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-Assay zum Nachweis von Atemwegsviren

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 im Rampenlicht: Fortschritte bei der Multiplex-Detektion von Atemwegsviren 

Entwicklung von Multiplex-Real-Time-RT-qPCR-Assays zum Nachweis von SARS-CoV-2, Influenza A/B und 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  

Vorbereitung und Prüfung von Komponenten des Multiplex R3T One-Step RT-qPCR Kits

Bereiten Sie zunächst den 2X-Puffer für die RT-qPCR in DNase/RNase-freiem Wasser vor. Lagern Sie den Puffer bis zur weiteren Verwendung bei minus 20 Grad Celsius. Als nächstes mischen Sie die Primer und die Sonden in einem 1,5-Milliliter-Röhrchen.Füllen Sie das Volumen mit Elutionspuffer auf. Lagern Sie das Röhrchen dann bei minus 20 Grad Celsius. Bereiten Sie eine Enzymmischung mit Taq-Polymerase und M-MLV RT in einem 1,5-Milliliter-Röhrchen auf Eis vor.Füllen Sie das Volumen mit Taq-Polymerase-Speicherpuffer auf. Suspendieren Sie nun die viralen synthetischen RNAs in separaten Röhrchen mit 100 Mikrolitern Tris-EDTA-Puffer, um Vorräte von 10 bis der sechsten RNA-Kopie pro Mikroliter herzustellen. Um die Virusvorräte zu mischen, fügen Sie je fünf Mikroliter SARS-CoV-2 Influenza A und B zu 50 Mikrolitern Tris-EDTA-Puffer hinzu.In ähnlicher Weise mischen Sie SARS-CoV-2 und MERS-CoV, um eine zweite Virusmischung zu erhalten. Beide Mischungen werden verzehnfacht, um eine endgültige Verdünnung von 10 RNA-Kopien pro Mikroliter zu erhalten. In jede Vertiefung einer 96-Well-Platte pipettieren Sie 2X Puffer, Primer, Enzym, DNase/RNase-freies Wasser und die entsprechenden RNA-Mischungen.Versiegeln Sie die Platte mit einer selbstklebenden Plattendichtung. Zentrifugieren Sie dann die Platte eine Minute lang, um die Flüssigkeit am Brunnenboden aufzufangen. Als Nächstes programmieren Sie das PCR-Gerät so, dass es den schnellen 96-Well-Blocktyp akzeptiert, wobei der experimentelle Typ auf die Standardkurve eingestellt ist.Stellen Sie die Reagenzien auf TaqMan-Reagenzien ein und wählen Sie die Eigenschaften des Standardlaufs aus. Definieren Sie die Genziele und den Reporterfarbstoff. Definieren Sie dann die Probennamen für jede zu testende Reaktion und weisen Sie dem Plattenlayout Targets und Proben zu.Geben Sie nun das Zyklusprogramm in das Gerät ein. Übertragen Sie die Platte in der richtigen Ausrichtung auf das real-time qPCR-Gerät und drücken Sie auf Lauf, um den Zyklus zu starten. Wählen Sie den Speicherort der Datei aus, um die experimentellen Daten zu speichern.Untersuchen Sie dann die vom qPCR-Programm bereitgestellten Amplifikationsdiagramme. Die beiden entwickelten Kits waren in der Lage, alle drei Zielgene gleichzeitig mit nur 10 RNA-Kopien pro Reaktion erfolgreich zu amplifizieren. Die Amplifikationseffizienzen für alle viralen Titrationen lagen bei über 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 ...