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Color-Coding of Microchip RT-PCR Test System for SARS-CoV-2 Detection

Journal of Biosciences and Medicines 2021年第5期9卷 94-119页

作者： Rajwant K. Gill Sikander S. Gill irina gelimson Maxim Slyadnev Gabriel Martinez Michelle Gaines Ryan Nunley Timea Majoros Lumex Instruments Canada Mission BC Canada 1CARE Medical Diagnostics Benicia CA USA

An RT-PCR based microchip test system for the detection of SARS-CoV-2 offers pre-loaded and lyophilized reagents in the microchip. However, the 30- and 48-microwell formats of the microchip being miniaturized and performing 1.2 μl reaction, seek visual attention during sample addition. Therefore, adding colorants as color indicator in the lyophilized matrix in the microchips or adding to sample or master mix can impart not only user-friendliness to the task of liquid handling but also precision, and color-codes for easy identification of multiple kits in the layout of the microchip without compromising PCR data quality. A panel of colorants was screened for their background intensity, spectral inertness towards detection channels of AriaDNATM analyzer, interference with the reporter dyes (FAM, Cy5 and ROX), and visibility of optimal concentration in the microwell. The concentration of the colorant displaying insignificant impact on the quality of the amplification (Ct, fluorescence, and sensitivity) in comparison to no-colorant control was chosen for inclusion in the test kit. Tartrazine, Acid Red, Brilliant Blue and FAST Green colorants lyophilized with the reagents in the SARS-CoV-2 microchips were found to be stable and suitable. Storage of microchips with Fast Green colorant was tested at 40°C, 22°C, 4°C, and -20°C for 70 days and was found to be suitable and compatible with different master mixes available as liquid or lyophilized. Additionally, the microchips pre-loaded with lyophilized reagents in the presence and absence of two colorants Tartrazine and Fast Green were validated with clinical samples of SARS-COV-2. No significant impact of these colorants both intra- and inter-microchips was observed on the Ct and intensity of amplification for the tested samples in comparison to no-colorant control. The data su

关键词： SARS-CoV-2 Colorants Microchip PCR 2-Plex

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Validation of Microchip Based RT-PCR ABC Test (InfA/B &COVID-19) in Clinical Samples

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Journal of Biosciences and Medicines 2022年第8期10卷 172-187页

作者： Gabriel Martinez Ryan Nunley Michelle Gaines Timea Majoros Rajwant K. Gill irina gelimson Natallia Varankovich Maxim Slyadnev Sikander S. Gill 1CARE Medical Diagnostics Benicia CA USA Lumex Instruments Canada Mission Canada

To contain the rapid and global spread of SARS-CoV-2, it is essential to develop an accurate and sensitive test system to address pandemic bottlenecks, simplified sample collection, and no sample prep. While meeting the demand of testing large populations, the miniaturized volume of assay reagents and offering rapid results is the need in such scenarios. Moreover, in view of the reports of co-infections and overlapping symptoms of Influenza caused by Influenza A or Influenza B, and COVID-19 caused by SARS-CoV-2, a test system with three targets can be supportive for accurate clinical diagnosis. In this presentation, we evaluated the performance of a test comprising Microchip RT-PCR Influenza and COVID-19 Detection System for identifying these three viral pathogens in nasal swabs and saliva specimens. A rapid and simplified total nucleic acid extraction method was developed and validated for the reliable, high-throughput simultaneous detection of respiratory viruses causing Influenza (type A and type B viruses) and COVID-19 (SARS-CoV-2 virus) using the microchip-based AriaDNATM platform deriving the name ABC Test. The test system was evaluated using 81 nasal swab samples, 77 clinical saliva samples, 5 blind CAP reference samples, and RNA standards. The limit of detection (LoD) was assessed using SARS-CoV-2, Influenza A, and Influenza B RNA standards. The multiplex ABC Test microchip displayed LoD of 14 copies/μL for SARS-CoV-2 and approximately 26 copies/μL for Influenza A, and 140 copies/μL for Influenza B, respectively. The ABC Test offers rapid multiplex one-step RT-PCR in 32 minutes for 45 cycles as the miniaturized reaction of 1.2 μL offering a highly sensitive, robust, and accurate assay for the detection of Influenza A/B, and SARS-CoV-2.

关键词： SARS-CoV-2 Influenza Multiplex Microchip PCR Nasal Swab and Saliva

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Validation of Microchip RT-PCR COVID-19 Detection System

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Journal of Biosciences and Medicines 2021年第9期9卷 8-24页

作者： Kelsey de Campos-Stairiker Asha Shravanthi Pidathala Rajwant K. Gill irina gelimson Natallia Varankovich Sikander S. Gill Maxim Slyadnev Sonia Kapur Ayumetrix Lake Oswego OR USA Lumex Instruments Canada Mission BC Canada

While meeting the pandemic demand of SARS-CoV-2 testing, clinical laboratories worldwide tend to adopt new test systems offering cost-effective and faster test outcomes. However, the reliability of SARS-CoV-2 test results has paramount importance in the management of such a health crisis. Therefore, this study sought to determine the accuracy of the test results from a novel duplex Microchip RT-PCR test system using patient saliva samples and nasal swabs stabilized in Viral Transport Medium (VTM) with reference threshold Cycle Values (Ct). The VTM used to stabilize these samples during transport was found to be inhibitory to the RT-PCR. Therefore, all the samples were subjected to spin column purification of total RNA to remove the influence of VTM. A total of 70 patient samples, including 24 positive- and 31 negative-saliva in VTM samples and 15 positive nasal swab samples, were tested. Results obtained from both the sample types were compared to their reference values and no false positive or false negatives were observed. From this data, accuracy, specificity, and sensitivity were determined to be 100% applying the corresponding formulae. The limit of detection with 95% confidence probability was determined to be 2.5 copies/μl in the original sample.

关键词： SARS-CoV-2 Microchip RT-PCR Duplex Nasal Saliva LoD

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