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

Multiplex RT-qPCR-test voor detectie van respiratoire virussen

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.

Auteur in de schijnwerpers: Vooruitgang in multiplexdetectie van respiratoire virussen 

Ontwikkeling van multiplex real-time RT-qPCR-assays voor de detectie van SARS-CoV-2, influenza A/B en 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  

Voorbereiding en testen van Multiplex R3T One-Step RT-qPCR Kit-componenten

Bereid om te beginnen de 2X-buffer voor RT-qPCR voor in DNase/RNasevrij water. Bewaar de buffer bij min 20 graden Celsius tot verder gebruik. Meng vervolgens de primers en de sondes in een tube van 1,5 milliliter.Vul het volume aan met elutiebuffer. Bewaar de tube vervolgens bij min 20 graden Celsius. Bereid een enzymmengsel met Taq-polymerase en M-MLV RT in een buis van 1,5 milliliter op ijs.Vul het volume aan met Taq-polymerase-opslagbuffer. suspendeer nu de virale synthetische RNA's opnieuw in aparte buisjes met 100 microliter Tris-EDTA-buffer om voorraden van 10 tot de zesde RNA-kopieën per microliter te maken. Om de virusvoorraden te mengen, voegt u vijf microliter SARS-CoV-2 influenza A en B toe aan 50 microliter Tris-EDTA-buffer.Meng op dezelfde manier SARS-CoV-2 en MERS-CoV om een tweede virale mix te verkrijgen. Verdun beide mengsels tienvoudig om een uiteindelijke verdunning van 10 RNA-kopieën per microliter te verkrijgen. Naar elk putje van een plaat met 96 putjes, pipet 2X buffer, primer, enzym, DNase/RNasevrij water en de bijbehorende RNA-mengsels.Verzegel de plaat met een zelfklevende plaatafdichting. Centrifugeer vervolgens de plaat gedurende een minuut om de vloeistof op de bodem van de put op te vangen. Programmeer vervolgens de PCR-machine om het snelle bloktype met 96 putjes te accepteren, waarbij het experimentele type is ingesteld op de standaardcurve.Stel de reagentia in op TaqMan-reagentia en selecteer standaardrun-eigenschappen. Definieer de gendoelen en de kleurstof van de reporter. Definieer vervolgens de monsternamen voor elke te testen reactie en wijs doelen en monsters toe aan de plaatlay-out.Voer nu het cyclusprogramma in het instrument in. Breng de plaat over naar de real-time qPCR-machine in de juiste richting en druk op run om de cyclus te starten. Kies de bestandslocatie om de experimentele gegevens op te slaan.Bestudeer vervolgens de amplificatieplots die door het qPCR-programma worden geleverd. De twee ontwikkelde kits waren in staat om alle drie de doelgenen tegelijkertijd te amplificeren met slechts 10 RNA-kopieën per reactie. De amplificatie-efficiëntie voor alle virale titraties lag boven de 99%

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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 ...