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

用于呼吸道病毒检测的多重 RT-qPCR 检测

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.

作者聚焦：呼吸道病毒多重检测的进展 

开发用于检测SARS-CoV-2、甲型/乙型流感和MERS-CoV的多重实时RT-qPCR检测

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  

多重R3T一步法RT-qPCR试剂盒组分的制备和检测

首先，在不含 DNase/RNase 的水中制备用于 RT-qPCR 的 2X 缓冲液。将缓冲液储存在零下 20 摄氏度直至进一步使用。接下来，将引物和探针混合在 1.5 毫升管中。用洗脱缓冲液补足体积。然后将试管存放在零下 20 摄氏度。在冰上的1.5毫升管中制备含有Taq聚合酶和M-MLV RT的酶混合物。用Taq聚合酶储存缓冲液补足体积。现在将病毒合成RNA重新悬浮在含有100微升Tris-EDTA缓冲液的单独试管中，以使每微升10至6个RNA拷贝的储备液。要混合病毒储备，将 SARS-CoV-2 甲型和乙型流感各 5 微升加入 50 微升 Tris-EDTA 缓冲液中。同样，将 SARS-CoV-2 和 MERS-CoV 混合以获得第二种病毒混合物。将两种混合物稀释十倍，以获得每微升 10 个 RNA 拷贝的最终稀释度。向 96 孔板的每个孔中，移液 2X 缓冲液、引物、酶、不含 DNase/RNase 的水和相应的 RNA 混合物。用粘性板密封板密封板。然后将板离心一分钟，将液体收集在井底。接下来，对PCR仪进行编程，使其接受快速96孔块类型，实验类型设置为标准曲线。将试剂设置为 TaqMan 试剂，然后选择标准运行特性。定义基因靶标和报告染料。然后定义要测试的每个反应的样品名称，并将目标和样品分配给板布局。现在，将循环程序输入仪器。将板以正确的方向转移到实时荧光定量qPCR仪上，然后按运行键启动循环。选择文件位置以保存实验数据。然后检查qPCR程序提供的扩增图。开发的两种试剂盒能够成功地同时扩增所有三个靶基因，每次反应的RNA拷贝低至10个。所有病毒滴定的扩增效率均高于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 ...