key: cord-1012103-va4t15mb authors: Yang, Weihua; Dang, Xiaofei; Wang, Qingxi; Xu, Mingjie; Zhao, Qianqian; Zhou, Yunying; Zhao, Huailong; Wang, Li; Xu, Yihui; Wang, Jun; Han, Shuyi; Wang, Min; Pei, Fengyan; Wang, Yunshan title: Rapid Detection of SARS-CoV-2 Using Reverse transcription RT-LAMP method date: 2020-03-02 journal: nan DOI: 10.1101/2020.03.02.20030130 sha: 267729947ca478946d5a4bffb8e13d50c3545120 doc_id: 1012103 cord_uid: va4t15mb Corona Virus Disease 2019 (COVID-19) is a recently emerged life-threatening disease caused by SARS-CoV-2. Real- time fluorescent PCR (RT-PCR) is the clinical standard for SARS-CoV-2 nucleic acid detection. To detect SARS-CoV-2 early and control the disease spreading on time, a faster and more convenient method for SARS-CoV-2 nucleic acid detecting, RT-LAMP method (reverse transcription loop-mediated isothermal amplification) was developed. RNA reverse transcription and nucleic acid amplification were performed in one step at 63 ℃ isothermal conditions, and the results can be obtained within 30 minutes. ORF1ab gene, E gene and N gene were detected at the same time. ORF1ab gene was very specific and N gene was very sensitivity, so they can guarantee both sensitivity and specificity for SARS-CoV-2. The sensitivity of RT-LAMP assay is similar to RT-PCR, and specificity was 99% as detecting 208 clinical specimens. The RT-LAMP assay reported here has the advantages of rapid amplification, simple operation, and easy detection, which is useful for the rapid and reliable clinical diagnosis of SARS-CoV-2. Corona Virus Disease 2019 (COVID-19) is a recently emerged life-threatening viral pneumonia disease caused by SARS-CoV-2 (named 2019-nCoV before February 11, 2020) (1) . COVID-19 is spreading worldwide, and as of February 29, 2020, approximately 80,000 confirmed cases had been identified in the world. SARS-CoV-2 belongs to the beta coronavirus genus and its genome is single-stranded, non-segmented positive-sense RNA, which is 29,881 nucleotides in length (2) . It has 84% nucleotide homology with bat SARS-like coronavirus, 78% homology with human SARS virus, and approximately 50% homology with MERS virus (3, 4) . So far, the full-length genomic sequences between virus samples are almost identical (3) , suggesting that no significant mutations have occurred in the virus (5) . There is no suitable antiviral drug or effective vaccine for the SARS-CoV-2 virus currently. There may be cross-reactions in serum antibody test (6) . Therefore, reliable and rapid laboratory diagnosis of SARS-CoV-2 infection is critical to managing patient care and preventing hospital transmission. For clinical diagnosis of COVID-19, nucleic acid detection or sequencing is currently used in conjunction with pulmonary CT (7) . Real-time reverse-transcription fluorescent PCR (RT-PCR) is the clinical standard for SARS-CoV-2 nucleic acid detection (8) . Now, in order to detect SARS-CoV-2 early and control the disease spreading on time, we developed a faster and more convenient method for detecting SARS-CoV-2 nucleic acid, named RT-LAMP (reverse transcription loop-mediated isothermal amplification). ORF1ab gene, E gene and N gene were selected and primers for them were designed. We have used the RT-LAMP method to detect pathogenic microorganisms and achieved good results (9, 10) . In 2003, RT-LAMP had been reported as a quick and easy diagnostic All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.03.02.20030130 doi: medRxiv preprint method for SARS coronavirus (11, 12) . LAMP is a kind of isothermal amplification method that can amplify nucleic acid with high specificity, sensitivity, and rapidity at 60-65 ℃ and does not require special instruments such as a thermal cycler. The amplification efficiency of the LAMP method is extremely high, the reaction can be completed within one hour. This method relies on self-recycling strand displacement DNA synthesis by Bst DNA polymerase, and previous studies have described detailed amplification mechanisms (13) . This reaction relies on the recognition of DNA targets by six independent sequences, making this assay highly specific. When the template is RNA, the amplification reaction can be occurred in one step by adding reverse transcriptase, and called RT-LAMP. Magnesium pyrophosphate is produced as a by-product during DNA amplification. Because the amplification efficiency of LAMP is higher than that of RT-PCR, a large amount of magnesium pyrophosphate causes the reaction solution to become cloudy. Besides, the added dye calcein can change the mixed solution from orange to green, so the results can not only be observed by a turbidimeter, but also by the naked eye. Because RT-LAMP is convenient and fast, it shows significant advantages in SARS-CoV-2 detection in clinic (14) . Sensitivity, specificity and application potential of RT-LAMP in the clinical diagnosis of SARS-CoV-2 were reported and discussed here. Our hospital is one of the six hospitals for SARS-CoV-2 nucleic acid testing approved by our city. We have five positive specimens and more than 4,000 negative specimens. Positive samples from our hospital were used for the method developing. Specimens from our hospital, Infectious disease hospital of Jinan and Jinan Center for Disease Control and Prevention (Jinan CDC) were used in the clinical verification stage. From January 30 to February 27, during the SARS-CoV-2 epidemic, a total of 208 RNA from throat swab specimens were collected and 17 were SARS-CoV-2 positive. All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.03.02.20030130 doi: medRxiv preprint RNA was extracted using Liferiver (Shanghai Liferiver Biotechnology Co., Ltd.) nucleic acid extraction kit by Nucleic acid extraction instrumentin a tertiary protection laboratory. In order to identify SARS-CoV-2, the species-specific ORF1ab gene, E gene and N gene were selected as the target gene (GenBank accession No.MN988669.1) after comparing the gene sequences of CoVs from human and bat origins. Oligonucleotide primers for LAMP were designed using Primer Explorer V5 software (http://primerexplorer.jp/e/), and synthesized commercially by invitrogen biotech (Invitrogen, Carlsbad, CA, USA). All primers were compared with other coronaviruses and published SARS-CoV-2 sequences by genebank using BLAST alignment tools. Results showed the sequences of the selected primers were specific. Ten sets primers for ORF1ab gene, 12 sets primers for E gene and 13 sets primers for N were designed and synthesized. Each set composed by four primers (F3, forward outer primer; B3, backward outer primer; FIP, forward inner primer; BIP, backward inner primer) targeting six distinct regions (Tsugunori, 2000) . After the suitable primer sets were screened out, loop primers (LF/LP) of them were designed to accelerate the amplification. Primer screening is divided into three steps. The first step is to screen primers that can react. The primers were screened using two positive samples, and the primers that could react within 60 minutes were selected. The second step is to screen primers without false positives reaction. Ten negative samples were used for the reaction. The primers without false positive reactions were selected and others were discarded. The third step is to screen the loop primers. Loop primers of the selected primers were designed. Then the loop primers which can react within 30 minutes were selected. RT-LAMP was carried out using a Loopamp reverse transcription nucleic acid amplification kit (Eiken China Co., Ltd., Shanghai, China) according to the manufacturers' instructions. Brifely, a 25µL total reaction mixture containing 12.5 µL All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is to screen out the optimal primer sets. All the samples we used had been tested by a RT-PCR kit (Jiangsu Bioperfectus Technologies Co.,Ltd.) and confirmed by clinical symptoms and epidemiology of patients. The kit had been registered and it detects ORF1ab gene, E gene and N gene too. RT-PCR was carried out according to the manufacturers' instruction. Briefly, a 25 µL total reaction mixture containing 19µL detection mixture, 1µL RT-PCR enzyme and 5 µL target RNA template were mixed in a reaction tube. Then RT-PCR was performed (45 ℃ for 10 min, 95 ℃ for 3 min, 95 ℃ for 15 sec,58 ℃ for 30 sec, 45 cycles) in ABI 7500 Real-time PCR system (Applied Biosystems, USA). The real-time amplification by RT-LAMP assay was monitored through spectrophotometric analysis by recording the optical density at 400 nm every 6 s with the help of the Loopamp real-time turbidimeter. Besides,the LAMP products can be detected based on color change. The color of the reaction mixture turned green in the presence of LAMP amplification, while the color remained orange in negative amplification tubes. RT-LAMP products were 2-fold serial diluted, and then observed electrophoresed. Because the amount of amplification products is very large, aerosols will be formed when open the reaction tube cover and will pollute the environment. It is not recommended to open the cover and observe results by electrophoresis. Electrophoresis was not used as a way to judge results no matter when, and here, we All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.03.02.20030130 doi: medRxiv preprint just for illustration. As reaction time increases, false positives will appear. In order to determine the timing for judging results, five positive samples and ten negative samples were tested, and the performance of three genes at different time points was compared. When five samples were positive for all three genes, and false positives did not appear, was the time to judge the results. To determine the sensitivity of the RT-LAMP method using the screened primers to identify ORF1ab gene, E gene and N gene of SARS-CoV-2, RNA No. 101 was serially 2-fold diluted, ranging from 10,20,40,80 to 160 folds, RT-LAMP and RT-PCR were performed at the same time using these diluted RNA. The minimum concentration of their positive reaction was observed and recorded. The sensitivity of RT-LAMP was determined by comparison with PCR. The detection limit of the PCR kit used is 1000 copies/ml. To validate the specificity of the RT-LAMP assays, RNA of 10 Influenza A, Influenza B, Respiratory Syncytial Virus positive samples and 20 SARS-CoV-2 negative samples was amplified by RT-LAMP as mentioned above. The RT-PCR method was used for comparison. The RT-LAMP products were assayed by 3% agarose gel electrophoresis and visualized under UVI gel-image analysis system (UVI, UK). DNA fragments were recovered by gel cutting, and the specificity of the fragments was verified by sequencing. During the outbreak of SARS-CoV-2, influenza A virus, influenza B virus and respiratory syncytial virus were detected in our hospital to rule out COVID-19. To the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is In order to compare the amplification efficiency of RT-LAMP and RT-PCR, the same RNA was amplified by RT-LAMP and RT-PCR, respectively, and the products were 2-fold serial diluted, and then electrophoresed. Compare the maximum dilution multiples of them. RT-LAMP detected RNA from a total of 208 nasopharyngeal swab specimens. These We initially screened out one set of optimal RT-LAMP primers for ORF1ab, E and N gene of SARS-CoV-2 respectively. The positions and sequences (Supplemental Table 1 ) of the optimal primer sets are shown in Figure1. All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is (Table 2) . the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.03.02.20030130 doi: medRxiv preprint Figure 1 ). That is to say, the amplification efficiency of RT-LAMP is higher than that of RT-PCR. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is We designed primers for detecting ORF1ab gene, E gene, and N gene of SARS-CoV-2. About Ten sets of primers were designed for each gene. The optimal primers which can cause specific amplification were selected from these primers. And then loop primers of these optimal primers were designed to shorten the reaction time. Primer design and screening are difficult aspects of LAMP, which takes us a lot of time and energy. After screening out the best primers, we did experiments such as sensitivity, specificity, cross-reaction, and clinical validation. To compare sensitivity of RT-LAMP and RT-PCR, they were carried out at the same time using the diluted RNA. Results showed that their sensitivity was similar for SARS-CoV-2 judgment. However, for ORF1ab gene, E gene and N gene, RT-LAMP showed different sensitivities. The sensitivity of the N gene is 80 times than the overall sensitivity. Furthermore, products of RT-LAMP and RT-PCR were 2-fold diluted, and were observed by electrophoretic. RT-LAMP is a number of ladder-like bands between 200 bp-2000 bp. Although the RT-PCR kit used also detects three genes, one band appears because the three gene fragments are all about 100 bp. RT-LAMP has one more dilution than RT-PCR, and because there are several bright bands in one electrophoretic lane, it indicates that the amount of RT-LAMP products is very large, which is the reason that the RT-LAMP results are visible by naked eyes. It was verified that influenza A, influenza B, and respiratory syncytial virus-positive specimens did not interfere SARS-CoV-2 detection by RT-LAMP. RNAs extracted All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.03.02.20030130 doi: medRxiv preprint from 208 specimens were used for clinical application verification. The 208 specimens had undergone multiple diagnose in clinical such as RT-PCR and clinical symptoms, epidemiological investigations, etc., and had been diagnosed as positive or negative. The accuraty rate of RT-LAMP assay is 99%. The ORF1ab gene is very specific, the N gene is very sensitive, and the E gene is in between. If using thees genes alone, the accuracy rates of the ORF1ab gene, E gene and N gene are 99%, 98.5%, and 92.3%, respectively, and the accuracy rate of the combined detection results is 99%. SARS-CoV-2 is highly infectious. In order to do not miss suspected cases, we tested the N gene, which is not specific enough but highly sensitive. However, the N gene alone cannot be judged as positive for SARS-CoV-2, and it needs to be re-examined in order to determine with other genes. Through the combined detection of the three genes, the N gene can detect a low concentration of the virus, so that possible positives are not missed, and the ORF1ab gene guarantees that no false positives appear, which also ensures the sensitivity and specificity for SARS-CoV-2 test. However, if one of the three genes is used alone, it can reduce the sensitivity or specificity of the test, causing false positives or false negatives. In summary, the single-step RT-LAMP measurement reported in this paper is simple, fast, and accurate, and has practicality for SARS-CoV-2 diagnosis in clinical. 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No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.03.02.20030130 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.03.02.20030130 doi: medRxiv preprint