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

호흡기 바이러스 검출을 위한 멀티플렉스 RT-qPCR 분석

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.

저자 스포트라이트: 호흡기 바이러스의 다중 검출 발전 

SARS-CoV-2, 인플루엔자 A/B 및 MERS-CoV 검출을 위한 다중 Real-Time RT-qPCR 분석 개발

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  

멀티플렉스 R3T 원스텝 RT-qPCR 키트 구성 요소의 준비 및 테스트

시작하려면 DNase/RNase 유리수에서 RT-qPCR용 2X 완충액을 준비합니다. 더 사용할 때까지 버퍼를 섭씨 영하 20도에서 보관하십시오. 다음으로, 프라이머와 프로브를 1.5 밀리리터 튜브에 혼합합니다.용리 버퍼로 부피를 구성합니다. 그런 다음 튜브를 섭씨 영하 20도에서 보관하십시오. 얼음 위의 1.5밀리리터 튜브에 Taq 중합효소 및 M-MLV RT가 포함된 효소 혼합물을 준비합니다.Taq 중합효소 저장 완충액으로 부피를 구성합니다. 이제 100 마이크로리터의 Tris-EDTA 완충액이 있는 별도의 튜브에 바이러스 합성 RNA를 다시 현탁시켜 마이크로리터당 10개에서 6번째 RNA 사본의 스톡을 만듭니다. 바이러스 스톡을 혼합하려면 SARS-CoV-2 인플루엔자 A와 B를 각각 5마이크로리터의 Tris-EDTA 완충액 50마이크로리터에 추가합니다.마찬가지로 SARS-CoV-2와 MERS-CoV를 혼합하여 두 번째 바이러스 혼합물을 얻습니다. 두 혼합물을 10배 희석하여 마이크로리터당 10개의 RNA 복제의 최종 희석을 얻습니다. 96웰 플레이트의 각 웰, 피펫 2X 완충액, 프라이머, 효소, DNase/RNase 자유수 및 해당 RNA 혼합물.접착판 씰로 플레이트를 밀봉합니다. 그런 다음 플레이트를 1분 동안 원심분리하여 웰 바닥에 액체를 수집합니다. 다음으로, 실험 유형이 표준 곡선으로 설정된 고속 96웰 블록 유형을 수용하도록 PCR 기계를 프로그래밍합니다.시약을 TaqMan 시약으로 설정하고 표준 실행 속성을 선택합니다. 유전자 표적과 리포터 염료를 정의합니다. 그런 다음 테스트할 각 반응에 대한 샘플 이름을 정의하고 플레이트 레이아웃에 표적과 샘플을 할당합니다.이제 사이클 프로그램을 기기에 입력합니다. 플레이트를 올바른 방향으로 Real-Time qPCR 기계로 옮기고 실행을 눌러 사이클을 시작합니다. 실험 데이터를 저장할 파일 위치를 선택합니다.그런 다음 qPCR 프로그램에서 제공하는 증폭 플롯을 검사합니다. 개발된 두 키트는 반응당 최소 10개의 RNA 복제로 세 가지 표적 유전자를 동시에 성공적으로 증폭할 수 있었습니다. 모든 바이러스 적정에 대한 증폭 효율은 99% 이상이었습니다

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 ...