PMC:7668588 / 5604-10725 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/7668588","sourcedb":"PMC","sourceid":"7668588","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7668588","text":"Materials and methods\n\nEthics statement\nThis study was approved by the Institutional Review Boards of Nagasaki University (approval no. 200409234).\n\nViral RNA and synthesized standard RNA\nViral RNA of SARS-CoV-2 and SARS-CoV were kindly provided by the Japanese National Institute of Infectious Diseases (NIID), and Dr. Koichi Morita from the Nagasaki University, Japan, respectively. To calculate the copy number of viral genome detection in both LAMP and quantitative PCR (qPCR) assays, synthesized RNAs were prepared using T7 RiboMAX Express Large Scale RNA Production System (Promega) with the artificially synthesized DNA of the viral target sequence conjugated with the T7 promoter sequence.\n\nLAMP primer design\nLAMP primers for SARS-CoV-2 detection were designed based on the sequences of the ORF1b region of the virus. The SARS-CoV-2 sequences available in GenBank and GISAID were aligned using BioEdit 7.0.5.3 software (http://www.mbio.ncsu.edu/BioEdit/bioedit.html) to identify conserved regions. A consensus sequence of the ORF1b region was used to design LAMP primers through Primer Explorer V5 software (Eiken; http://primerexplorer.jp/). The RT-LAMP assay requires a set of 6 primers: 2 outer primers (F3 and B3), 2 inner primers (FIP and BIP), and 2 loop primers (LF and LB). FIP consists of a complementary sequence of F1 and a sense sequence of F2, whereas BIP includes a complementary sequence of B1 and a sense sequence of B2 (10). The detailed primer sequences used for SARS-CoV-2 amplification are shown in Table 1.\nTable 1 Sequences of LAMP primers.\nName Type Sequence (5’–3’)\nLAMP_ORF1b-1_F3 F3 AACCTGAGTTTTATGAGGCT\nLAMP_ORF1b-1_B3 B3 TCCTAAGTAAAGTTGAGTCACA\nLAMP_ORF1b-1_FIP FIP TGCAAGCACCACATCTTAATGAAGTCGCATACAGTCTTACAGGCT\nLAMP_ORF1b-1_BIP BIP ACGACCATGTCATATCAACATCACAACATCACAACCTGGAGCAT\nLAMP_ORF1b-1_LF LF CAAAGAACACAAGCCCCAAC\nLAMP_ORF1b-1_LB LB GTCTTGTCTGTTAATCCGTATGTTTG\nLAMP_ORF1b-2_F3 F3 GGTTTTTTCACTTACATTTGTGG\nLAMP_ORF1b-2_B3 B3 TCCTCCAAAATATGTAATTTGCA\nLAMP_ORF1b-2_FIP FIP GCGAAGTGTCCCATGAGCTTATAAACTAGCTCTTGGAGGTTCCG\nLAMP_ORF1b-2_BIP BIP AATGCGTCATCATCTGAAGCATTTTCATAACCATCTATTTGTTCGCG\nLAMP_ORF1b-2_LF LF TCAGCATTCCAAGAATGTTCTGT\nLAMP_ORF1b-2_LB LB ATTGGATGTAATTATCTTGGCAAACC\n\nRT-LAMP assay\nRT-LAMP was performed with Isothermal Master mix reagent ISO-004 (Canon medical systems) using the Genelyzer FIII real-time fluorescence detection platform (Canon medical systems). The reaction mixture (total volume = 25 μL) contained 15 μL of Isothermal Master Mix, 1 U of AMV Reverse Transcriptase (Nippon gene), 20 pmol (each) of FIP and BIP primers, 5 pmol (each) of F3 and B3 outer primers, 10 pmol (each) of F and B loop primers, and 5 μL of RNA sample (template). The reaction was performed at the following conditions: for the ORF1b-1 primer set, 68°C for 20 min, followed by a dissociation analysis at 95°C–75°C with the temperature change rate of 0.1°C/s; for the ORF1b-2 primer set, 67°C for 20 min, followed by a similar dissociation analysis. Extracted RNA from samples or heat inactivated swab samples were used as a template. Synthesized RNAs containing the target sequence of the LAMP assay were used as positive controls. Nonspecific amplification was excluded by comparing the melting temperature to that of the positive control [13].\n\nRT-qPCR assay\nRT-qPCR was performed using the One Step PrimeScript III RT-qPCR Mix (Takara Bio) as reported previously [14]. The reaction mixture (total volume = 20 μL) contained 10 μL of 2× One Step PrimeScript III RT-qPCR Mix, 0.4 μL of ROX Reference Dye, 2 μL of 10× primer-probe mixture, 2 μL of RNA sample, and 5.6 μL of RNase-free water. Two different sets of primer-probe mixture were used in this study. The first set was developed by NIID (NIID-qPCR) and used with the final concentration of 500 nM, 700 nM, and 200 nM of the forward primer, reverse primer, and FAM-labeled probe, respectively [15]. The second primer-probe set was reported by WHO for detection of E gene of SARS-CoV-2 and SARS (WHO-qPCR), and used 400 nM of each primer and 200 nM of the probe [9]. The NIID-qPCR reaction was performed using the StepOnePlus instrument (Applied Biosystems) with a thermal cycle program of 52°C for 5 min, 95°C for 10 sec, followed by 45 cycles of 95°C for 5 sec, and 60°C for 30 sec WHO-qPCR was performed with the same program. Cut-off values were set at the threshold cycle (Ct) value of 40. To quantify viral RNA, a standard curve was generated with 10-fold serial dilutions of synthesized standard RNA of the qPCR target sequences.\n\nValidation of the assays using clinical specimens\nA validation study was performed using 224 nasal swab samples collected in Nagasaki prefecture, Japan, without any clinical information. For RT-LAMP and RT-qPCR, RNA was extracted using QIAamp Viral RNA Mini Kit (QIAGEN) according to the instructions of the manufacturer. For Direct RT-LAMP, the samples were heated at 95°C for 10 min for inactivation of SARS-CoV-2. Aliquots of 5 μL of RNA samples or heat inactivated swab samples were used for the LAMP assay with ORF1b-1 primer set and RT-qPCR with NIID primer 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