagnosis, the assay obtained almost 99.99% accuracy and specificity. We anticipate that the newly established low-cost amplification-free detection of SARS-CoV-2 RNA will aid in the development of a platform technology for rapid and POC diagnosis of COVID-19 and other pathogens.
登录
查看更多内容
影响因子：
9.5
作者：
Srivastava I;Misra SK;Bangru S;Boateng KA;Soares JANT;Schwartz-Duval AS;Kalsotra A;Pan D
通讯作者：
Pan D
影响因子：
17.1
作者：
Lukas, Heather;Xu, Changhao;Gao, Wei
通讯作者：
Gao, Wei
影响因子：
14.8
作者：
Alafeef, Maha;Moitra, Parikshit;Pan, Dipanjan
通讯作者：
Pan, Dipanjan
影响因子：
12.6
作者：
Alafeef, Maha;Moitra, Parikshit;Pan, Dipanjan
通讯作者：
Pan, Dipanjan
影响因子：
3.3
作者：
Gearheart, LA;Ploehn, HJ;Murphy, CJ
通讯作者：
Murphy, CJ