key: cord-334518-mjr6u7ak authors: Hu, X.; Deng, Q.; Li, J.; Chen, J.; Wang, Z.; Fang, Z.; Li, H.; Zhao, Y.; Yu, P.; Li, W.; Wang, X.; Li, S.; Zhang, L.; Hou, T. title: Development and clinical application of a rapid and sensitive loop-mediated isothermalamplification test for SARS-CoV-2 infection date: 2020-05-23 journal: nan DOI: 10.1101/2020.05.20.20108530 sha: doc_id: 334518 cord_uid: mjr6u7ak Background The outbreak of SARS-CoV-2 urgently requires sensitive and convenient COVID-19 diagnostics to assure the containment and timely treatment of patients. We aimed to develop and validate a novel reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to detect SARS-CoV-2 in both qualified laboratories and point-of-care settings. Methods Patients with suspected COVID-19 and close contacts between Jan 26 and April 8, 2020, were recruited from two hospitals. Respiratory samples were collected and tested with LAMP and the results were compared with those obtained by RT-qPCR. The inconsistent samples between these two methods were subjected to next-generation sequencing for confirmation. In addition, we tested the RT-LAMP on an asymptomatic COVID-19 carrier and patients with other respiratory viral infections. Results We finally collected a cohort of 129 cases (329 nasopharyngeal swabs) and the independent cohort of 76 patients (152 nasopharyngeal swabs and sputum samples). RT-LAMP was validated to be accurate (overall sensitivity and specificity: 88.89% and 99.00%; positive and negative predictive values: 94.74% and 97.78%) and diagnostically useful (positive and negative likelihood ratios: 88.89 and 0.11). RT-LAMP showed an increased sensitivity (88.89% vs 81.48%) and high consistency (kappa 0.92) compared with RT-qPCR for SARS-CoV-2 screening while requiring only constant temperature heating and visual inspection. The median time required for RT-LAMP was less than 1 h from sample to result. Further analyses indicated that RT-LAMP was feasible for asymptomatic patients and did not cross-react with other respiratory pathogen infections. Conclusion The RT-LAMP assay offers a rapid, sensitive and straightforward detection for SARS-CoV-2 infection, which could aid the expansion of COVID-19 testing in the public domain and hospitals. The skyrocketing COVID-19 outbreak has become a public health emergency of international concern. A total of 4,618,821 confirmed cases and 311,847 deaths have been reported in 216 countries as of May 18, 2020, since early December of 2019, according to the WHO COVID-19 report. 1 At present, there are still no effective drugs or vaccines reported for COVID-19, and prompt diagnosis, close contact tracking and quarantine management are the hallmarks for the containment of this new pandemic. Accurate early diagnosis of SARS-CoV-2 infection is crucial to prevent virus transmission and provide appropriate treatment for patients. Due to its nonspecific symptoms and radiological features overlapping with those of the common cold and influenza, the confirmation of SARS-CoV-2 infection entirely depends entirely on viral RNA detection. [2] [3] RT-qPCR is the standard and widely used method for SARS-CoV-2 RNA detection in clinical laboratories. 4 Despite its outstanding analytical performance, RT-qPCR detection for COVID-19 still suffers from many limitations, such as long turnaround times (more than 2 h), poor availability (it is currently restricted to public health laboratories), requirement of expensive instrumentation, and high proportion of false negatives or equivocal values (up to 38%) [5] [6] in upper respiratory samples due to insufficient viral materials. These limitations make the RT-qPCR test far from adequate to meet the current challenge of a tremendous undocumented infected population, asymptomatic transmission 7 and convalescence with viral RNA conversion, 8 which highlights the pressing need for a more rapid, simple and sensitive approach to quickly identify infected patients in different settings. Loop-mediated isothermal amplification (LAMP) is regarded as a promising point-of-care test (POCT) assay due to its advantages of high sensitivity and specificity, rapid reaction and low laboratory infrastructure requirements. 9 Reverse transcription-LAMP (RT-LAMP) is a kind of LAMP method to detect target RNA using the AMV reverse transcriptase. This approach allows reverse transcription and DNA amplification to be accomplished rapidly at a 60-65 ℃ constant temperature in less than an hour and in one step, and detailed amplification mechanisms have been reported previously. 10 RT-LAMP can be detected by visual turbidity or fluorescence in real time, which makes this method a practical near-patient assay. In recent years, RT-LAMP has been widely used in specialized laboratory testing as well as field surveys to identify various pathogens, including Mycobacterium tuberculosis, 11 Zika virus, 12 MERS-CoV, 13 and SARS-CoV. 14 Shirato et al. 13 reported a useful RT-LAMP assay for the diagnosis of MERS that was developed in this way, with a detection limit of 3.4 copies per reaction and no cross-reaction with other respiratory viruses. Hong et al. 14 developed a real-time quantitative RT-LAMP for the early and rapid diagnosis of SARS-CoV, which demonstrated 100-fold greater sensitivity than conventional RT-qPCR. To accelerate clinical diagnostic testing for COVID-19, we conducted a prospective cohort study to develop and validate a novel RT-LAMP assay capable of detecting SARS-CoV-2 RNA for potential use in centralized facilities and point-of-care settings. Moreover, we compared RT-qPCR and RT-LAMP on clinical samples and demonstrated that RT-LAMP produced a higher sensitivity and cost effectiveness for SARS-CoV-2 detection. To the best of our knowledge, this study is the first to comprehensively assess a rapid RT-LAMP test for both COVID-19 patients and an asymptomatic carrier, with improved diagnostic value in addition to current diagnostics for SARS-CoV-2 infection. This study was designed as a prospective observational cohort study with three sequential phases. In the initial stage, we developed a visual and rapid RT-LAMP assay for SARS-CoV-2 detection and assessed its anti-cross interface ability, stability and detection limit. Subsequently, we evaluated the RT-LAMP and standard RT-qPCR assays on 329 nasopharyngeal swabs from a cohort of 129 suspected COVID-19 patients and on the serial upper respiratory samples from an asymptomatic carrier, and the insistent samples between RT-LAMP and RT-qPCR were further subjected to next-generation sequencing (NGS) for SARS-CoV-2 confirmation. Finally, we analyzed an additional 20 patients with other viral infections, 20 healthy individuals, and an independent cohort of 76 cases suspected of having COVID-19 to further validate the detective captivity of RT-LAMP for SARS-CoV-2. The overall study strategy is shown in Figure 1 . Cohort I Inpatients with clinical-radiological suspicion of COVID-19 presenting to Guangdong Provincial People's Hospital between January 26 to April 8, 2020 were eligible for inclusion. Close contacts with exposure to confirmed COVID-19 cases were simultaneously enrolled in the present study. Every participant underwent a standard SARS-CoV-2 set of investigations testing for COVID-19. The patients' demographic, clinical, laboratory and radiological findings were collected from their medical records. Serial nasopharyngeal swabs were collected from patients during hospitalization and close contact screening. Sample sizes for swabs were defined by their availability. At least one nasopharyngeal swab from suspected patients was simultaneously sent to the CDC for double checking as required, where RT-qPCR was standardly utilized for SARS-CoV-2. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 23, 2020. Cohort II We enrolled an independent cohort of suspected patients from Guangdong Second Provincial General Hospital for validation. SARS-CoV-2 set testing and the diagnosis procedure for COVID-19 were identical in the two hospitals. A nasopharyngeal swab and 5 mL of morning sputum were collected from suspected patients to validate the diagnostic performance of RT-LAMP for SARS-CoV-2. In addition, nasopharyngeal swab samples obtained from 20 healthy subjects and 20 patients with other respiratory virus infections were used to test the specificity of RT-LAMP for SARS-CoV-2 detection. Swabs were preserved in 500 μL of virus preservation solution (TianLong, China), which virtually inactivates the virus and preserves all RNA in the specimen. The sputum samples were preprocessed by a standard NALC-NaOH digestion. Total RNA was extracted from specimens within 2 h using a magnetic bead-based viral RNA isolation kit on the DA3200 system . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 23, 2020. . https://doi.org/10.1101/2020.05.20.20108530 doi: medRxiv preprint instrument (Daan Gene, China) according to the manufacturer's instructions. The extracts were stored at -70°C until use. The same extracted RNA of each specimen was submitted in parallel to RT-qPCR and RT-LAMP in a double-blind manner for testing SARS-CoV-2 in a biosafety level 2 laboratory. The inconsistent samples between these two methods were further analyzed with NGS for verification. RT-qPCR was carried out using an officially approved clinical RT-qPCR kit for the ABI COVID-19 QuantStudio Dx TM real-time PCR system (Applied Biosystems, USA) following the manufacturer's protocol (Daan Gene). Primer and probe sets targeting the ORF1ab and N genes of SARS-CoV-2 are provided in Table S1 . A final 25-μL-volume reaction mixture for RT-qPCR included 17 μL of reaction buffer, 3 μL of enzyme solution, and 5 μL of template RNA. The cycling program started at 50°C for 15 min for reverse transcription, followed by 95°C for 15 minutes for PCR initial activation and 45 cycles consisting of 94°C for 15 s and 55℃ for 45 s. A cycle threshold value less than 40 was defined as a positive test. Patient were defined as having a laboratory-confirmed COVID-19 when both targets (ORF1a/b and N gene) were positive, and repeated tests using another approved RT-qPCR kit were necessary for single-target-positive (ORF1a/b or N positive) samples. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 23, 2020. . https://doi.org/10.1101/2020.05.20.20108530 doi: medRxiv preprint was selected as the target to design our RT-LAMP primers because it is highly homologous among various COVID-19 sequences and highly divergent from those of other coronaviruses examined. We designed 4 sets of RT-LAMP primers targeting the SARS-CoV-2 S gene sequence (No. MN908947.3) using the online PrimerExplorer V5 software (available at: https://primerexplorer.jp/e/). One set of RT-LAMP primers with the best parameters was chosen, including two outer primers, F3 and B3; two inner primers, forward inner primer (FIP) and backward inner primer (BIP); and two loop forward (LF) and backward (LB) primers ( Figure S1 ), and synthesized by Invitrogen (Shanghai, China). Primer specificity was verified with a BLAST search of the GenBank nucleotide database via comparison with other coronavirus and published SARS-CoV-2 sequences, and the percent mismatch result is offered in Table S2 . . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 23, 2020. potential cross-reactivity for RT-LAMP. Their RT-LAMP products were assayed by 3% agarose gel electrophoresis. To determine the lower detection limit of the RT-LAMP method for COVID-19, a 10-fold gradient dilution series of synthetic SARS-CoV-2 S gene cDNA (1.5×10 2 -1.5×10 -9 ng/reaction) was tested as a template for amplification with RT-LAMP. The minimum concentration of the positive reaction was recorded. This dilution series was run in parallel with RT-qPCR using primers that targeted this same region of the COVID-19 genome. The detection limit of RT-LAMP was determined by comparing the lowest concentration of the positive reaction with that of RT-qPCR. The inconsistent samples between RT-LAMP and RT-qPCR and samples from COVID-19 patients that were RT-qPCR negative were further analyzed with multiplex PCR-based enrichment plus NGS to detect the SARS-CoV-2 genome. Briefly, total RNA was reverse transcribed to synthesize first-strand cDNA with random hexamers and Superscript III reverse transcriptase kit (Vazyme, China). Two-step SARS-CoV-2 genome amplification was performed with two pooled mixtures of primer sets (designed by Genskey Medical Technology Co., Ltd.). The pooled primer sets were designed to cover the entire SARS-CoV-2 genome. cDNA was . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 23, 2020. . https://doi.org/10.1101/2020.05.20.20108530 doi: medRxiv preprint mixed with the components of the first PCR following the manufacturer's instructions. The 2nd PCR was performed using the index primer and the constructed libraries were sequenced on the Illumina NovaSeq PE 150 platform. Analysis was carried out based mainly on an in-house pipeline produced by Genskey Medical Technology. Raw sequencing data was quality trimmed and subsequently filtered if shorter than 130 bases by fastp v0.19.5. Sequence reads were first filtered against the human reference genome and then aligned to a reference genome of SARS-CoV-2 (NC_045512.2) using Bowtie v2.2.4. The mapped reads were assembled with SPAdes v3.14.0 with kmers ranging from 19 to 109 to obtain the coronavirus genome sequences. The sensitivity, specificity, positive and negative predictive values, likelihood ratios and their respective 95% confidence intervals for RT-LAMP and RT-qPCR testing of nasopharyngeal specimens were calculated, and agreement analysis was computed using kappa concordance coefficients (a value ≥ 0.75 was deemed good) and percentage agreement (≥ 0.9 was considered good). 15 Statistical analyses were performed in the R programming environment. Written informed consents were obtained from all participants before the study, and the study was approved by the ethics committee of each participating institution. The analysis was conducted on samples collected during standard COVID-19 tests, with no extra burden on patients. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 23, 2020. . https://doi.org/10.1101/2020.05.20.20108530 doi: medRxiv preprint As described in the Materials and Methods, we developed a rapid and simple RT-LAMP assay to detect SARS-CoV-2 RNA, and positive reactions resulted in a color change from purple to blue due to decreased magnesium concentration in the presence of extensive Bst DNA polymerase activity, while negative reactions retained the purple color. Figure S3 shows the overall procedure of the RT-LAMP assay. RT-LAMP primers for COVID-19 were specific and had a 9.14-37.56% nucleotide mismatch with SARS, MERS and other coronavirus sequences (Table S2 ), and a cross-interface experiment further demonstrated that RT-LAMP did not cross-react with other human-pathogenic coronaviruses and common virus pathogens, supporting the specificity of this assay for COVID-19 ( Figure S4 ). Our dilution experiments of the synthetic SARS-CoV-2 S gene showed shown the analytic limit of detection (LOD) of RT-LAMP relative to that of RT-qPCR for the detection of SARS-CoV-2 ( Figure S5 ). The resulting LOD was approximately 1.5×10 -8 ng per 25-μL reaction solution (i.e., 4.23 copies/reaction) for RT-LAMP and 1.5×10 -7 ng/reaction (i.e., 42.3 copies/reaction) for RT-qPCR. RT-LAMP exhibited a 10-fold higher sensitivity than the RT-qPCR used in the current clinical test, similar to previous LAMP studies. [16] [17] Characteristics of the subjects is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 23, 2020. . https://doi.org/10.1101/2020.05.20.20108530 doi: medRxiv preprint presenting primarily with fever, cough/expectoration, and muscle pain/fatigue ( Table 1) Table 1 . We first evaluated the clinical application of the RT-LAMP assay on 329 nasopharyngeal specimens from Cohort I. Of these 329 nasopharyngeal swabs, 35 swabs were confirmed as SARS-CoV-2 positive according to the combined criteria of RT-qPCR positive results (28 samples) and NGS confirmation (7 samples) (see Table 2 , Table S3 and Figure S6 ). Thirty-one out of 35 clinically positive samples were determined to be positive using the RT-LAMP assay, (Table 2) . Compared with RT-qPCR, RT-LAMP had a significantly better sensitivity (88.57% vs 80.00%) and comparable specificity (98.98% vs 100%) for the diagnosis of SARS-CoV-2 infection ( Table . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 23, 2020. . https://doi.org/10.1101/2020.05.20.20108530 doi: medRxiv preprint 2). Detection results obtained using RT-LAMP were in good concordance with those obtained using RT-qPCR, with a Cohen's kappa of 0.89 (0.79-1.00), 100% positive predictive agreement and 98.01% negative predictive agreement. These observations are in line with data reported by the studies from Baek et al. [16] [17] [18] In addition to exploring the diagnostic potential of RT-LAMP on active COVID-19 patients, we also tested the RT-LAMP assay on an asymptomatic COVID-19 carrier. RT-LAMP has a higher sensitivity in detecting SARS-COV-2, particularly for those samples with a low viral load, and also suggested that RT-LAMP can be used for the diagnosis of asymptomatic COVID-19 carriers. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 23, 2020. . https://doi.org/10.1101/2020.05.20.20108530 doi: medRxiv preprint We next validated the RT-LAMP assay on an independent Cohort II of 28 COVID-19 patients and 48 COVID-19 exclusion cases. Both one nasopharyngeal swab and one sputum specimen were collected from every participant in Cohort II. A total of 152 samples comprised of 46 positive samples (28 swabs and 18 sputum) and 106 negative samples ( Table 2 and Table S3 ). Table 2 ), and the agreement between the two assays was excellent (kappa 0.93 (0.77-1.00), Table 2 ). These observations corroborate with the results from Cohort I as well as the previous RT-LAMP findings, [16] [17] [18] suggesting that RT-LAMP may improve the sensitivity of pathogenic diagnosis for COVID-19. To further assess whether the RT-LAMP assay was COVID-19 specific, 40 swab specimens from 20 patients with influenza (n = 9) or respiratory viral infections (n = 11, representing Mycoplasma pneumoniae, HPIV-1/2/3, RSV-A/B, RSV, and HAdV-B/E) and 20 healthy individuals were subjected to the RT-LAMP assay. No positive results were observed, which demonstrated that RT-LAMP-based detection can distinguish SARS-CoV-2 with no crossreactivity for other respiratory viruses, similar to reports in recent studies. [16] [17] [18] We summarized the RT-LAMP assay reported here for SARS-CoV-2 detection in two cohorts. RT-LAMP exhibited an overall sensitivity of 88.89% (higher than the 81.48% for RT-qPCR), an overall specificity of 99.00%, high consistency (kappa 0.92) with the RT-qPCR, and a median turnaround time less than 1 h from sample to result in the detection of 481 clinical specimens . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 23, 2020. . https://doi.org/10.1101/2020.05.20.20108530 doi: medRxiv preprint from two cohorts (Figure 3 ). Additional advantages of RT-LAMP include cost effectiveness, simple operation and visual determination capability, which facilitate SARS-CoV-2 screening in well-equipped labs as well as in the field (Figure 3 ). Rapid and reliable diagnosis is of particular importance for the containment of COVID-19 outbreak. We described a simple and sensitive RT-LAMP approach to rapidly diagnose SARS-CoV-2 infection. The robustness of the present study was demonstrated, as this RT-LAMP assay was useful to diagnose active COVID-19 patients and asymptomatic carriers and generally not confounded by other respiratory pathogen infections using clinical samples from two hospitals. Existing methods to detect SARS-CoV-2 are based mainly on RT-qPCR, NGS and IgM and IgG immunological tests. Comparing the results between RT-LAMP and RT-qPCR, RT-LAMP provided a better sensitivity (88.89% vs 81.48%) than RT-qPCR for SARS-CoV-2. This added sensitivity is important in consideration of a significant number of COVID-19 patients that have presented with negative qPCR 7 results or the "relapse after negative" phenomenon 8 due to potentially large variability between clinical samples, low-virus-titer samples and even disrupted binding of RT-qPCR primers due to variation in the viral genome. 19 We used the self-developed Bst DNA polymerase in this RT-LAMP assays, which was demonstrated a higher polymerization activity than the commercial Bst DNA polymerase 20 and ensured the high sensitivity of this RT-LAMP method. Based on these findings, we propose that the RT-LAMP assay was able to detect viral RNA not only in RT-qPCR positive samples but also in those inconclusive samples. We found that RT-LAMP was less sensitive and informative than NGS in our study and other literature. [21] [22] [23] [24] NGS is a robust tool for obtaining extensive genetic information and completing whole-genome sequence with the LOD as low as 10 copies/mL for SARS-CoV-2 detection, . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 23, 2020. . https://doi.org/10.1101/2020.05.20.20108530 doi: medRxiv preprint ranking as one reference test for COVID-19, especially for those challenging samples containing low viral content. 2, [23] [24] However, compared to the complex and costly NGS platform, RT-LAMP had the advantage of low-threshold of infrastructure, data processing requirement and cost effectiveness, which enabled this friendly assay to be immediately deployed in hospitals and communities. RT-LAMP also showed no cross-reactivity with other viruses that manifest similar respiratory diseases, and thus, the specificity of RT-LAMP was higher than that reported for the IgM and IgG detection. 25 In addition, we described the accuracy of COVID-19 RT-LAMP by means of likelihood ratios. Likelihood ratios are not affected by disease prevalence, and their values higher than ten and lower than one strongly support the diagnostic value of a test. 26 Based on this metric, the near-patient LAMP assay used in this study is diagnostically useful for COVID-19. Overall, the established RT-LAMP in this study could be a powerful complementary method for monitoring massive numbers of exposed individuals as well as aiding with screening efforts in hospitals and public domains, especially in areas with limited laboratory capacities. Additionally, nasopharyngeal swabs from COVID-19 patients had a higher positive rate than sputum specimens in both the RT-qPCR and LAMP assays. Liu et al. 27 reported that the detection rate of SARS-CoV-2 RNA in nasopharyngeal swabs was lower than that in bronchoalveolar lavage fluid and sputum. This inconsistency is most likely due to poor sputum quality and fluctuation of virus RNA during different stages of the disease course. 28 Despite this inconsistency, nasopharyngeal swabs are noninvasive and easy to acquire, and evidence has shown that SARS-CoV-2 replicates actively in upper respiratory tissue. 29 Therefore, we argue that nasopharyngeal swabs are suitable for the detection of SARS-CoV-2 at an early stage of infection. We note that four samples from non-COVID-19 cases were RT-LAMP positive but RT-qPCR negative (see Table 1 ), as reported previously. 18 The four false positive results by RT-LAMP . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 23, 2020. . were caused by aerosol contaminants, as we retested these samples in another clean room and obtained the negative RT-LAMP results as expected. Contaminant issues are not rare for nucleic acid testing, even when the best available reference laboratory tests are used. Precautions to prevent cross-contamination or aerosol contaminants during assay are highly recommended, including using a spray solution for the elimination of potential RNA fragments and changing gloves frequently. The RNA extraction-free RT-LAMP assay could address this important question. 18 Since this study was completed, the SARS-CoV-2 RT-LAMP test has been optimized further, with the use of lyophilized reagents and direct detection of SARS-CoV-2 without any need to conduct RNA extraction. This one-step single-tube RT-LAMP hastens reaction time and minimizes false positive reactions and would be an ideal POCT for COVID-19 if validated in future studies. One limitation of our study was the relatively small sample size of positive COVID-19 cases, which has resulted in widened confidence intervals for our estimates of diagnostic accuracy. We tested the samples using RT-LAMP in a blind manner, and the designation of the real status of SARS-CoV-2 infection in clinical samples was based on a set of combined criteria of RT-qPCR and subsequent NGS confirmation to obviate potential false negative or positive results. We further validated the diagnostic potential of RT-LAMP in another independent cohort with nasopharyngeal swabs and sputum samples. Therefore, despite our small sample size, our study provided sufficient robustness for the RT-LAMP assays. In conclusion, we developed a simple and rapid RT-LAMP assay for SARS-CoV-2 detection and demonstrated its high diagnostic sensitivity and specificity among clinical samples. Our findings suggest that RT-LAMP can be an appropriate auxiliary assay for the diagnosis and epidemiologic surveillance of COVID-19 in different hospital and community settings. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 23, 2020. . . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 23, 2020. . . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 23, 2020. . Notes: Sen, sensitivity; Spe, specificity; PPV, positive predictive value; NPV, negative predictive value; PLR, positive likelihood ratio; NLR, negative likelihood ratio. Inconsistent samples between RT-LAMP and RT-qPCR assays were further determined by next-generation sequencing . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 23, 2020. . https://doi.org/10.1101/2020.05.20.20108530 doi: medRxiv preprint • positive • negative NGS positive . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 23, 2020. . https://doi.org/10.1101/2020.05.20.20108530 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 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Notes: PPV, positive predictive value; NPV, negative predictive value; PLR, positive likelihood ratio; NLR, negative likelihood ratio.In Cohort I, 35 out of 37 nasopharyngeal swabs from 24 COVID-19 patients were confirmed as SARS-CoV-2 positive according to the criteria of RT-qPCR (28 samples) and NGS confirmation (7 samples, Table S3 ). In Cohort II, 46 out of 56 samples (paired nasopharyngeal swabs and sputum samples) from 28 COVID-19 patients were determined as SARS-CoV-2 positive accordingly (38 were RT-qPCR-positive and 8 were NGS-positive, Table S3 ).