key: cord-0883609-4m9p717r
authors: Yan, Chao; Cui, Jinghua; Huang, Lei; Du, Bing; Chen, Lu; Xue, Guanhua; Li, Shaoli; Zhang, Weiwei; Zhao, Linqing; Sun, Yu; Yao, Hailan; Li, Nannan; Zhao, Hanqing; Feng, Yanling; Liu, Shiyu; Zhang, Qun; Liu, Di; Yuan, Jing
title: Rapid and visual detection of 2019 novel coronavirus (SARS-CoV-2) by a reverse transcription loop-mediated isothermal amplification assay
date: 2020-04-08
journal: Clin Microbiol Infect
DOI: 10.1016/j.cmi.2020.04.001
sha: a6009259cceb933dbbaa6456be281cf953d5137d
doc_id: 883609
cord_uid: 4m9p717r

OBJECTIVE: To evaluate a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for detection of SARS-CoV-2, and compare it with RT polymerase chain reaction (RT-PCR). METHODS: We designed primers specific to the orf1ab and S genes of SARS-CoV-2. Total viral RNA was extracted using the QIAamp Viral RNA Mini Kit. We optimized the RT-LAMP assay. And, this assay was evaluated for its sensitivity and specificity of detection using real-time turbidity monitoring and visual observation. RESULTS: The primer sets orf1ab-4 and S-123 amplified the genes in the shortest times, the mean (±SD) time was 18 ± 1.32 min and 20 ± 1.80 min, respectively, and 63°C was the optimum reaction temperature. The sensitivity was 2×10(1) copies and 2×10(2) copies per reaction with primer sets orf1ab-4 and S-123, respectively. This assay showed no cross-reactivity with other 60 respiratory pathogens. To describe the availability of this method in clinical diagnosis, we collected 130 specimens from patients with clinically suspected SARS-CoV-2 infection. Among them, 58 were confirmed to be positive and 72 were negative by RT-LAMP. The sensiticity was 100% (95% CI 92.3% - 100%), specificity 100% (95% CI 93.7% - 100%). This assay detected SARS-CoV-2 in the mean (±SD) time of 26.28 ± 4.48 min and the results can be identified with visual observation. CONCLUSION: These results demonstrate that we developed a rapid, simple, specific, and sensitive RT-LAMP assay for SARS-CoV-2 detection among clinical samples. It will be a powerful tool for SARS-CoV-2 identification, and for monitoring suspected patients, close contacts, and high-risk groups.

(RT-LAMP) assay for detection of SARS-CoV-2, and compare it with RT polymerase 23 chain reaction (RT-PCR).

Methods. We designed primers specific to the orf1ab and S genes of SARS-CoV-2. 25 Total viral RNA was extracted using the QIAamp Viral RNA Mini Kit. We optimized 26 the RT-LAMP assay. And, this assay was evaluated for its sensitivity and specificity of 27 detection using real-time turbidity monitoring and visual observation. The outbreak of a cluster of respiratory infections designated as coronavirus disease 46 2019 (COVID- 19) , caused by severe acute respiratory syndrome coronavirus 2 47 (SARS-CoV-2), had a significant impact on both the health and economy of the China 48 [1] [2] [3] [4] . To date (1 April 2020), up to 754, 948 SARS-CoV-2 cases have been confirmed, 49 including more than 36, 571 deaths (https://www.who.int/home). Human-to-human 50 transmission was confirmed through a case of five patients in a family cluster [5] . The 51 virus transmitted rapidly via aerial droplets, contact, and fomites [6] . 52 SARS-CoV-2 has been classified as a beta-coronavirus of group 2B with higher 53 similarity, over 96% identical at the whole-genome level, to two bat-derived The extracted RNAs were stored at -80°C. (Xiamen Zeesan Biotech Co., Ltd.) were also used. The copy number of the 98 pseudo-viruses was calculated using the following formula: copies/µL = 6.02× 10 23 99 ×10 -9 × concentration(ng/µL) / (fragment length × 340). Then, 10-fold serial dilutions 100 of the pseudo-viruses ranging from 1×10 8 copies/µL to 1 copy/µL were prepared. (Table S1 ).

To screen for the optimum temperature, the reaction was incubated at five different 166 temperatures (60-64°C) for 60 min. As presented in Figure 1 , the highest 167 amplification efficiency occurred at 63°C. Therefore, 63°C was confirmed as the 168 optimum reaction temperature for this assay.

Sensitivity test for the RT-LAMP assay 170 The sensitivity of the RT-LAMP assay using primer sets orf1ab-4 and S-123 was 171 evaluated using turbidity monitoring and visual observation. Ten-fold serial dilutions 172 of the pseudo-viruses, ranging from 1×10 8 copies/µL to 1 copy/µL (concentration of 173 template input) were detected by the RT-LAMP assays. As illustrated in Figure 2 taken for positive detection ranged from 20 min at 1×10 8 copies /µL to 48 min at 177 1×10 2 copies/µL . Thus, sensitivity of the assays was 2×10 1 copies and 2×10 2 copies 178 per reaction at 63 within 60 min with primer sets orf1ab-4 and S-123, respectively. The main findings of this study is that we established a rapid, sensitive, and 199 specific assay for SARS-CoV-2 detection by RT-LAMP. To improve the sensitivity, preparation [19] . The sensitivity of this assay was 20 copies per reaction, similar to 216 that reported for the RT-PCR assay in a previous study [20] . The RT-LAMP assay 217 showed no cross-reactivity with other respiratory pathogens, so the diagnostic 218 specificity of this method was higher than that reported for the serology test [21] .

To assess the applicability of the assay for the clinical diagnosis of SARS-CoV-2, The main limitation of the current study is that we aligned only 103 complete 235 genomes of SARS-CoV-2 obtained from four databases when design the primers.

With the spread of this virus, the accuracy of this RT-LAMP assay will be affected by 237 the mutations occurring in the primers sequence region of target gene. So, it is 238 necessary to monitor the mutant sites of virus genome by whole genome sequencing.

The established RT-LAMP assay has important implications for clinical practice. The detection was monitored by turbidity using a Loopamp real-time turbidimeter, 17 and was judged by the naked eye depending on a color change from orange to green.

In B, 1-9: 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 , and 10 0 copies/µL. 19 In E, 1-8: 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 , and 10 0 copies/µL.

In C and F, 1-8: 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 , and 10 0 copies/µL.

The reaction volume of 25 µL contained 2 µL RNA template, and the template 22 concentration was 1ng/µL. In the sensitivity test, 60 min can be used as the cut-off for 23 the visual detection. 

The first two cases of 278 2019-nCoV in Italy: Where they come from

Transmission of 2019-nCoV Infection from an Asymptomatic Contact in Germany

Washington State 2019-nCoV Case Investigation Team. First Case of

Coronavirus in the United States

COVID-19) in France: surveillance, 287 investigations and control measures

A familial cluster of infection associated 290 with the 2019 novel coronavirus indicating potential person-to-person transmission 291 during the incubation period

World Health Organization. Novel coronavirus (2019-nCoV) situation report-7: 27 293

Genomic characterisation and 295 epidemiology of 2019 novel coronavirus: implications for virus origins and receptor 296 binding

The global 298 spread of 2019-nCoV: a molecular evolutionary analysis

Divergence of the Novel Coronavirus (2019-nCoV) Originating in China

Laboratory readiness and response for novel coronavirus (2019-nCoV) in expert 305 laboratories in 30 EU/EEA countries

Loop-mediated isothermal amplification of DNA

Rapid and sensitive detection of 311 novel avian-origin influenza A (H7N9) virus by reverse transcription loop-mediated 312 isothermal amplification combined with a lateral-flow device

A Rapid and Specific Assay 315 for the Detection of

Visual detection of west nile 317 virus using reverse transcription loop-mediated isothermal amplification combined 318 with a vertical flow visualization strip

Rapid and specific detection of Asian-and African-lineage Zika 321 viruses

Survey and Visual Detection of 323

Zaire ebolavirus in Clinical Samples Targeting the Nucleoprotein Gene in Sierra 324

A real-time 326 reverse transcription loop-mediated isothermal amplification assay for the rapid 327 detection of yellow fever virus

Loop-mediated isothermal amplification 329 (LAMP): principle, features, and future prospects

Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR

Molecular 334 Diagnosis of a Novel Coronavirus (2019-nCoV) Causing an Outbreak of Pneumonia

Molecular and 337 serological investigation of 2019-nCoV infected patients: implication of multiple 338 shedding routes

Clinical features of patients 340 infected with 2019 novel coronavirus in Wuhan, China

A Novel Coronavirus 343 from Patients with Pneumonia in China

Genetic Diagnostic Methods for Novel Coronavirus 2019 (nCoV-2019) in Japan

RNA based mNGS approach 349 identifies a novel human coronavirus from two individual pneumonia cases

Wuhan outbreak

Load in Upper Respiratory Specimens of Infected Patients

HCoV-NL63-3; S6: HCoV-OC43-1; S7: HCoV-OC43-2; S8: HCoV-HKU1-1

ADV-1-2; S29: ADV-2-1

A-2; S41: RSV B-1; S42: RSV B-2; S43: HMPV-1; S44: HMPV-2; S45: BoV-1

BoV-2; S47: Rh A-1; S48: RhA-2; S49: Rh B-1; S50: RhB-2; S51: Rh C-1

S56: Klebsiella pneumoniae; S57: Streptococcus pneumoniae; S58: Pseudomonas 45 aeruginosa

Figure 1 Sequence comparison and gene analysis of the orf1b and S genes 49

Sequence comparison and gene mutation analysis of the orf1b gene (8084bp)

Sequence comparison and gene mutation analysis of the S gene (3822bp)

F3: outer forward primer; B3: outer backward primer; FIP: forward inner primer; BIP: 52 backward inner primer; LF: loop forward primer

Bat severe acute respiratory syndrome like coronavirus 55 MERS: Middle East respiratory syndrome coronavirus

GenBank Accession No. MG772933; Bat-SL-CoVZC45

GenBank Accession No. NC_019843.3