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. 2024 Jan 25:1287:342072.
doi: 10.1016/j.aca.2023.342072. Epub 2023 Nov 29.

Development of an integrated sample amplification control for salivary point-of-care pathogen testing

Navaporn Sritong  1 , Winston Wei Ngo  1 , Karin F K Ejendal  1 , Jacqueline C Linnes  2
Affiliations

Development of an integrated sample amplification control for salivary point-of-care pathogen testing

Navaporn Sritong et al. Anal Chim Acta. .

Abstract

Background: 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.

Results: 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 min 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.

Significance: 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.

Keywords: Diagnostic; Internal amplification control; Lateral flow immunoassay; Reverse transcription loop-mediated isothermal amplification; SARS-CoV-2; Saliva.

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Conflict of interest statement

Declaration of competing interest Jacqueline C. Linnes is co-founder of EverTrue LLC, a diagnostics company developing paper-based POC NAATs, and co-founder of OmniVis Inc. NAAT company developing POC diagnostics. Navaporn Sritong, Winston Wei Ngo, and Karin F. K. Ejendal have declared that they have no competing interests.

Figures

Figure 1.
Figure 1.
Analytical sensitivity of SARS-CoV-2 RT-LAMP assay in saliva. Ct value of SARS-CoV-2 RT-LAMP in various concentrations (A). RT-LAMP based detection of inactivated viral particles as visualized on gel electrophoresis (B) LFIA (C), and corresponding test band intensity analysis (D). n = 3; **** indicates p value ≤ 0.0001. The ladder-like bands on gel electrophoresis indicating successful amplification.
Figure 2.
Figure 2.
Specificity of IAC primer. Amplification plot (A) and gel electrophoresis (B) of human 18S rRNA RT-LAMP using different templates. The ladder-like bands indicating successful amplification are only present when saliva and human RNA were used as templates. N = 2.
Figure 3.
Figure 3.
One-pot duplex RT-LAMP of SARS-CoV-2 and human 18S rRNA. (A) Amplification plot of duplex RT-LAMP using different templates. (B) Gel electrophoresis and (C) corresponding LFIAs of optimized assay (n=3). Integration of 18S rRNA human sample control into duplex RT-LAMP assay demonstrating differentiation between saliva (with 18S rRNA) and water matrix (without 18S rRNA) and SARS-CoV-2 spiked into each matrix. W- and W+ represent water with and without SARS-CoV-2, respectively. S- and S+ represent saliva sample with and without SARS-CoV-2, respectively. (D) Restriction enzyme digestion of duplex RT-LAMP of SARS-CoV-2 and human 18S rRNA visualized on the gel. Red boxes indicate BstYI digested amplicon with the characteristic band at 110 bp and blue boxes indicate DdeI digested amplicon the characteristic band at 150 bp. Ø indicate non-digested amplicons.
Figure 4.
Figure 4.
Analytical sensitivity of duplex RT-LAMP assay of SARS-CoV-2 and IAC in saliva visualized on (A) LFIA and (B) corresponding test band intensity analysis. n = 3; */# indicates p value ≤ 0.05; **/## indicates p ≤ 0.01; ***/### indicates p value ≤ 0.001; compared to equivalent bands at 0 viral copies/μL.
Schematic 1:
Schematic 1:. Detection of duplex RT-LAMP amplicons on LFIA.
The stem-loop structure of SARS-CoV-2 amplicons and human 18S rRNA amplicons displayed different regions in DNA templates. The loop primers of SARS-CoV-2 RT-LAMP were labeled with FITC and biotin, while the loop primers of human 18S rRNA RT-LAMP were labeled with DIG and biotin (Top). After the amplification, amplicons were added to LFIA to visualize. SARS-CoV-2 amplicon with FITC and biotin labelling bound to anti-FITC antibody on the first test line, while human 18S rRNA amplicons labeled with DIG and biotin bound to anti-DIG antibody on the second test line. The excess streptavidin coated AuNPs were wicked to flow control line where biotinylated anti-mouse IgGs were deposited.
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