key: cord-0916512-s6ky3fco authors: Tran, Diem Hong; Cuong, Hoang Quoc; Tran, Hau Thi; Le, Uyen Phuong; Do, Hoang Dang Khoa; Bui, Le Minh; Hai, Nguyen Duc; Linh, Hoang Thuy; Thao, Nguyen Thi Thanh; Anh, Nguyen Hoang; Hieu, Nguyen Trung; Thang, Cao Minh; Vu, Van Van; Phung, Huong Thi Thu title: A comparative study of isothermal nucleic acid amplification methods for SARS-CoV-2 detection at point of care date: 2020-05-25 journal: bioRxiv DOI: 10.1101/2020.05.24.113423 sha: b4c897a1e0156e6b64f2d88fd2e4222677ec3b2b doc_id: 916512 cord_uid: s6ky3fco The COVID-19, caused by the novel coronavirus SARS-CoV-2, has broken out of control all over the globe and put the majority of the world under lockdown. There have been no specific antiviral medications for SARS-CoV-2 while vaccines are still under development. Thus, rapid diagnosis and necessary public health measures are currently key parts to contain the pandemic. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is the gold standard method for SARS-CoV-2 detection. However, this method is not suitable for point-of-care (POC) diagnosis because of the timeconsuming procedure, the requirements of biosafety conditions and expensive equipment. In this study, the colorimetric isothermal nucleic acid amplification tests (iNAATs) for SARS-CoV-2 based on loop-mediated isothermal amplification (LAMP), cross-priming amplification (CPA), and polymerase spiral reaction (PSR) were developed and compared. The three methods exhibited similar performance with the limit of detection (LOD) as low as just 1 copy per reaction when evaluated on the synthetic DNA fragments. The results can be read with naked eyes within 30 minutes without crossreactivity to closely related coronaviruses. When tested with SARS-CoV-2 extracted genomic-RNA, LAMP outperformed both CPA and PSR assays. Moreover, the direct detection of SARS-CoV-2 in simulated patient samples (oropharyngeal and nasopharyngeal swabs) by colorimetric iNAATs was also successful. Further preparation of the lyophilized reagents for LAMP reactions revealed that the freeze-dried, ready-to-use kit maintained the sensitivity and LOD value of the liquid assays. These results strongly indicate that the colorimetric lyophilized LAMP test kit developed herein is highly suitable for detecting SARS-CoV-2 at POC. Coronavirus is a large family of RNA viruses, including the human coronavirus 229E, OC43, NL63, and HKU1, which often lead to respiratory illnesses with mild cold symptoms [1, 2] . The two exceptions of human coronavirus that cause severe diseases including the fatal Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) [3] and the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) [4] that caused two global outbreaks in the last two decades. Very recently, the mortal pneumonia disease caused by a novel a coronavirus called SARS-CoV-2, named "COVID-19" by the World Health Organization (WHO), started in Wuhan, China and rapidly spread all over the globe [5] . In March 2020, WHO has classified the COVID-19 outbreak as "Global Pandemic" [6] . As of May 24 th , 2020, COVID-19 has spread to over 210 countries and regions worldwide with more than 5.4 million confirmed cases and 344,000 casualties [7] . The diagnostic standard of SARS-CoV-2 involves clinical symptoms and molecular methods. For an accurate diagnosis, the molecular methods include metagenomic nextgeneration sequencing (mNGS) and quantitative reverse transcription PCR (qRT-PCR) are required. mNGS is the most precise technique, but it is extremely expensive, timeconsuming, and reliant on highly skillful personnel. qRT-PCR is sensitive for the detection of SARS-CoV-2, which was introduced by WHO [8]. The test can be carried out using respiratory samples such as nasopharyngeal or oropharyngeal swabs, which is currently widely used as the gold standard method for SARS-Cov-2 diagnosis. Nevertheless, qRT-PCR requires high-cost equipment and results are only available within a few hours to 2 days, limiting its application at resource-limited settings. Different virological and serological approaches for rapidly detecting SARS-CoV-2 at POC have been introduced. Virological diagnosis directly detects the viral nucleic acids via isothermal nucleic acid amplification tests (iNAATs) [9] [10] [11] [12] and CRISPR Cas12 based method [13] . Serological tests detect the rising titers of antibody between acute and convalescent stages of infection or detect IgM in primary infection [14] [15] [16] [17] . However, serological diagnosis usually shows lower sensitivity especially in the early stage of infection [18] . Meanwhile, like PCR, iNAATs that amplify the viral nucleic acids at a constant temperature are expected to determine the presence of infectious viruses even in the patient without clinical symptoms. Loop-mediated isothermal amplification (LAMP) was introduced in 2000 and broadly utilized nowadays [19] . LAMP was shown to be rapid, specific, and remarkably sensitive compared to conventional PCR [20, 21] . LAMP uses only one kind of DNA polymerase possessing the strand displacement activity and no modified/labeled DNA probes, simplifying the preparation procedure and significantly saving the cost [22] . Other isothermal DNA amplification methods developed later, which also depend on the use of a DNA polymerase strand displacement activity, include cross-priming amplification (CPA) [23, 24] and polymerase spiral reaction (PSR) [25] . While LAMP requires two to three primer pairs, PSR needs only one primer pair, and CPA uses multiple cross-linked primers (six to eight primers). CPA and PSR amplicon can also be visualized via detection methods that have been used for LAMP, such as using SyBr Green or a pH-sensitive indicator [26] . Both PSR and CPA methods have similar benefits to LAMP, including easy operation, low-cost equipment, and simple and fast readout of the results, which makes them ideal for resource-restricted settings. Various studies indicated that PSR and CPA performance regarding sensitivity and specificity was comparable to that of LAMP [27] [28] [29] [30] [31] . In this study, we developed and compared colorimetric LAMP, CPA, and PSR for SARS-CoV-2 utilizing a pH-sensitive dye for readout visualization. LAMP exhibited the best LOD value with respect to the identification of SARS-CoV-2 viral genomic-RNA. The ready-to-use lyophilized LAMP kit could detect SARS-CoV-2 genomic-RNA directly in clinical samples, which is suitable and convenient for POC diagnostics. Primers for LAMP, CPA, and PSR assays targeting the N and Orf1ab sequences of SARS-CoV-2 (GenBank accession number MN908947) were designed using the free online software Primer Explorer V5 (https://primerexplorer.jp/e/). Primer selection was carried out as instructed (https://primerexplorer.jp/e/v4_manual/). Two sets of primer pairs for LAMP (targeting the N and Orf1ab genes), two sets of primer pairs for PSR (targeting the N and Orf1ab genes), and one set of primer pairs for CPA (targeting the Orf1ab gene) were selected. Primers were synthesized by Phu Sa Biochem (Can Tho, Vietnam) and their sequences are listed in Table 1 . The sequences from 282740 -28516 of the N gene and from 2853 -2452 of the Orf1ab gene of SARS-CoV-2 (GenBank MN908947) were selected to serve as the control templates for iNAATs. The N and Orf1ab sequences were obtained from Phu Sa Biochem, (Can Tho, Vietnam). The DNA templates of MERS-CoV, SARS-CoV, and bat SARS-like-CoV were prepared similarly. The sequences of synthesized DNA templates are listed in Table 1 . The reference genomes of SARS-CoV-2 and related species were downloaded from NCBI (https://www.ncbi.nlm.nih.gov). The primer sequences were aligned to genomes of different coronaviruses to calculate the number of mismatches using Geneious Prime 2020.0.3 (https://www.geneious.com). The percentage of mismatch was calculated by dividing the total number of different bases between primers and genome sequences to the total length of primers. The software FastPCR available at http://primerdigital.com/fastpcr.html was used for in silico PCR analysis. The genomic RNA of SASR-CoV-2 was prepared in a biosafety level 3 laboratory Hilden, German) following the manufacturer's instruction. The copy number of extracted genomic RNAs of SASR-CoV-2 was calculated using the standard curve based on qRT-PCR Ct-value as described in the previous study [33] . Nasopharyngeal and oropharyngeal swab specimens from volunteer nurses and doctors were collected at a local hospital (Ho Chi Minh City, Vietnam). The volunteers had been already diagnosed negative with SARS-CoV-2 by qRT-PCR. All participants gave their informed consent to participate. Oropharyngeal and nasopharyngeal specimens were collected using a sterile flocked plastic swab which was then soaked into 400 µl of nuclease-free water. Fresh samples were kept on ice until analysis or frozen for subsequent assays. To prepare the simulated clinical specimens, various concentrations of SARS-CoV-2 synthesized DNA or extracted viral genomic-RNA were spiked into the nasopharyngeal and oropharyngeal swab collected. Next, the simulated swab samples containing the spiked synthetic-DNA were 10-fold diluted in the nuclease-free water and 1 µl of the diluted sample was added to the iNAAT reactions. The simulated swab samples containing the viral RNA were 50-fold diluted in the nuclease-free water and 5 µl of the diluted sample were added to the LAMP reactions. Non-spiked specimens were used as negative samples. The sample collection was approved by Hospital Management. The internal use of samples was agreed under the medical and ethical rules of each participating individuals. The PCR assays were carried out in a 20 The amplification products were analyzed by electrophoresis using a 2% agarose gel. WarmStart® °C for LAMP and 63 °C for CPA and PSR reactions. The amplification products were detected by the color shifting from red to yellow of the test reaction, which is based on the use of phenol red, a pH-sensitive indicator as instructed by the manufacturer. The products were also analyzed by electrophoresis on a 2% agarose gel when necessary. One nanogram of the synthesized DNA template was used to perform the optimization experiment. Regarding the identification of optimal incubation time, the iNAAT reactions were incubated from 5 to 70 min at 60 °C for LAMP and 63 °C for CPA and PSR reactions. As for temperature optimization, the reaction mixtures were incubated at different temperatures from 50 °C to 70 °C for 30 min. The iNAAT reactions were carried out in the duration and at the temperature optimized using synthetic-DNA templates. The synthesized sequences of the Orf1ab and N gene of SARS-CoV-2 were utilized as the template to perform the LAMP, CPA, and PSR reactions in the presence of the pHsensitive indicator phenol red. The results indicated that the color change from red to yellow of the iNAAT reactions corresponding to the amplified products generated only when the relevant DNA templates were present (Fig. 1 ). Next, the optimal temperature and required time of iNAAT reactions for the detection of SARS-CoV-2 were defined. For LAMP reactions, 15 min was the minimum time required for the readout of positive amplification judged by eye ( Fig. 2A, left panel) . Interestingly, Orf1ab-targeting primers performed the amplification process faster than the N-targeting primers as the product could be observed just after 10 min of incubation based on the gel electrophoresis result (data not shown). LAMP amplicons were produced from 52 to 70 , however, the clearest color changes were observed at 60 °C and higher temperature ( Fig. 2A, right panel) . Therefore, to guarantee the outcome signal, the conditions of LAMP reaction were set at 60 °C for 30 min. Regarding CPA and PSR reactions, amplified products could be observed the best after 30 min ( Fig. 2B and C, left panels). Meanwhile, 61 and 63 °C were the minimal temperatures for CPA and PSR amplified products, respectively, to be visualized by the color change ( Fig. 2B and C, right panels). Thus, 30 min and 63 °C conditions were selected to perform CPA and PSR reactions. The sequences of primers used for iNAATs were aligned to genome sequences of and NC045512) was observed, suggesting that the developed iNAATs could detect different strains of SARS-CoV-2 (Table 2 ). In contrast, except for bat SARS-like-CoV 2015 and 2017 strains, most of the other coronaviruses gave nucleotide mismatch higher than 20% with our designed primers. Thus, it is likely that the designed primer sets would not amplify those sequences, ensuring the specificity of iNAATs for SARS-CoV-2 (Table 2 ). in silico PCR results also support the high specificity of primer sets used (SI 1 file). Further data demonstrated that the iNAATs primer sets used selectively detected the presence of SARS-CoV-2 DNA while no cross-reactivity was observed with DNA of SARS-CoV, MERS-CoV, and bat SARS-like-CoV (Fig. 3) , confirming the absolute specificity of the assay. The LOD value of iNAAT reactions was evaluated using a serial-dilution of the synthesized DNA templates in nuclease-free water. As shown in Fig. 4A and B, roughly a single copy of the synthesized targeted gene per reaction was the lowest amount that LAMP and CPA assays could detect. As for PSR, LOD of the primer set targeting the Orf1ab sequence was 10 3 copies/reaction while N-primer set succeeded to detect a single copy of the synthesized N-sequence (Fig. 4C ). The obtained results indicated that primer pairs designed for LAMP, CPA and PSR targeting N-sequence performed outstandingly and highly promising for a real diagnosis. Thus, the PSR primer set targeting the Orf1ab sequence was herein eliminated from further investigation. The presence of non-target DNA in samples was shown to not interfere with the sensitivity of LAMP [19, 21, 34] negligible. Therefore, we used nuclease-free water instead of the viral transport medium to store the collected nasopharyngeal and oropharyngeal swabs. reactions required 30 minutes to produce the best-visualized results (Fig. 5) . 43.14 (N) viral-RNA copies/reaction ( Table 3 ). The obtained values were outstanding compared to 431.47 and 862.9 copies/reaction of CPA and PSR, respectively (Table 3) . Therefore, the LAMP assays were used for further evaluation with simulated clinical samples containing the different amounts of spiked viral-RNAs. The results indicated that the LAMP assays could directly detect SARS-CoV-2 genomic-RNA in crude samples without the requirement of RNA extraction (Fig. 6) . For better on-site testing, lyophilized reagents which are ready-to-use without strict storage conditions at low temperature would be highly advantageous. Thus, we made an attempt to verify the LAMP kit performance using the dried reagents. Accordingly, the reaction mixture containing the enzyme, primers, and dye was first lyophilized. LODs of the lyophilized LAMP kits for the simulated nasopharyngeal and oropharyngeal swab samples containing the spiked synthetic DNA template were examined. Consistently, the lyophilized kit targeting Orf1ab-sequence exhibited the faster amplification process than the one targeting N-sequence. As for the Orf1ab-sequence set, results could be read after 30 min with the LOD value of 1 DNA copy/reaction, which is equivalent to 10 DNA copies/µl in simulated specimens (Fig. 7A, upper panel) . In contrast, the N-sequence set expressed the slower reaction, which required 45 min for the color change to be clearly observed. The N-sequence set also exhibited a LOD value of 1 DNA copy/reaction corresponding to 10 DNA copies/µl in simulated samples (Fig. 7A, lower panel) . Subsequently, the LOD values of lyophilized LAMP kit for SARS-CoV-2 genomic RNA were also evaluated. Both primer sets targeting Orf1ab-and N-sequence could identify approximately 43.14 copies of viral RNA per reaction ( of the dried-reagent LAMP assay ( Fig. 7B and C) . Accordingly, all nasopharyngeal and oropharyngeal specimens without spiked DNA or viral RNA generated negative results, ensuring the low false-positive occurrence of the assay ( Fig. 7B and C) . It is worth noting that the direct addition of unprocessed clinical specimens to the reaction is greatly convenient for diagnosis at POC, markedly reducing the time required for sample preparation and thus, simplifying operation procedure. When all those features are taken into account, the lyophilized LAMP kit is highly suitable for POC diagnosis. The pneumonia outbreak COVID-19 has been announced by WHO as the global pandemic. Even though each country has its own strategy to prevent the spread of the disease, identifying the person who contracts the SARS-CoV-2 is one of the most essential actions. While the gold standard for identifying the patients is real-time RT-PCR (qRT-PCR), which shows remarkably high accuracy, sensitivity and specificity, it is not portable and only specific high-biosafety laboratories are able to perform the test. Epidemiology and clinical presentations of the four human coronaviruses 229E, HKU1, NL63, and OC43 detected over 3 years using a novel multiplex real-time PCR method A Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2 Rapid and visual detection of 2019 novel coronavirus (SARS-CoV-2) by a reverse transcription loop-mediated isothermal amplification assay CRISPR-Cas12-based detection of SARS-CoV-2 Evaluation of Nucleocapsid and Spike Protein-based ELISAs for detecting antibodies against SARS-CoV-2 Severe Acute Respiratory Syndrome Coronavirus 2-Specific Antibody Responses in Coronavirus Disease Serological survey of SARS CoV 2 for experimental, domestic, companion and wild animals excludes intermediate hosts of 35 different species of animals Antigen Production, and Test Setup Laboratory testing of SARS CoV Loop-mediated isothermal amplification of DNA Optimization of the incubation time and temperature for iNAATs. The LAMP (in A), CPA (in B) and PSR (in C) reactions were incubated from 10 to 60 min at °C (LAMP) and 63 °C (CPA and PSR) (left panels) and at different temperatures from 50 to 70 °C for 30 min (right panels). One ng of the synthesized DNA template was used The authors declare that they have no conflicts of interest.