key: cord-0961631-63higujj
authors: Li, Junmin; Hu, Xuejiao; Wang, Xiaoming; Yang, Jianing; Zhang, Lei; Deng, Qianyun; Zhang, Xiqin; Wang, Zixia; Hou, Tieying; Li, Shan
title: A Novel One-Pot Rapid Diagnostic Technology for COVID-19
date: 2021-02-11
journal: Anal Chim Acta
DOI: 10.1016/j.aca.2021.338310
sha: 31e59024b428d831d9842447a2cc4db46b419fb4
doc_id: 961631
cord_uid: 63higujj

Novel coronavirus disease (COVID-19) caused by SARS-CoV-2 is an ongoing global pandemic associated with high rates of morbidity and mortality. RT-qPCR has become the diagnostic standard for the testing of SARS-CoV-2 in most countries. COVID-19 diagnosis generally relies upon RT-qPCR-mediated identification of SARS-CoV-2 viral RNA, which is costly, labor-extensive, and requires specialized training and equipment. Herein, we established a novel one-tube rapid diagnostic approach based upon formamide and colorimetric RT-LAMP (One-Pot RT-LAMP) that can be used to diagnose COVID-19 without the extraction of specific viral RNA. The technique could visually detect SARS-CoV-2 within 45 minutes with a limit of detection of 5 copies per reaction in extracted RNA, and about 7.66 virus copies per μL in viral transport medium. The One-Pot RT-LAMP test showed a high specificity without cross-reactivity with 12 viruses including SARS-CoV, MERS-CoV, and human infectious influenza virus (H1N1/H3N2 of influenza A and B virus, ect. We validated this One-Pot RT-LAMP approach by its successful use for the analysis of 45 clinical nasopharyngeal swab samples, yielding results identical to those of traditional RT-qPCR analyses, while achieving good selectivity and sensitivity relative to a commercial RT-qPCR approach. As such, this One-Pot RT-LAMP technology may be a valid means of conducting high-sensitivity, low-cost and rapid SARS-CoV-2 identification without the extraction of viral RNA.

The novel coronavirus SARS-CoV-2 [1] , which causes a novel coronavirus disease , has rapidly become a global pandemic [2] . As of December 17, 2020, over 70 million confirmed cases and over 1.6 million COVID-19-related deaths have been confirmed worldwide [3] . A rapid and accurate diagnosis of COVID-19 is essential to control this deadly pandemic disease.

To date, approaches for diagnosing COVID-19 based on RNA amplification include RT-qPCR [4] [5] [6] , reverse transcription recombinase polymerase amplification [7] [8] [9] , CRISPR-based method [10, 11] , and RT-LAMP [12] [13] [14] , etc. RT-PCR is the most commonly used technology for pathogen nucleic acid detection and has been considered as a gold standard for infectious disease diagnostics including SARS-CoV-2 [5, 15] . However, this approach is costly, labor-extensive, and requires specialized training and equipment.

RT-qPCR analyses also require roughly 1.5 hours to yield results and are associated with false-negative rates as high as 30-40% [16] [17] [18] . These limitations mean that RT-qPCR approaches are a suboptimal means of accurately identifying patients with suspected COVID-19, testing their close contacts, or assessing individuals with potentially

A SARS-CoV-2 N fragment (28274-29533 nt) in the SARS-CoV-2 complete genome, (accession nos. MN908947.3) was generated in Sangon BiotechCo., Ltd. (Shanghai, China).

RT-LAMP assays were conducted using the synthesized nucleotide plasmid (PUC57-SARS-CoV-2 N) as a positive control. Furthermore, a 2019-nCov-N pseudovirus (Da'an Gene Co., Ltd. of Sun Yat-Sen University, China) was used to assess the relative sensitivity of RT-LAMP and RT-qPCR analyses. A QIAamp Viral RNA Minikit (QIAGEN, CA, USA) was used to extract viral RNA from 200 μL of viral samples, after which a Nanodrop instrument was used to evaluate RNA concentration and quality. Nucleotide copy number was assessed with the underlined formula: RNA copies µL -1 = (concentration of nucleotide (g µL -1 ) / length of nucleotide × 340) ×6.022×10 23 .

Primers specific for the SARS-CoV-2 N gene were designed for this assay using the Primer Explorer v3.0.0 software (http://primerexplorer.jp/elamp3.0.0/index.html) based on sequences in GenBank (MN908947. 3) in accordance with reported recommendations [39] .

The six designed primers included two loops (forward loop primer LF, backward loop primer LB), two inner (forward inner primer FIP, backward inner primer BIP), and two outer (forward primer F3, backward primer B3) primers. The FIP primer consisted of the F1 complementary sequence (F1C) and the F2 sense sequence, while the BIP primer consisted of B1C and the B2 sense sequence. The respective sites of primers and sequences synthesized in Sangon Biotech Co., Ltd. (Shanghai, China) were shown in Figure 1 and Table 1, respectively.

All RT-LAMP assays were conducted in a designated room with RNase-free pipettes and tubes. Analysis and imaging procedures were conducted in a separate room to avoid potential contamination. Individual RT-LAMP reaction containing 6% formamide (Sigma-Aldrich, USA), a 5 μL NP swab sample was used as a template, the concentrations of all other reactions' components mentioned above were maintained for the 25 μL reaction ( Figure 5 ). Reaction tubes were assembled on ice, after which they were incubated for 45 minutes at 65 °C. Reaction tubes were then transferred to 80°C for 5-10 minutes to inactivate the enzyme and cease the reaction. All RT-LAMP reactions were run in triplicate or more.

RT-LAMP assay results were visually evaluated based on the color change of the solution of the reaction. The color of the negative reaction remained violet, while the positive reaction turned sky blue. In addition, agarose gel electrophoresis was also used to confirm the RT-LAMP amplification products. Agarose gel (2%, W/V) was prepared with agarose G-10 powder (Biowest) using 1× TAE buffer (20 mM Acetic acid, 40 mM Tris, 1 mM EDTA) stained with the SYBR®Green I dye (Invitrogen TM , UK). The RT-LAMP products (3 μL) were run on these gels for 40 minutes at 120V and 400 mA. Gels were then imaged using a serving as a negative control.

The low limit of detection for this RT-LAMP reaction was analyzed using 10-fold gradient dilutions of SARS-CoV-2 RNA extracted from 2019-nCov-N pseudovirus (5×10 -1 -5×10 5 copies per reaction). Diluted samples were analyzed in parallel via both RT-LAMP and RT-qPCR. Comparing the lowest concentration that resulted in a positive reaction to RT-qPCR results, the sensitivity of RT-LAMP analysis was defined.

An RT-qPCR kit (Da'an Gene Co., Ltd.) that was confirmed to detect the ORF1ab gene and the N gene of SARS-CoV-2 was used for RT-qPCR analyses based on provided instructions.

Briefly, individual RNA-free tubes used for these analyses contained reaction buffer A (17 μL), reaction buffer B (3 μL), and target RNA template (5 μL). RT-qPCR was then run by the authorized commercial RT-qPCR kit in ABI 7500 Real-time PCR system (Applied Biosystems, USA). 

All participants in the present study provided written informed consent, and the appropriate committees of the participating institutions approved this study. Samples used for these analyses were isolated from standard COVID-19 tests, and thus did not impose any additional burden on these patients.

To design RT-LAMP primers, we began with the conserved SARS-CoV-2 nucleocapsid (N) gene sequence that is used for RT-PCR-based viral detection (MN908947.3) [41, 42] . Both the US CDC and the Chinese authorities recommend the use of primers targeting the viral N gene and the ORF1ab region [7, 43] . We, therefore, compared the homology of the SARS-CoV-2 N gene sequence to that of several other respiratory virus N genes, revealing 88% nucleotide similarity to SARS-CoV, 51% homology with MERS-CoV and lower 10% homology with RSV, HIPV, H1N1, and Influenza B virus ( Figure 1A ). Six primers specific for the SARS-CoV-2 N gene (nucleotides 28274-29533) were subsequently designed for this RT-LAMP assay ( Figure 1B and Table 1 ). RT-LAMP primer specificity was confirmed via aligning N gene sequences from different viruses ( Figure 1C ), and by using the BLAST Global Alignment tool to compare these sequences to those of other coronaviruses. This analysis revealed these primers to be mismatched by 17.09-53.80% with 14 other analyzed coronaviruses (Table S1), indicating that these coronaviruses are unlikely to yield positive RT-LAMP results [44] . These findings thus confirmed that the designed RT-LAMP primers were specific for the SARS-CoV-2 N gene.

The underlined RT-LAMP primers specificity was next tested via examining their ability to amplify nucleic acids from 12 common pseudoviruses including SARS-CoV, MERS-CoV, (Table S2 ) served as an RT-LAMP template, with color changes of the metal indicator HNB being used to gauge reaction outcomes. In the process of DNA amplification by LAMP, a large amount of by-product pyrophosphate ion was produced, which could bond with metals firmly and form insoluble salts [45] . As a metal indicator, the color of HNB changing from violet to sky blue indicated a positive reaction [25] . Reaction mixtures containing template SARS-CoV-2 N gene DNA yielded positive (sky blue) results, whereas all other reaction mixtures, including a DEPC water negative control, yielded negative (violet) results. Agarose gel electrophoresis was also used to assess these reaction products ( Figure 2 ), revealing that only samples containing the target gene yielded the amplified fragments, with no amplification being evident for other tested virus RNA. The result of no cross-reaction with these 12 common pseudoviruses indicates that the primers used for detection have high specificity and can be used for the detection of clinical samples.

In order to establish the sensitivity of RT-LAMP assay for SARS-CoV-2, a tenfold serial dilution of extracted SARS-CoV-2 RNA (5×10 -1 -5×10 5 copies per reaction) was used to conduct parallel RT-LAMP and RT-qPCR assays. The results indicated that the RT-LAMP assay successfully detected approximately 5 copies per reaction ( Figure 3A ), whereas RT-qPCR assays exhibited a LOD of ~50 copies per 25 μL reaction ( Figure 3B ). These findings thus indicated that this RT-LAMP approach was more sensitive than RT-qPCR as a means of detecting SARS-CoV-2.

To achieve a One-Pot RT-LAMP reaction, mild surfactants were identified as the optimal reagents for simultaneous viral lysis and disruption of protein-protein, protein-lipid, and lipid-lipid interactions. Most surfactants, however, facilitate viral lysis most effectively at high temperatures (~100°C). Commercial RT-LAMP buffers contain 0.1% Triton X-100 as a surfactant, but it is not an effective denaturant in the present assay context. We, therefore, J o u r n a l P r e -p r o o f evaluated the ability of a range of mild surfactants to lyse viral particles at 65 °C (data not shown). We ultimately found that a combination of Triton X-100 with the strong nucleic acid denaturant formamide [46] [47] [48] yielded optimal viral lysis under these conditions.

In addition, to determine the optimal formamide concentration for use in this assay context, we conducted this RT-LAMP procedure using a combination of Triton X-100 and a range of formamide concentrations (2, 4, 6, or 8% v/v). Only concentrations of 2-6% facilitated positive SARS-CoV-2 N detection ( Figure 4A ), whereas 8% of formamide yielded an erroneous negative result. This suggests that high formamide concentrations can adversely impact the RT-LAMP enzymatic reactions. We, therefore, tested this One-Pot RT-LAMP assay as a means of detecting the 2019-nCoV-N pseudovirus by adding 6 % formamide with RT-LAMP reaction system ( Figure 5 ), and we found that both high (8.38×10 5 copies μL -1 ) and low (9.8×10 2 copies μL -1 ) viral concentrations were readily detectable without the need for a devoted RNA step ( Figure 4B ). Together these results revealed that 6% formamide combined with 0.1% Triton X-100 degraded the virus strongly and effectively, and thereby promoted in situ reverse transcription.

In order to establish the sensitivity of the One-Pot RT-LAMP assay for SARS-CoV-2, Low-concentration 2019-nCoV-N pseudoviruses (9.8×10 2 -9.57×10 -1 copies μL -1 ) in 2-fold serial dilutions with VTM were used as templates for test. A 5 μL diluted pseudovirus was added to the reaction system containing 6% formamide. In these reactions, the concentrations of the buffer, primer, polymerase, and other reaction components were kept constant within the 25 μL reaction as mentioned above. As shown in Figure 

To test the clinical utility of our assay, 45 clinical NP swab samples, of which 30 and 15 had been found to be COVID-19 negative and positive by RT-qPCR, respectively, were analyzed in a double-blind manner using this One-Pot RT-LAMP approach. For RT-LAMP assays, NP samples (5 μL) were used as a template for direct detection of SARS-CoV-2 ( Figure 5 ). As shown in Figure 6 Compared with some detection methods based on RNA amplification, our method has competitive advantages (see Table2): 1) Simplicity and sensitivity. The One-Pot RT-LAMP assay does not require RNA extraction. Some RT-LAMP researches could rapidly detect SARS-CoV-2 with a low LOD (2 or 3 copies per reaction) [12, 51] , but require an RNA extraction step. Although some RT-LAMP methods have used unextracted swab samples to detect SARS-Cov-2 effectively, a pretreatment of samples lysed with a 95 °C lysis step in TCEP or VTM regent is required, and LOD of the assay is 50 copies per μL [35, 36] , which is higher than our work (5 copies per reaction in extracted RNA, and about 7.66 virus copies per μL in VTM); Other isothermal amplification technologies, such as improved RPA approach, report extraction-free SARS-CoV-2 with a low limit of detection ( 5 viral copies per reaction), however, it is necessary to manually insert the lateral flow strips into the open tube of each sample to visually interpret the results, which is tedious to achieve high J o u r n a l P r e -p r o o f throughput [21] . 2) One-tube reaction. Carry-over contamination usually leads to false-positive results in Lamp reactions [37, 38] . The reaction system of this method is adding 5 μL NP swab sample to the tube of RT-LAMP reaction solution containing 6% formamide ( Figure 5 ). This method detects SARS-CoV-2 from NP swabs to visualized results in a single 

Herein, we designed a One-Tube RT-LAMP approach to amplify the SARS-CoV-2 N gene using formamide and Triton X-100 as surfactants. By adding formamide to the lysis buffer, we were able to carry out all reaction's steps in a single closed tube without RNA extraction, enabling rapid, sensitive, and specific viral RNA detection. This reaction was relatively simple, economical, required just 45 minutes, and did not necessitate the use of complex or costly instrumentation. As such, this protocol may be of value for future efforts to diagnose and control the spread of COVID-19.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 

The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2

World Health Organization

COVID-19 Dashboard by the Center for Systems Science and Engineering

CDC 2019-novel coronavirus (2019-nCoV) real-time RT-PCR diagnostic panel for emergency use only instructions for use

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

National Health Commission of the People's Republic of China, Diagnosis and treatment of novel coronavirus pneumonia

Overcoming the bottleneck to widespread testing: A rapid review of nucleic acid testing approaches for COVID-19 detection

Rapid Detection of SARS-CoV-2 by Low Volume Real-Time Single Tube Reverse Transcription Recombinase Polymerase Amplification Using an Exo Probe with an Internally Linked Quencher (Exo-IQ)

Single-copy sensitive, field-deployable, and simultaneous dual-gene detection of SARS-CoV-2 RNA via modified RT-RPA

Ultrasensitive and visual detection of SARS-CoV-2 using all-in-one dual CRISPR-Cas12a assay

opvCRISPR: One-pot visual RT-LAMP-CRISPR platform for SARS-cov-2 detection

Development of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

Development of Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) Assays Targeting SARS-CoV-2

Variation in False-Negative Rate of Reverse Transcriptase Polymerase Chain Reaction-Based SARS-CoV-2 Tests by Time Since Exposure

Current and Perspective Diagnostic Techniques for COVID-19

Multiplex reverse transcription loop-mediated isothermal amplification combined with nanoparticle-based lateral flow biosensor for the diagnosis of COVID-19

Loop-mediated isothermal amplification of DNA

DNA Detection Using Recombination Proteins

An enhanced isothermal amplification assay for viral detection

Accelerated reaction by loop-mediated isothermal amplification using loop primers

Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products

Detection of Loop-Mediated Isothermal Amplification Reaction by Turbidity Derived from Magnesium Pyrophosphate Formation

Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue

Development of a loop-mediated isothermal amplification method for rapid mass-screening of sand flies for Leishmania infection

Visual detection of isothermal nucleic acid amplification using pH-sensitive dyes

Real-Time Reverse Transcription Loop-Mediated Isothermal Amplification for Rapid Detection of West Nile Virus

One-Pot Reverse Transcriptional Loop-Mediated Isothermal Amplification (RT-LAMP) for Detecting MERS-CoV

A Simple and Multiplex Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of SARS-CoV

Rapid Detection of Influenza Virus Subtypes Based on an Integrated Centrifugal Disc

Rapid Molecular Detection of SARS-CoV-2 (COVID-19) Virus RNA Using Colorimetric LAMP, medRxiv

SARS-CoV-2 RNA in clinical samples

SARS-CoV-2 detection using isothermal amplification and a rapid, inexpensive protocol for sample inactivation and purification

Rapid isothermal amplification and portable detection system for SARS-CoV-2

Simultaneous elimination of carryover contamination and detection of DNA with uracil-DNA-glycosylase-supplemented loop-mediated isothermal amplification (UDG-LAMP)

A novel method to control carryover contamination in isothermal nucleic acid amplification

The detection and identification of saliva in forensic samples by RT-LAMP

Enhancement of polymerase activity of the large fragment in DNA polymerase I from Geobacillus stearothermophilus by site-directed mutagenesis at the active site

A pneumonia outbreak associated with a new coronavirus of probable bat origin

A new coronavirus associated with human respiratory disease in China

Diagnosing COVID-19: The Disease and Tools for Detection

Rapid detection of novel coronavirus (COVID-19) by reverse transcription-loop-mediated isothermal amplification

Loop-mediated isothermal amplification (LAMP) dingof gene sequences and simple visual detection of products

Thermodynamic Effects of Formamide on DNA Stability

Rates of formation and thermal stabilities of RNA:DNA and DNA:DNA duplexes at high concentrations of formamide

One-step hot formamide extraction of RNA from Saccharomyces cerevisiae

Viral load of SARS-CoV-2 in clinical samples

Detection of SARS-CoV-2 in Different Types of Clinical Specimens

RT-LAMP for rapid diagnosis of coronavirus SARS-CoV-2

Streamlined inactivation, amplification, and Cas13-based detection of SARS-CoV-2

CRISPR-based surveillance for COVID-19 using genomically-comprehensive machine learning design

Comparison of SARS-CoV2 N gene real-time RT-PCR targets and commercially available mastermixes

Massive and rapid COVID-19 testing is feasible by extraction-free SARS-CoV-2 RT-qPCR, medrxiv

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

This work was financially supported by Guangdong Science and Technology Project