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

בדיקת RT-qPCR מולטיפלקס לזיהוי וירוסים נשימתיים

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.

מחבר זרקור: התקדמות בזיהוי מולטיפלקס של וירוסים נשימתיים 

פיתוח מבחני RT-qPCR בזמן אמת לזיהוי SARS-CoV-2, שפעת A/B ו-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  

הכנה ובדיקה של רכיבי ערכת RT-qPCR מולטיפלקס R3T בצעד אחד

כדי להתחיל, הכן את מאגר 2X עבור RT-qPCR במים נטולי DNase/RNase. אחסנו את המאגר בטמפרטורה של מינוס 20 מעלות צלזיוס עד לשימוש נוסף. לאחר מכן, מערבבים את הפריימרים ואת הבדיקות בצינור 1.5 מיליליטר.השלם את עוצמת הקול עם חיץ אלוציה. לאחר מכן אחסנו את הצינור במינוס 20 מעלות צלזיוס. הכינו תערובת אנזימים עם Taq פולימראז ו-M-MLV RT בצינור של 1.5 מיליליטר על קרח.השלם את עוצמת הקול עם מאגר אחסון Taq פולימראז. כעת השהו מחדש את הרנ"א הסינתטי הנגיפי בצינורות נפרדים עם 100 מיקרוליטר של חיץ Tris-EDTA כדי ליצור מלאי של 10 עד עותקי RNA שישי למיקרוליטר. כדי לערבב את מלאי הנגיף, הוסף חמישה מיקרוליטרים כל אחד של שפעת SARS-CoV-2 A ו- B ל- 50 מיקרוליטר של מאגר Tris-EDTA.באופן דומה, ערבבו SARS-CoV-2 ו-MERS-CoV כדי לקבל תערובת נגיפית שנייה. לדלל את שתי התערובות פי עשרה כדי לקבל דילול סופי של 10 עותקי RNA למיקרוליטר. לכל באר של צלחת 96 בארות, פיפטה 2X חיץ, פריימר, אנזים, מים נטולי DNase/RNase, ותערובות RNA המתאימות.אוטמים את הצלחת באטם צלחת דביק. לאחר מכן צנטריפוגו את הצלחת למשך דקה אחת כדי לאסוף את הנוזל בתחתית הבאר. לאחר מכן, תכנת את מכונת ה- PCR לקבל את סוג הבלוק המהיר של 96 בארות עם סוג הניסוי מוגדר לעקומה סטנדרטית.הגדר את הריאגנטים לריאגנטים TaqMan ובחר מאפייני הפעלה סטנדרטיים. הגדירו את מטרות הגן ואת צבע המדווח. לאחר מכן הגדר את שמות הדגימות עבור כל תגובה שתיבדק והקצה מטרות ודגימות לפריסת הלוחות.כעת, הזן את תוכנית המחזור לתוך המכשיר. העבר את הצלחת למכונת qPCR בזמן אמת בכיוון הנכון ולחץ על הפעל כדי להתחיל את המחזור. בחר את מיקום הקובץ כדי לשמור את הנתונים הניסיוניים.לאחר מכן בחן את חלקות ההגברה המסופקות על ידי תוכנית qPCR. שתי הערכות שפותחו הצליחו להגביר בהצלחה את כל שלושת גני המטרה בו זמנית עם עד 10 עותקי רנ"א לכל תגובה. יעילות ההגברה עבור כל הטיטרציות הנגיפיות הייתה מעל 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 ...