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The Role of New Technologies for Diagnosis of Coronavirus Disease 2019 in Near Future
Journal of Pediatric Infectious Diseases ( IF 0.2 ) Pub Date : 2020-06-17 , DOI: 10.1055/s-0040-1713594
Bahadır Feyzioğlu 1 , Mehmet Özdemir 1
Affiliation  

Since there is as yet no specific drug or vaccine for coronavirus disease 2019 (COVID-19), early diagnosis is very important in controlling the outbreak by preventing transmission of the virus. Conventional cell culture for viruses is not easy or widely available, meaning that routine virologic diagnosis for many years has been mainly made by serological tests. However, molecular tests—including classic polymerase chain reaction (PCR), multiplex PCR, and syndromic PCR—have advantages such as rapid results turnaround and high sensitivity, and they are widely used in virologic diagnosis in recent years. Although reliable instruments are available in well-equipped laboratories for the diagnosis of symptomatic patients, other methods are required to screen asymptomatic people in the incubation phase or to determine viral shedding in patients in recovery. In addition, to control the pandemic globally, solutions for less-equipped laboratories and for portable use are needed. Development of fast, simple, low cost, and portable tests that can be used in airports at border gates and in rural areas is one of the important targets in the control of COVID-19 pandemic.

The Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) method, a new technology that operates on the basis of gene regulation and significantly simplifies nucleic acid-based viral detection, promises new horizons in COVID-19 laboratory diagnosis.[1] The method has a highly analytically sensitive mechanism that includes viral nucleic acid specific guide RNA (called sgRNA) and CRISPR effector enzymes such as Cas9, Cas13a, or Cas13B. CRISPR modifications such as Specific High Sensitivity Enzymatic Reporter UnLOCKING (SHERLOCK) and DNA endonuclease-targeted CRISPR trans reporter (DETECTR) were shown to be able to detect Zika virus, dengue virus and human papillomavirus with high sensitivity in recent publications.[2] [3] This method can be transferred on paper and the cost of reagents is very low. Adaptations of the method with lateral flow reading were successful in the diagnosis of COVID-19 with 10 copies/µL sensitivity and result time under 1 hour. The system also allows the creation of multiple infection panels (all in one). As a striking example, in a recently published article, it was stated that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and HIV were simultaneously detected quickly and efficiently.[4] Thermal cycles are not required for these methods. Thus, testing time and equipment costs are significantly reduced.

Isothermal amplification methods that allow the amplification of nucleic acids at constant temperatures are increasingly used as an alternative to PCR. A commercial form of loop-mediated isothermal amplification (LAMP), targeting the SARS-CoV-2 RdRp Gen region, has received an emergency use permit from the Food and Drug Administration. It has been reported SARS-CoV-2 could be detected with 0.125 copies/µL sensitivity in just 10 minutes by this method. SARS-CoV-2 was detected with 100% analytical sensitivity and specificity only 1 hour after sampling in a study using oropharyngeal swabs from patients diagnosed with COVID-19, and healthy controls.[5] With all these advantages, we can predict that isothermal amplification options—especially a reverse transcriptase-LAMP—will be used more in the coming periods as point of care applications.

Luciferase-labeled antigens and high quantitative immunoprecipitation systems have been used to provide virus identification, monitor antiviral treatments, and categorize virus-related infections. This new method allows the evaluation of immunoreactivity against full or partial proteomes of viruses. Extracts containing recombinant luciferase-labeled viral antigen are incubated with serum/plasma or other body fluids containing antibodies. Immune complexes consisting of antiviral antibodies bound to luciferase-labeled viral antigen are precipitated with protein A/G-coated beads and washed. Finally, luciferase activity is measured using a luminometer.[6] It has been revealed that antibodies specific to ORF3b and ORF8 antigens can be detected relatively early in the infection, and a predominance of the N antigen in COVID-19 patients, through a recent study on the use of luciferase immunoprecipitation system (LIPS) in the diagnosis of COVID-19.[7] The authors reported that the S (spike) antigen could not produce an adequate antibody response in the early stage of the disease, but antibodies against ORF3b, ORF8, and N antigens can be used for diagnostic purposes. This research has provided crucial results for COVID-19 and immunoreactivity. These results also provide important information regarding the benefits of LIPS applications in terms of diagnostic and immunological perspectives. Preliminary studies involving multiple simultaneous analysis of antibodies against different antigens of SARS-CoV-2 have revealed which antibodies can be predominant or insufficient against the target antigen. It is likely that this method will be at the center of antibody-based research for SARS-CoV-2 in the coming months.

VirScan is a new method for cataloging viral exposure based on antibodies. It is based on a comprehensive T7 phage library that encodes specific viral peptides for each virus on their surface. Thus, it is possible to detect all viruses that can cause infection in a person and to identify humoral responses to dozens of different viruses at once.[8] The misdiagnosis of COVID-19 infection caused by cross-reactions or other viral infections that may occur in individuals during the pandemic are important potential problems . Although its widespread use seems not yet possible technically, experiences that can be obtained during the COVID-19 pandemic with this method may shed light on future virologic diagnostic applications.

Third-generation sequencing technologies that enable real-time sequencing by directly targeting single DNA molecules has resulted in a new revolution that can examine genomes, transcriptomes and metagenomes with unprecedented resolution. It has advantages such as long-reading length, shorter sequencing time, and freedom from sequencing deviations caused by PCR. Oxford Nanopore MinION commercial sequencer, one of the most important representatives of the third generation sequencing, has been introduced recently.[9] This system has been widely used to identify the virus and map its mutations since the beginning of COVID-19 outbreak, with the advantage of producing highly specific data. SARS-CoV-2 data obtained from all over the world through nanopore technology called “laboratory in a suitcase” can be monitored centrally via ARTIC-network.[10] We have expected that third-generation sequencing technologies will be more widely used in the next a few years.

The scientific world is experiencing a dynamic pandemic process. In this period new, accessible, portable, and cost-effective identification methods that promise reduced times— without compromising analytical sensitivity—will be most useful and indeed are seriously necessary. The presence of a large number of trials in the development process for these methods provides strong evidence that SARS-CoV-2 diagnostic algorithms will have richer and creative solutions in the near future.[11] We also look forward to publishing scientific studies for this purpose in our journal.



中文翻译:

2019年新技术在冠状病毒疾病诊断中的作用

由于目前还没有针对2019年冠状病毒疾病的特定药物或疫苗(COVID-19),因此早期诊断对于通过防止病毒传播来控制暴发非常重要。常规的病毒细胞培养方法不容易或不能广泛获得,这意味着多年以来的常规病毒学诊断主要是通过血清学检测进行的。但是,分子检测(包括经典的聚合酶链反应(PCR),多重PCR和综合PCR)具有诸如快速结果转换和高灵敏度等优点,并且近年来已广泛用于病毒学诊断。尽管设备齐全的实验室中有可靠的仪器可用于诊断有症状的患者,在孵化阶段还需要其他方法来筛查无症状的人,或者确定康复中患者的病毒脱落情况。此外,要在全球范围内控制流感大流行,需要针对设备不足的实验室和便携式应用的解决方案。可以在边境口岸的机场和农村地区使用的快速,简单,低成本和便携式测试的开发,是控制COVID-19大流行的重要目标之一。

簇状规则散布的短回文重复序列(CRISPR)方法是一种新技术,该新技术在基因调节的基础上运行,并大大简化了基于核酸的病毒检测,为COVID-19实验室诊断开辟了新领域。[1] 该方法具有高度分析敏感性的机制,其中包括病毒核酸特异的指导RNA(称为sgRNA)和CRISPR效应酶,例如Cas9,Cas13a或Cas13B。最近的出版物显示,CRISPR修饰,例如特异性高灵敏度酶报告基因解锁(SHERLOCK)和靶向DNA核酸内切酶的CRISPR反转录报告基因(DETECTR),能够以高灵敏度检测寨卡病毒,登革热病毒和人乳头瘤病毒。[2] [3]该方法可以在纸上转移,试剂成本非常低。该方法与侧流读数相适应,成功诊断COVID-19,灵敏度为10拷贝/ µL,结果时间不到1小时。该系统还允许创建多个感染面板(全部合为一体)。作为一个引人注目的例子,在最近发表的一篇文章中指出,可以同时快速有效地检测出严重急性呼吸系统综合症冠状病毒2(SARS-CoV-2)和HIV。[4] 这些方法不需要热循环。因此,大大减少了测试时间和设备成本。据指出,重症急性呼吸综合征冠状病毒2(SARS-CoV-2)和HIV被同时快速有效地检出。[4] 这些方法不需要热循环。因此,大大减少了测试时间和设备成本。据指出,重症急性呼吸综合征冠状病毒2(SARS-CoV-2)和HIV被同时快速有效地检出。[4] 这些方法不需要热循环。因此,大大减少了测试时间和设备成本。

允许在恒定温度下扩增核酸的等温扩增方法越来越多地用作PCR的替代方法。靶向SARS-CoV-2 RdRp Gen区域的商业化形式的环介导的等温扩增(LAMP)已获得美国食品药物管理局的紧急使用许可。据报道,该方法仅需10分钟即可检测到SARS-CoV-2,灵敏度为0.125拷贝/ µL。在一项使用来自诊断为COVID-19的患者和健康对照组的口咽拭子的研究中,采样后仅1小时就检测到SARS-CoV-2具有100%的分析灵敏度和特异性。[5] 凭借所有这些优点,我们可以预测,等温扩增选项(尤其是逆转录酶LAMP)将在未来一段时间内用作护理点。

荧光素酶标记的抗原和高度定量的免疫沉淀系统已用于提供病毒鉴定,监测抗病毒治疗以及对与病毒相关的感染进行分类。这种新方法可以评估针对全部或部分病毒蛋白质组的免疫反应性。将含有重组荧光素酶标记的病毒抗原的提取物与血清/血浆或其他含有抗体的体液一起孵育。由结合有萤光素酶标记的病毒抗原的抗病毒抗体组成的免疫复合物用蛋白A / G包被的珠沉淀并洗涤。最后,使用发光计测量萤光素酶活性。[6] 已经发现,可以在感染的相对早期检测到针对ORF3b和ORF8抗原的特异性抗体,并且在COVID-19患者中N抗原占优势,通过最近关于使用萤光素酶免疫沉淀系统(LIPS)诊断COVID-19的研究[7]。作者报告说,S(穗状)抗原不能在疾病的早期阶段产生足够的抗体应答,但是针对ORF3b,ORF8和N抗原的抗体可以用于诊断。这项研究为COVID-19和免疫反应性提供了关键的结果。这些结果还提供了有关LIPS应用在诊断和免疫学方面的益处的重要信息。涉及多个同时分析抗SARS-CoV-2不同抗原的抗体的初步研究表明,哪些抗体对靶抗原的优势或不足。

VirScan是一种基于抗体对病毒暴露进行分类的新方法。它基于全面的T7噬菌体文库,该文库为每种病毒在其表面上编码特定的病毒肽。因此,有可能检测出所有可能导致人感染的病毒,并立即识别出对数十种不同病毒的体液反应。[8] 在大流行期间可能由个体发生的交叉反应或其他病毒感染引起的COVID-19感染的误诊是重要的潜在问题。尽管从技术上来看,它的广泛使用似乎还不可能,但是使用此方法在COVID-19大流行期间获得的经验可能会为将来的病毒学诊断应用提供启示。

通过直接靶向单个DNA分子实现实时测序的第三代测序技术引发了一场新革命,该革命可以以前所未有的分辨率检查基因组,转录组和元基因组。它具有读取长度长,测序时间短和不受PCR引起的测序偏差等优点。最近推出了牛津纳米孔MinION商业测序仪,它是第三代测序最重要的代表之一。[9] 自COVID-19爆发以来,该系统已被广泛用于识别病毒并绘制其突变图,具有产生高度特异性数据的优势。通过称为“手提箱实验室”的纳米孔技术从世界各地获得的SARS-CoV-2数据可以通过ARTIC网络进行集中监控。

科学界正在经历一个动态的大流行过程。在此期间,可以保证减少时间而又不影响分析灵敏度的新颖,可访问,便携式且具有成本效益的识别方法将是最有用的,而且确实非常必要。这些方法在开发过程中的大量试验提供了有力证据,表明SARS-CoV-2诊断算法将在不久的将来提供更丰富和创新的解决方案。[11] 我们也期待为此目的在我们的期刊中发表科学研究。

更新日期:2020-06-18
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