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

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.

Development of an Integrated Sample Amplification Control for Salivary Point-of-Care Pathogen Testing.

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 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. 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.

Promise and perils of paper-based point-of-care nucleic acid detection for endemic and pandemic pathogens.

From HIV and influenza to emerging pathogens like COVID-19, each new infectious disease outbreak has highlighted the need for massively-scalable testing that can be performed outside centralized laboratory settings at the point-of-care (POC) in order to prevent, track, and monitor endemic and pandemic threats. Nucleic acid amplification tests (NAATs) are highly sensitive and can be developed and scaled within weeks while protein-based rapid tests require months for production. Combining NAATs with paper-based detection platforms are promising due to the manufacturability, scalability, and simplicity of each of these components. Typically, paper-based NAATs consist of three sequential steps: sample collection and preparation, amplification of DNA or RNA from pathogens of interest, and detection. However, these exist within a larger ecosystem of sample collection and interpretation workflow, usability, and manufacturability which can be vastly perturbed during a pandemic emergence. This review aims to explore the challenges of paper-based NAATs covering sample-to-answer procedures along with three main types of clinical samples; blood, urine, and saliva, as well as broader operational, scale up, and regulatory aspects of device development and implementation. To fill the technological gaps in paper-based NAATs, a sample-in-result-out system that incorporates the integrated sample collection, sample preparation, and integrated internal amplification control while also balancing needs of users and manufacturability upfront in the early design process is required.

Light emitting diode irradiation induced shape conversion of DNA-capped silver nanoparticles and their antioxidant and antibacterial activities.

Due to size- and shape-dependent properties, a shape-controlled synthesis of silver nanoparticles (AgNPs) is one of the research challenging topics for their production and potential applications. This work reported the simple eco-friendly syntheses of different shaped AgNPs controlled by the plasmid DNA content and light emitting diodes (LEDs) irradiation. The synthesized AgNPs appeared as yellow, orange and green colloidal AgNPs, which transmission electron microscope (TEM) images revealed their different shapes; spherical AgNPs, a mixture of spherical and hexagonal AgNPs, and a mixture of spherical, hexagonal and corner-truncated triangle AgNPs, respectively. The average sizes of spherical, hexagonal and corner-truncated triangle AgNPs in the green colloidal solution were 12.32 ± 2.22, 23.03 ± 6.62 and 15.84 ± 4.31 nm, respectively. The analyses of X-ray diffraction, selected area electron diffraction and high-resolution TEM indicated the crystalline nature of the synthesized particles as the face-centred cubic silver. All synthesized AgNPs exhibited antioxidant activities similarly, whereas the yellow colloidal AgNPs exhibited the strongest antibacterial activity against both Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus as compared with the green and orange colloidal AgNPs.