Development and validation of a rapid, single-step reverse transcriptase loop-mediated isothermal amplification (RT-lamp) system potentially to be used for reliable and high-throughput screening of COVID-1.
Development and validation of a rapid, single-step reverse transcriptase loop-mediated isothermal amplification (RT-lamp) system potentially to be used for reliable and high-throughput screening of COVID-1.
复制标题
开发和验证快速、单步逆转录酶环介导的等温扩增 (RT-LAMP) 系统，有望用于可靠和高通量的 COVID-19 筛查
DOI：
10.3389/fcimb.2020.00331
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发表时间：
2020-01-01
影响因子：
5.7
通讯作者：
Li, X. J.
中科院分区：
文献类型：
--
作者：
Jiang, Ming-hua;Pan, Wei-hua;Li, X. J.
目的：开发和验证一种单步和准确的逆转录酶环介导等温扩增技术（RT-LAMP），相对于商业定量逆转录酶实时PCR（qRT-PCR）测定法，用于快速鉴定SARS-CoV-2，以迅速启动适当的医疗护理和遏制病毒传播。研究方法：对显示最佳计算机模拟特征的引物进行分析灵敏度和特异性，以评估检测限（LOD）以及与密切相关和远距离相关的病毒种属以及临床上突出的细菌和真菌种属的交叉反应。为了评估临床实用性，我们的RT-LAMP进行了大量的临床样本，包括213阴性和47阳性患者，相对于两个商业定量RT-PCR检测。结果如下：当在水和痰液中进行连续稀释时，我们的检测方法的分析特异性和灵敏度为100%和500拷贝/ml。将我们的RT-LAMP测定用于临床样品显示出相对于qRT-PCR使用时的高度特异性（99.5%）、灵敏度（91.4%）、阳性预测值（97.7%）和阴性预测值（98.1%）。我们的RT-LAMP测定比qRT-PCR快两倍，并且可在室温下储存。使用我们的RT-LAMP和两种qRT-PCR检测试剂盒，随后变为阳性的疑似病例检测结果均为阳性，这表明我们的检测试剂盒具有筛查目的的能力。结论：我们提出了一种快速的RT-LAMP检测方法，可以扩展实验室的能力，相对于qRT-PCR处理两倍以上的临床样本，并可能用于高通量筛选目的时，需求在关键情况下增加。
Objectives: Development and validation of a single-step and accurate reverse transcriptase loop-mediated isothermal amplification technique (RT-LAMP) for rapid identification of SARS-CoV-2 relative to commercial quantitative reverse transcriptase real-time PCR (qRT-PCR) assays to allow prompt initiation of proper medical care and containment of virus spread. Methods: Primers showing optimal in-silico features were subjected to analytical sensitivity and specificity to assess the limit of detection (LOD) and cross-reaction with closely- and distantly-related viral species, and clinically prominent bacterial and fungal species. In order to evaluate the clinical utility, our RT-LAMP was subjected to a large number of clinical samples, including 213 negative and 47 positive patients, relative to two commercial quantitative RT-PCR assays. Results: The analytical specificity and sensitivity of our assay was 100% and 500 copies/ml when serial dilution was performed in both water and sputum. Subjecting our RT-LAMP assay to clinical samples showed a high degree of specificity (99.5%), sensitivity (91.4%), positive predictive value (97.7%), and negative predictive value (98.1%) when used relative to qRT-PCR. Our RT-LAMP assay was two times faster than qRT-PCR and is storable at room temperature. A suspected case that later became positive tested positive using both our RT-LAMP and the two qRT-PCR assays, which shows the capability of our assay for screening purposes. Conclusions: We present a rapid RT-LAMP assay that could extend the capacity of laboratories to process two times more clinical samples relative to qRT-PCR and potentially could be used for high-throughput screening purposes when demand is increasing at critical situations.