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Aug 2020
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A Protocol for Simple, Rapid, and Direct Detection of SARS-CoV-2 from clinical samples, using Reverse Transcribed Loop-Mediated Isothermal Amplification (RT-LAMP)
使用逆转录环介导等温扩增(RT-LAMP)从临床样本中简单、快速、直接检测SARS-CoV-2   

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Abstract

SARS-CoV-2 has quickly spread all around the globe causing illness and wide damages. Most countries were unprepared for such a rapid spread and crisis. This led to various strategies for effective control of the new pandemic. A key aspect in all countries was to effectively test the population for the virus. Most countries chose a lockdown strategy in which many workplaces and activities are completely closed, leading to substantial economy costs. Here, we present a protocol we recently developed that allows rapid and simple detection of SARS-CoV-2 for the large population, eliminating costs and involvement of professional teams and laboratories. This protocol is based on Reverse Transcribed Loop-Mediated Isothermal Amplification (RT-LAMP). We tested this protocol directly on patient samples, both nasal and throat clinical swabs as well as saliva. Notably, this protocol is simple, cheap and can be easily applied to other pathogens as well.

Keywords: SARS-CoV-2 (SARS-CoV-2), Covid-19 (新型冠状病毒肺炎), RT-LAMP (逆转录环介导等温扩增), Pandemic (流行病), Rapid molecular Detection (快速分子检测), Colorimetric (比色法), Isothermal (等温的), Saliva (唾液)

Background

The Covid-19 pandemic, caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is affecting large populations, and has been declared a pandemic by the World Health Organization (WHO).

Mass surveillance of the population and quarantine proved to be effective strategies in dealing with this crisis. The main key for detection is the reverse transcription quantitative polymerase chain reaction (RT-qPCR) test. While this test is effective, it requires professional experience both in sampling and in performing the test. Furthermore, the reagents and lab equipment are expensive (Bruce et al., 2020). Altogether, these created a bottleneck for mass scale testing.

Fortunately, to date, alternative molecular biology methods can overcome such limitations. One of these methods is colorimetric Loop-Mediated Isothermal Amplification (LAMP) (Notomi et al., 2000). LAMP is performed at a single and constant temperature (i.e., isothermal), and allows a one-step reverse transcription. Its results can be visualized by color change with the naked eye. This method is cheap and requires little to no lab equipment (Wang et al., 2016; Yu et al., 2020; Zhang et al., 2020). It can be widely used in points of care such as the workplace and schools, which can increase the number of tested subjects tremendously. We see this method as a surveillance tool to markedly increase the total number of tests per day and identify patients for further tests in hospital settings.

Here we present a protocol for applying one step reverse transcribed LAMP (RT-LAMP) detection method on clinical samples from nasal, throat swabs and from saliva. The primers we used were previously designed by Zhang et al., 2020 on synthetic and purified RNA (Table 1). The protocol we present (Ben-Assa*, Naddaf* et al., 2020), while with these same primers, does not require RNA purification steps and can be conducted directly on clinical samples. This protocol requires no professional experience and results can be obtained within an hour. The results of this protocol were compared to the approved RNA purification and quantification RT-qPCR method at the Rambam Health Care Campus (RHCC) hospital. The downside of this method is its detection sensitivity, which is considerably lower than the standard RT-qPCR method (Ben-Assa*, Naddaf* et al., 2020). Therefore, it is applicable for a large-scale surveillance tool rather than a replacement of the current gold-standard detection method. While this protocol was validated on SARS-CoV-2, it can be adjusted to other pathogens too (bacteria or viruses).

Material and Reagents

  1. Personal protective equipment (PPE) appropriate for working with SARS-CoV-2
  2. 0.2 ml PCR tube strips (Labcon, catalogue number: LC 3927-550)
  3. Pipette tips
  4. WarmStart® Colorimetric LAMP 2x Master Mix (New England BioLabs Inc., catalog number: M1800 ) (aliquot and store at -20 °C)
  5. Proteinase K (Seegene, catalog number: 744300.4.UC384, store at -20 °C)
  6. Guanidine hydrochloride (Sigma, catalog number: G4505 )
  7. DNase RNase free water (Biological Industries, catalog number: 01-869-1B)
  8. Primers for SARS-CoV-2 (Table 1) (Zhang et al., 2020)
  9. Primers for pop7–positive internal processing and amplification control (Table 2) (Curtis et al., 2018)
  10. Primer mix (see Recipes)
  11. Proteinase K (see Recipes)
  12. Guanidine hydrochloride (see Recipes)

    Table 1. SARS-CoV-2 Primers for RT-LAMP



          Table 2. RNaseP pop7 Primers for RT-LAMP–Positive Control

Equipment

  1. 1 µl-10 µl, 10 µl-50 µl, 20 µl-200 µl pipettes
  2. PCR Thermocycler as heat source (Biometra, catalog number: T3000 )

Procedure

  1. Prepare primer mix. (see Recipes)
    Prepare two mixes: (1) For SARS-Cov-2 test. (2) For pop7 positive internal processing and amplification control.

  2. Sample lysis and preparation (see Figure 1A)
    1. For saliva samples: Resuspend in 1 ml of DNase RNase free water (in the same container of the saliva sample). For throat/nasal swab samples: Use the standard universal transfer media (UTM) for this test.
    2. Add 5 µl of the sample (from Step B1) to a PCR tube containing 40 µl of DNase RNase free water.
    3. Include a non-template control. Add 5 µl DNase RNase free water instead of the sample.
    4. Add 2 µl proteinase K (final concentration 1.22 mg/ml).
    5. Incubate at room temperature for 15 min.
    6. Incubate samples at 95 °C for 5 min in a PCR machine or any other heat source.
      Note: We have successfully used thermal cup and hot water.
    7. Cool your sample down to room temperature.

  3. RT-LAMP reaction (see Figure 1A)
    Prepare three mixes: (1) For SARS-CoV-2 test. (2) For pop7 gene as positive control. For each mix use the relevant primers. (3) For non-template control (see Step B3) using the SARS-CoV-2 primers.
    1. Prepare reaction:
      LAMP mix                                                           10 µl
      Primer mix                                                           2 µl
      Guanidine hydrochloride                                   1 µl
      Sample lysate (product of Procedure B)          7 µl
    2. Incubate at 65 °C for 30-40 min.
    3. Read results:
      If the mix color changes from pink to yellow, the sample has tested positive (see Figure 1B).
      If the mix maintains its pink color, the sample has tested negative (see Figure 1B).
      Please note that the results are binary as positive or negative. Only bright yellow results are considered positive. Pink is considered negative, and any other range or gradient of color is considered negative.
      Positive control results were published at Ben-Assa*, Naddaf* et al., 2020 (Figure 3a).


      Figure 1. Procedure and results of SARS-CoV-2 RT-LAMP detection test. A. An illustration of the steps of the protocol: this illustration summarizes the main steps of the protocol. Illustration was created with BioRender.com. B. One negative sample (Neg S.) and one positive sample (Pos S.) before and after incubation. At t = 0 min (left panel), all samples were pink. After incubation of 30min (right panel), positive samples turned yellow while negative samples retained their pink color. Pictures are representative RT-LAMP test results of clinical diagnostic nasal and throat swabs of data published in Ben-Assa*, Naddaf* et al., 2020. Results were confirmed by conventional RT-qPCR clinical test for SARS-CoV-2 following RNA extraction and purification step.

Notes

  1. Alternative to a PCR machine, any heating source (e.g., water bath) can be used for Steps B5 and C2. We have successfully used a thermos cup and a thermometer to adjust water temperature.
  2. Longer incubation than 40 min may result in non-specific color change and interpreted as false positive results.
  3. It is very important to use proper personal protective equipment when handling the samples starting from step B and throughout the rest of the protocol.
  4. The color change in the protocol is an indication for a binary positive/negative result and does not represent any gradient indication of the clinical status of the patient.

Recipes

  1. Primer mix
    1. Prepare each one of the primers at 100 µM
    2. Primer mix:
      F3                    2 µl (2 µM final concentration)
      B3                    2 µl (2 µM final concentration)
      LF                    4 µl (4 µM final concentration)
      LB                    4 µl (4 µM final concentration)
      FIP                   16 µl (16 µM final concentration)
      BIP                   16 µl (16 µM final concentration)
    3. DNase RNase free water 56 µl
    4. Aliquot and store at -20 °C
  2. Proteinase K at stock concentration 28.67 (mg/ml)
    Soluble in DNase RNase free water
    Store at -20 °C
  3. Guanidine hydrochloride
    Soluble in DNase RNase free water
    Stock concentration, 800 mM
    Store at room temperature

Acknowledgments

We thank the Geva-Zatorsky lab for fruitful discussions and contributions. We would like to thank the Rambam hospital for their support and for hosting us, especially to the infectious disease and virology teams. We would also like to thank Dr. Rich Roberts and Dr. Nathan Tanner for their valuable support, as well as Prof. Daniel King, Prof. Yehuda Chowers, Dr. Ronit Almog, Dr. Yuval Geffen, Dr. Dani Zvi-Bar and Prof. Oded Lewinson for their crucial help. In addition, we would like to thank Dima Abdu for preparing the figure for this paper. The illustration in this in Figure 1B was created with BioRender.com.
   This work was supported by the Technion Integrated Cancer Center, the Technion – Israel Institute of Technology, “Keren Hanasi”, Alon Fellowship for Outstanding Young Researchers, Horev Fellow (Taub Foundation). NGZ is an Azrieli Global Scholar at the Canadian Institute for advanced research (CIFAR).

Competing interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. While performing this research, no company had invested in the research, and no commercialization was intended.

Ethics

This study was granted exemption from IRB approval of the Rambam Health Care Campus (# 0244-20-RMB) for use of de-identified COVID-19 tests performed for the purpose of the standard testing, and for 4 volunteers.

References

  1. Ben-Assa, N., Naddaf, R., Gefen, T., Capucha, T., Hajjo, H., Mandelbaum, N., Elbaum, L., Rogov, P., King, D. A., Kaplan, S., Rotem, A., Chowers, M., Szwarcwort-Cohen, M., Paul, M. and Geva-Zatorsky, N. (2020). Direct on-the-spot detection of SARS-CoV-2 in patients. Exp Biol Med (Maywood) 245(14): 1187-1193.
  2. Bruce, E. A., Huang, M. L., Perchetti, G. A., Tighe, S., Laaguiby, P., Hoffman, J. J., Gerrard, D. L., Nalla, A. K., Wei, Y., Greninger, A. L., Diehl, S. A., Shirley, D. J., Leonard, D. G. B., Huston, C. D., Kirkpatrick, B. D., Dragon, J. A., Crothers, J. W., Jerome, K. R. and Botten, J. W. (2020). DIRECT RT-qPCR DETECTION OF SARS-CoV-2 RNA FROM PATIENT NASOPHARYNGEAL SWABS WITHOUT AN RNA EXTRACTION STEP. bioRxiv. doi: 10.1101/2020.03.20.001008.
  3. Curtis, K. A., Morrison, D., Rudolph, D. L., Shankar, A., Bloomfield, L. S. P., Switzer, W. M. and Owen, S. M. (2018). A multiplexed RT-LAMP assay for detection of group M HIV-1 in plasma or whole blood. J Virol Methods 255: 91-97.
  4. Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., and Hase, T. (2000). Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28(12): e63-e63.
  5. Wang, X., Yin, F., Bi, Y., Cheng, G., Li, J., Hou, L., Li, Y., Yang, B., Liu, W. and Yang, L. (2016). Rapid and sensitive detection of Zika virus by reverse transcription loop-mediated isothermal amplification. J Virol Methods 238: 86-93.
  6. Yu, L., Wu, S., Hao, X., Dong, X., Mao, L., Pelechano, V., Chen, W. H. and Yin, X. (2020). Rapid Detection of COVID-19 Coronavirus Using a Reverse Transcriptional Loop-Mediated Isothermal Amplification (RT-LAMP) Diagnostic Platform. Clin Chem 66(7): 975-977.
  7. Zhang, Y., Odiwuor, N., Xiong, J., Sun, L., Nyaruaba, R. O., Wei, H., and Tanner, N. A. (2020). Rapid Molecular Detection of SARS-CoV-2 (COVID-19) Virus RNA Using Colorimetric LAMP. MedRxiv doi: https://doi.org/10.1101/2020.02.26.20028373.

简介

[摘要] SARS-CoV-2在全球迅速蔓延,导致疾病和广泛的损害。大多数国家对如此迅速的蔓延和危机毫无准备。这导致了有效控制这一新流行病的各种战略。所有国家的一个关键方面是对人口进行有效的病毒检测。大多数国家选择了一种封锁战略,即许多工作场所和活动完全关闭,从而导致巨大的经济成本。在这里,我们介绍了一个我们最近开发的协议,它允许对大量人群进行快速和简单的SARS-CoV-2检测,省去了成本和专业团队和实验室的参与。该方案基于反向转录环介导的等温扩增(RT-LAMP)。我们直接在患者样本上测试了该方案,包括鼻腔和喉咙的临床拭子以及唾液。值得注意的是,该方案简单、廉价,并且可以很容易地应用于其他病原体。

[背景] 由新型严重急性呼吸综合征冠状病毒2型(SARS-CoV-2)引起的Covid-19大流行正在影响大量人群,世界卫生组织(WHO)已宣布为大流行。
大规模的人口监测和检疫被证明是应对这场危机的有效策略。检测的关键是逆转录定量聚合酶链反应(RT-qPCR)试验。虽然这项测试是有效的,但它需要在抽样和执行测试方面的专业经验。此外,试剂和实验室设备昂贵(Bruce等人,2020年)。总之,这给大规模测试带来了瓶颈。
幸运的是,到目前为止,其他的分子生物学方法可以克服这些局限性。其中一种方法是比色环介导的等温扩增(LAMP)(Notomi等人,2000年)。LAMP是在一个单一且恒定的温度(即等温)下进行的,并且允许一步反转录。它的结果可以用肉眼观察到颜色的变化。这种方法成本低廉,几乎不需要实验室设备(Wang等人,2016年;Yu等人,2020年;Zhang等人,2020年)。它可以广泛应用于工作场所和学校等护理点,这可以极大地增加测试对象的数量。我们将这种方法视为一种监测工具,可以显著增加每天的检查总数,并确定患者是否需要在医院进行进一步的检查。
我们从一个临床样品中转录了一个样品的鼻拭子(RT-LAMP)检测方法。我们使用的引物先前是由Zhang等人于2020年在合成和纯化RNA上设计的(表1),我们提出的方案(Ben Assa*,Naddaf*等人,2020年)使用这些相同的引物,不需要RNA纯化步骤,可以直接在临床样本上进行。本方案无需专业经验,一小时内即可得出结果。该方案的结果与兰巴姆卫生保健院(RHCC)医院批准的RNA纯化和定量RT-qPCR方法进行了比较。该方法的缺点是其检测灵敏度大大低于标准RT-qPCR方法(Ben-Assa*,Naddaf*等人,2020)。因此,它适用于大型监控工具而不是取代现行金标准的检测方法。虽然该方案在SARS-CoV-2上得到了验证,但它也可以适用于其他病原体(细菌或病毒)。.

关键字:SARS-CoV-2, 新型冠状病毒肺炎, 逆转录环介导等温扩增, 流行病, 快速分子检测, 比色法, 等温的, 唾液

材料及相关试剂
 
1. 适用于SARS-CoV-2的个人防护装备(PPE)
2. 0.2 ml PCR管条(Labcon,产品编号:LC 3927-550)
3. 吸管尖头
4. WarmStart®比色灯2x主混料(新英格兰生物实验室公司,目录号:M1800)(等分并在-20°C下储存)
5. 蛋白酶K(见基因,目录号:744300.4.UC384,储存温度-20°C)
6. 盐酸胍(西格玛,目录号:G4505)
7. 脱氧核糖核酸酶游离水(生物工业,目录号:01-869-1B)
8. SARS-CoV-2的引物(表1)(Zhang等人,2020年) 
9. 阳性内部处理和扩增控制引物(表2)(Curtis等人,2018年)流行音乐7 
10. 底漆混合物(见配方)
11. 蛋白酶K(见食谱)
12. 盐酸胍(见配方)
 
表1。用于RT-LAMP的SARS-CoV-2引物
底漆名称 序列
基因-A-F3 TGG CTA CTA CCG AAG AGC T公司
基因-A-B3 TGC AGC ATT GTT AGG在
GeneN-A-LF(循环前进) GGA CTG AGA TCT TTC附件TTA CCG T
GeneN-A-LB(回路向后) 表演GAG GGA GCC TTG AAT ACA
GeneN-A-FIP(前内侧底漆) TCT GGC CCA GTT CCT AGG标签TCC AGA CGA ATT CGT GGT GG
GeneN-A-BIP(反向内部底漆) AGA CGG目录目录ATG GGT TGC ACG GGT GCC AAT GTG ATC T
 
表2。RT-LAMP用RNaseP pop7引物-阳性对照
底漆名称 序列
pop7型-三层 TTG ATG AGC TGG AGC CA公司
pop7型-B3 CAC CCT CAA TGC AGA GTC公司
pop7型-LF(循环前进) ATG TGG ATG GCT堵头TTG TT
pop7型-LB(向后循环) 类别GCT ACC GAG
pop7型-FIP(前内侧底漆) GTG-TGA-CCC-TGA-AGA-CTC-GGT-TTT-AGC-TGA-CTC-GGA-TC
pop7型-BIP(反向内部底漆) CCT CCG TGA TAT GGC TCT TCG TTT TTT TCT TAC ATG GCT CTG GTC
 
设备
 
1. 1µl-10µl、10µl-50µl、20µl-200µl移液管
2. PCR热循环器作为热源(Biometra,目录号:T3000)
 
程序
 
A、 准备底漆混合物。(参见配方)准备两种混合物:(1)用于SARS-Cov-2测试。(2) 用于pop7阳性的内部处理和扩增控制。
 
 
B、 样品分析和制备(见图1A)
1.     对于唾液样品:在1毫升无脱氧核糖核酸酶核糖核酸酶的水(在唾液样品的同一容器中)中再悬浮。对于喉部/鼻腔拭子样本:使用标准通用转移介质(UTM)进行此测试。
2.     将5µl样品(步骤B1)添加到含有40µl DNase RNase游离水的PCR管中。
3.     包含非模板控件。加入5µl无DNase RNase的水代替样品。
4.     添加2µl蛋白酶K(最终浓度1.22 mg/ml)。
5.     室温下培养15分钟。
6.     在PCR机器或任何其他热源中于95℃孵育5分钟。摄氏度
注:我们成功地使用了热水杯。
7.     把样品冷却到室温。
 
C、 RT-LAMP反应(见图1A)准备三种混合物:(1)用于SARS-CoV-2试验。(2) 以pop7基因为阳性对照。对于每种混合物,使用相关底漆。(3) 对于使用SARS-CoV-2引物的非模板控制(见步骤B3)。
1.     准备反应:
混合灯10µl
底漆混合物2µl
盐酸胍1µl
样品裂解液(程序B的产物)7µl
2.     在65°C下培养30-40分钟。
3.     读取结果:
如果混合物颜色从粉红色变为黄色,则样品测试呈阳性(见图1B)。如果混合物保持粉红色,则样品测试为阴性(见图1B)。请注意,结果是二进制的,可以是正数,也可以是负数。只有亮黄色的结果被视为阳性。粉红色被认为是负的,任何其他范围或颜色的渐变都被认为是负的。阳性对照结果发表于Ben Assa*、Naddaf*等人,2020年(图3a)。
 
 
图1。SARS-CoV-2rt-LAMP检测方法及结果。A、 步骤的说明协议:这个例子总结了协议的主要步骤。插图是用BioRender.com网站. B、 培养前后各一个阴性样品(阴性样品)和一个阳性样品(阳性样品)。t=0 min时(左面板),所有样本均为粉红色。孵育30分钟后(右图),阳性样品变为黄色,阴性样品保持粉红色。图片是临床诊断鼻和喉拭子的代表性RT-LAMP测试结果,发表于Ben Assa*,Naddaf*等人,2020年。用常规RT-2法和常规提取法对SARS-CoV进行RT-2纯化。
 
笔记
 
1.     除PCR机外,步骤B5和C2可使用任何加热源(例如水浴)。我们成功地用了一个保温杯和一个温度计来调节水温。
2.     培养时间超过40分钟可能会导致非特异性的颜色变化,并被解释为假阳性结果。
3.     从步骤B开始并在整个方案的其余部分处理样品时,使用适当的个人防护设备是非常重要的。
4.     方案中的颜色变化表示二元阳性/阴性结果,不代表患者临床状态的任何梯度指示。
 
食谱
 
1底漆混合物1底漆混合物
a、 在100µM处制备每种底漆
b、 底漆混合物:F3 2µl(2µM最终浓度)
B3 2µl(2µM最终浓度)
LF 4µl(4µM最终浓度)
LB 4µl(4µM最终浓度)
FIP 16µl(16µM最终浓度)
BIP 16µl(16µM最终浓度)
c、 脱氧核糖核酸酶游离水56µl
d、 等分份并在-20°C下储存
2.     储备浓度为28.67(mg/ml)的蛋白酶K
溶于无DNase-RNase的水
储存于-20°C
3.     盐酸胍
溶于无DNase-RNase的水
原料浓度,800 mM
室温下储存
 
致谢
 
我们感谢Geva-Zatorsky实验室富有成效的讨论和贡献。我们要感谢兰巴姆医院的支持和接待,特别是对传染病和病毒学小组的支持。我们还要感谢Rich Roberts博士和Nathan Tanner博士的宝贵支持,以及Daniel King教授、Yehuda Chowers教授、Ronit Almog博士、Yuval Geffen博士、Dani Zvi Bar博士和Oded Lewinson教授提供的重要帮助。此外,我们要感谢迪马·阿布杜为本文件编制了数字。图1B中的插图是用BioRender.com网站.
这项工作得到了Technion综合癌症中心、Technion-以色列理工学院、“Keren Hanasi”、Alon杰出青年研究员奖学金、Horev Fellow(Taub基金会)的支持。NGZ是加拿大高级研究所(CIFAR)的Azrieli全球学者。
 
 
 
相互竞争的利益
 
作者声明与本文的研究、作者身份和/或出版没有潜在的利益冲突。在进行这项研究时,没有任何公司对这项研究进行投资,也没有打算商业化。
 
伦理学
 
本研究中,志愿者被批准使用本研究的IRCOVAM-4标准。
 
工具书类
 
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引用:Naddaf, R., Ben-Assa, N., Gefen, T., Capucha, T., Hajjo, H., Mandelbaum, N., Elbaum, L., Kaplan, S., Rotem, A., Chowers, M., Szwarcwort-Cohen, M., Paul, M. and Geva-Zatorsky, N. (2020). A Protocol for Simple, Rapid, and Direct Detection of SARS-CoV-2 from clinical samples, using Reverse Transcribed Loop-Mediated Isothermal Amplification (RT-LAMP). Bio-protocol 10(20): e3789. DOI: 10.21769/BioProtoc.3789.
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