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BACKGROUND INFO AVAnT1A Study - short for "Antiviral Action against Type 1 diabetes Autoimmunity" - examines whether vaccination against COVID-19 at the age of six months can prevent the development of islet autoantibodies in babies at increased genetic risk of developing type 1 diabetes, thus reducing their risk of developing the condition. In addition, the researchers want to monitor in saliva and stool samples which viruses the children had contact with. This allows the researchers to clarify further connections between type 1 diabetes and viral infections in early childhood. Since many infections in young children occur almost without symptoms, participating families are asked to collect saliva weekly and stool samples monthly from their children. From these samples, researchers can identify which viruses the children had contact with. The viruses will be identified using multiplex RT-PCR methods: Saliva samples will be screened for SARS CoV-2, Enterovirus, Human Coronaviruses-NL63, -229E, -OC43, and -HUK1, influenza A, Rhinovirus, Adenovirus and Bocavirus, and similarly stool samples will be screened for enterovirus, rotavirus, adenovirus, rhinovirus, norovirus and astrovirus. NEED Due to high number of RT-PCR analysis multiple RNA and DNA viruses will be analyzed in same RT-PCR reaction using RT-PCR multiplex kit. The nucleic acid will be extracted using Sbeadex Pathogen Nucleic Acid Purification Kit - no Proteinase K (tendering process was done in Germany by the PI of AVAnT1A project). It is highly important that the RT-PCR kit is compatible with the nucleic acid extraction kit. In addition to the compatibility with the NA extraction kit it is extremely important that the multiplex RT-PCR kit is highly sensitive to detect also low copy numbers of viral nucleic acid in the RT-PCR reaction. Additional criteria are as follows. Performance requirements for the reagent: • Must amplify both viral RNA and DNA template • 4-plex capability to ensure effective multiplexing capacity • 4 x concentration to enable more sample to be run per reaction - Must be possible to add larger sample input amounts to standard reaction volumes for more accurate quantification of low-titer targets • The tolerance against RT-PCR inhibitors in the stool and saliva samples • Mitigation of inhibitors: - use of purification methods of extracted nucleic acids is not allowed due to higher costs. - no additives like BSA (bovine serum albumin) or PCR enhancers to neutralize their effects is allowed due to possibility of reduction of the detection sensitivity • Must have ultra pure DNA polymerase to reduce lot-to-lot variability and background amplification and increase accuracy • Must include hot-start polymerase to reduces cross-reactivity and enhances signal clarity for each target • The product must be available with or without ROX passive reference Requirements for the packing of the reagent: • Ready-made mastermix in at least 10ml bottle, to reduce spared reagents • Buffer solution that does not freeze when stored at -20oC, to ensure faster laboratory performance Compatibility with qPCR realtime machines: • Reagent must be evaluated on the QuantStudio series instruments that are available at TAU laboratory facilities • Reagent must be compatible with fast (45min) and standard cycling mode on Applied Biosystems Real-Time PCR instruments QuantStudio 5 and QuantStudio 12K Flex systems - Fast cycling protocols must have at least the same sensitivity as is expected from standard cycling qPCR MARKET ANALYSIS AND CONCLUSION FOR DIRECT AWARD Market research has been conducted by searching information about commercially available multiplex RT-PCR kits capable for analysis of both RNA and DNA viruses. Key reasons for the selection of the TaqMan™ Fast Virus 1-Step Multiplex Master Mix for qPCR (No ROX) reagent and ThermoFisher Scientific as a supplier: • Meets All Technical Specifications: High sensitivity, 4X concentration of mastermix, dual RNA/DNA amplification, multiplex (4-plex) capability, and the kit includes ultra-pure hot-start polymerase. • High Inhibitor Tolerance: Our test showed that the RT-qPCR kit amplified efficiently the spiked viruses in stool and saliva samples without the need for additional purification methods for the extracted nucleic acids or inhibitor elimination additives. • Optimized Packaging: Supplier has a kit of ready-to-use 10 mL bottles and the mastermix reagent does not freeze at -20°C, ensuring operational efficiency. • Instrument Compatibility: The kit has been validated and is fully compatible with Applied Biosystems QuantStudio 5 and 12K Flex systems, supporting both fast and standard cycling modes without loss of sensitivity. • Reliable Supplier Support: Finnish-language customer service and <24-hour response time for all inquiries is guaranteed. • Best Overall Value: The selected supplier provides the most technically robust and operationally suitable solution. Summary: Considering the overall goals of the study, the combination of Speadex extraction and TaqMan™ Fast Virus 1-Step Multiplex Master Mix for qPCR (No ROX) is the best choice for the detection of viruses in the AVAnT1A study. This combination showed excellent sensitivity to detect viruses and especially the tolerance for the RT-PCR inhibition in stool samples. TaqMan™ Fast Virus 1-Step Multiplex Master Mix for qPCR (No ROX) ensure high-quality RT-PCR workflows, reliable test performance, compatibility to laboratory facilities at TAU and efficient laboratory operations.