Name: Preparation and Testing of Multiplex R3T One-Step RT-qPCR Kit Components
Uploaded: 2023-11-10T14:50:00
Description: Watch this Scientific Journal Video about Preparation and Testing of Multiplex R3T One-Step RT-qPCR Kit Components at JoVE.com

preparation and testing of multiplex r3t one-step rt-qpcr kit components

Ensayo RT-qPCR multiplex para la detección de virus respiratorios

The pandemic of the ongoing coronavirus disease 2019 (COVID-19) is caused by the novel coronavirus known as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)1. Due to SAR-CoV-2&#39;s strong contagiousness and capacity for rapid transmission, the COVID-19 pandemic emerged in Wuhan City, China, and spread quickly throughout the world. This eventually led to the start of respiratory distress signs and even death2,3,4. COVID-19 has been declared a pandemic in more than 213 countries, expecting a steep increase in the number of confirmed cases, as evidenced by the papers published by different research studies3,5. COVID-19 is transmitted primarily by small respiratory droplets that infected individuals release into the environment and then get exposed to vulnerable individuals through inhalation or close contact with contaminated surfaces. When these droplets come into contact with the mucosa of the eyes, mouth, or nose, a person may become infected6. Statistics released by the World Health Organization (WHO) show that there have been more than 76 million confirmed cases of the pandemic worldwide, with a staggering 7 million deaths7. Thus, the United Nations classified the pandemic caused by COVID-19 disease as a disaster because of its direct impact on the lives of billions of people around the globe and had far-reaching economic, environmental, and social effects.
Public health initiatives including thorough testing, early detection, contact tracing, and case isolation have all been shown to be crucial in keeping this pandemic under control8,9,10,11. The winter months will increase the circulation of other respiratory viruses like Influenza A and B with COVID-19-like symptoms making it difficult to identify, track down, and isolate COVID-19 instances early on. Every year, Influenza A and B outbreak starts in the late fall or early January with a predictable seasonality12. Numerous epidemiological traits are shared by SARS-CoV-2 and Influenza viruses. Besides, sharing similarities in the susceptible populations which include children, the elderly, immunocompromised, and individuals with chronic comorbidities such as asthma, chronic obstructive pulmonary disease, cardiac and renal failure, or diabetes12,13. These viruses not only share vulnerable populations but also transmission routes of contact and respiratory droplets14. It is anticipated that patients may likely contract more than one of these respiratory viruses as flu season approaches14. For that, the screening of SARS-CoV-2 and the Influenza viruses needs to be done on symptomatic patients before they are isolated. Running separate tests for the three viruses (SARS-CoV-2, Influenza A, and Influenza B) is not possible due to the global lack of resources for nucleic acid extraction and diagnostics. In order to screen them all in one reaction, a method or test needs to be developed.
Middle East respiratory syndrome (MERS)-CoV is a human coronavirus (CoV) family member. The first MERS-CoV virus isolates came from a hospitalized patient in Saudi Arabia who had died in September 2012 due to acute respiratory issues15. There is evidence that suggests that a prominent reservoir host for MERS-CoV is dromedary camels. It has been proven that viruses from infected dromedary camels are zoonotic and thus can infect humans16,17. Humans infected with this virus can spread it to others through close contact18. As of January 26th,&#160;2018, there had been 2143 laboratory-confirmed cases of MERS-CoV infection including 750 deaths globally19. The most typical MERS-CoV symptoms are coughing, fever, and shortness of breath. MERS-CoV infections have also been reported to exhibit pneumonia, diarrhea and gastrointestinal sickness symptoms20. Currently, no commercial vaccine or specific treatment for MERS-CoV is available. Therefore, prompt and precise diagnosis is essential for preventing the widespread MERS-CoV outbreaks and differentiating MERS-CoV from SARS-CoV-2 disease.
To date, many approaches have been proposed to detect these viruses such as multiplex RT-PCR21,22,23,24,25, CRISPR/Cas1226,27, CRISPR/Cas928, and CRISPR/Cas329, lateral flow immunoassay30, paper-based biomolecular sensors31, SHERLOCK testing in one pot32, DNA aptamer33, loop-mediated isothermal amplification (LAMP)19,34, etc. Each of the aforementioned methods has unique benefits and drawbacks in terms of sensitivity and specificity. Among these methods, the nucleic acid amplification-based test: multiplex qRT-PCR, is the most common and is considered to be the gold standard for the diagnosis of SARS-CoV-2, Influenza A, Influenza B, and MERS-CoV.
In this study, we designed and assessed various primer combinations and probes for the effective, accurate, and simultaneous detection of SARS-CoV-2, Influenza A, Influenza B, and SARS-CoV-2, MERS-CoV utilizing standard twist synthetic viral RNAs. The multiplexed assays developed for either MERS-CoV or SARS-CoV-2 target genes are recommended by the World Health Organization (WHO). These genes generally encode proteins and complexes that contribute to the formation of a replication/transcription complex (RTC)35 such as the region within the open reading frame 1a (ORF1a) that is used for MERS-CoV assay. In addition, structural proteins are encoded by the genes utilized in diagnostic assays such as the upstream region of envelope gene (upE) and nucleocapsid gene (N) which are used for MERS-CoV and SARS-Cov-2 assays, respectively35,36. We used in-house R3T one-step RT-qPCR kit to establish the RT-qPCR for the detection of viruses37. Virus detection, sensitivity, specificity, and dynamic range of our R3T one-step RT-qPCR kit and primer sets were tested and evaluated using 10-fold serial dilutions of the standard twist synthetic RNAs. The lowest practical detection limit was approximately 10 transcripts copies per reaction. As a result, the in-house R3T one-step RT-qPCR kit and primer/probe sets can be successfully used and implemented for routine simultaneous diagnosis of SARS-CoV-2, Influenza A, Influenza B, and SARS-CoV-2, MERS-CoV.

Autor destacado: Avances en la detección múltiple de virus respiratorios 

Desarrollo de ensayos multiplex de RT-qPCR en tiempo real para la detección de SARS-CoV-2, influenza A/B y MERS-CoV

Watch this Scientific Journal Video about Preparation and Testing of Multiplex R3T One-Step RT-qPCR Kit Components at JoVE.com

Preparation and Testing of Multiplex R3T One-Step RT-qPCR Kit Components  

Preparación y prueba de los componentes del kit de RT-qPCR de un solo paso Multiplex R3T

Para comenzar, prepare el tampón 2X para RT-qPCR en agua libre de DNasa/RNasa. Guarde el tampón a menos 20 grados centígrados hasta su nuevo uso. A continuación, mezcle los cebadores y las sondas en un tubo de 1,5 mililitros.Compensar el volumen con tampón de elución. A continuación, guarde el tubo a menos 20 grados centígrados. Prepare una mezcla de enzimas con Taq polimerasa y M-MLV RT en un tubo de 1,5 mililitros sobre hielo.Recupere el volumen con el tampón de almacenamiento de polimerasa Taq. Ahora vuelva a suspender los ARN sintéticos virales en tubos separados con 100 microlitros de tampón Tris-EDTA para hacer existencias de 10 a la sexta copia de ARN por microlitro. Para mezclar las existencias de virus, agregue cinco microlitros de influenza A y B del SARS-CoV-2 a 50 microlitros de tampón Tris-EDTA.Del mismo modo, mezcle el SARS-CoV-2 y el MERS-CoV para obtener una segunda mezcla viral. Diluir ambas mezclas diez veces para obtener una dilución final de 10 copias de ARN por microlitro. A cada pocillo de una placa de 96 pocillos, pipetee tampón 2X, cebador, enzima, agua libre de DNasa/RNasa y las mezclas de ARN correspondientes.Selle la placa con un sello adhesivo para placas. Luego centrifuga la placa durante un minuto para recoger el líquido en el fondo del pozo. A continuación, programe la máquina de PCR para que acepte el tipo de bloque rápido de 96 pocillos con el tipo experimental establecido en la curva estándar.Establezca los reactivos en Reactivos TaqMan y seleccione las propiedades de ejecución estándar. Definir las dianas génicas y el colorante reportero. A continuación, defina los nombres de las muestras para cada reacción que se va a probar y asigne objetivos y muestras al diseño de la placa.Ahora, ingrese el programa de ciclo en el instrumento. Transfiera la placa a la máquina de qPCR en tiempo real en la orientación correcta y presione ejecutar para iniciar el ciclo. Elija la ubicación del archivo para guardar los datos experimentales.A continuación, examine los gráficos de amplificación proporcionados por el programa qPCR. Los dos kits desarrollados fueron capaces de amplificar con éxito los tres genes diana simultáneamente con tan solo 10 copias de ARN por reacción. Las eficiencias de amplificación para todas las titulaciones virales fueron superiores al 99%

Research

JoVE Journal

Biology

Preparation and Testing of Multiplex R3T One-Step RT-qPCR Kit Components

Development of Multiplex Real-Time RT-qPCR Assays for the Detection of SARS-CoV-2, Influenza A/B, and MERS-CoV

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes Coronavirus disease 2019 (COVID-19) is a serious threat to the general ...