Sritong Navaporn, Ngo Winston Wei, Ejendal Karin F K, Linnes Jacqueline C
Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN, USA.
Department of Public Health, Purdue University, West Lafayette, IN, USA.
medRxiv. 2023 Oct 3:2023.10.03.23296477. doi: 10.1101/2023.10.03.23296477.
The COVID-19 pandemic has led to a rise in point-of-care (POC) and home-based tests, but concerns over usability, accuracy, and effectiveness have arisen. The incorporation of internal amplification controls (IACs), essential control for translational POC diagnostics, could mitigate false-negative and false-positive results due to sample matrix interference or inhibition. Although emerging POC nucleic acid amplification tests (NAATs) for detecting SARS-CoV-2 show impressive analytical sensitivity in the lab, the assessment of clinical accuracy with IACs is often overlooked. In some cases, the IACs were run spatially, complicating assay workflow. Therefore, the multiplex assay for pathogen and IAC is needed.
We developed a one-pot duplex reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay for saliva samples, a non-invasive and simple collected specimen for POC NAATs. The ORF1ab gene of SARS-CoV-2 was used as a target and a human 18S ribosomal RNA in human saliva was employed as an IAC to ensure clinical reliability of the RT-LAMP assay. The optimized assay could detect SARS-CoV-2 viral particles down to 100 copies/μL of saliva within 30 minutes without RNA extraction. The duplex RT-LAMP for SARS-CoV-2 and IAC is successfully amplified in the same reaction without cross-reactivity. The valid results were easily visualized in triple-line lateral flow immunoassay, in which two lines (flow control and IAC lines) represent valid negative results and three lines (flow control, IAC, and test line) represent valid positive results. This duplex assay demonstrated a clinical sensitivity of 95%, specificity of 100%, and accuracy of 96% in 30 clinical saliva samples.
IACs play a crucial role in ensuring user confidence with respect to the accuracy and reliability of at-home and POC molecular diagnostics. We demonstrated the multiplex capability of SARS-COV-2 and human18S ribosomal RNA RT-LAMP without complicating assay design. This generic platform can be extended in a similar manner to include human18S ribosomal RNA IACs into different clinical sample matrices.
新型冠状病毒肺炎（COVID-19）大流行导致即时检测（POC）和居家检测增多，但人们对其可用性、准确性和有效性产生了担忧。内部扩增对照（IAC）作为转化型POC诊断的重要对照，可减少因样本基质干扰或抑制导致的假阴性和假阳性结果。尽管用于检测严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的新型POC核酸扩增检测（NAAT）在实验室中显示出令人印象深刻的分析灵敏度，但使用IAC进行临床准确性评估常常被忽视。在某些情况下，IAC是在空间上单独运行的，这使检测工作流程变得复杂。因此，需要针对病原体和IAC的多重检测方法。
我们开发了一种用于唾液样本的一锅式双链逆转录环介导等温扩增（RT-LAMP）检测方法，唾液是一种用于POC NAAT的非侵入性且采集简单的样本。以SARS-CoV-2的开放阅读框1ab（ORF1ab）基因为靶标，以人唾液中的人类18S核糖体RNA作为IAC，以确保RT-LAMP检测的临床可靠性。优化后的检测方法无需RNA提取，在30分钟内可检测低至100拷贝/μL唾液中的SARS-CoV-2病毒颗粒。用于SARS-CoV-2和IAC的双链RT-LAMP在同一反应中成功扩增，无交叉反应。有效的结果在三线侧向流免疫分析中易于观察，其中两条线（流动对照线和IAC线）代表有效的阴性结果，三条线（流动对照线、IAC线和检测线）代表有效的阳性结果。在30份临床唾液样本中，这种双链检测方法的临床灵敏度为95%，特异性为100%，准确性为96%。
IAC在确保用户对居家和POC分子诊断的准确性和可靠性的信心方面起着至关重要的作用。我们展示了SARS-CoV-2和人类18S核糖体RNA RT-LAMP的多重检测能力，且不使检测设计复杂化。这个通用平台可以以类似的方式扩展，将人类18S核糖体RNA IAC纳入不同的临床样本基质中。
