key: cord-0708192-blax9sz2 authors: Park, Gun-Soo; Ku, Keunbon; Baek, Seung-Hwa; Kim, Seong-Jun; Kim, Seung Il; Kim, Bum-Tae; Maeng, Jin-Soo title: Development of Reverse Transcription Loop-Mediated Isothermal Amplification Assays Targeting SARS-CoV-2 date: 2020-04-07 journal: J Mol Diagn DOI: 10.1016/j.jmoldx.2020.03.006 sha: 59f626cb155c19ab37ae6e06b2911dd26c01c005 doc_id: 708192 cord_uid: blax9sz2 Epidemics of coronavirus disease 2019 (COVID-19) now have >100,000 confirmed cases worldwide. Diagnosis of COVID-19 is currently performed by quantitative RT-PCR methods, but the capacity of quantitative RT-PCR methods is limited by their requirement of high-level facilities and instruments. Herein, reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays to detect genomic RNA of SARS-CoV-2, the causative virus of COVID-19, were developed and evaluated. RT-LAMP assays in this study can detect as low as 100 copies of SARS-CoV-2 RNA. Cross-reactivity of RT-LAMP assays to other human coronaviruses was not observed. A colorimetric detection method was adapted for this RT-LAMP assay so that the tests potentially performed in higher throughput. Targeting SARS-CoV-2 is the causative viral pathogen of coronavirus disease 2019, of which the outbreaks resulted in 857,641 confirmed cases involving 42,006 deaths over 206 countries, areas, or territories as of April 2, 2020 (https://www. who.int/emergencies/diseases/novel-coronavirus-2019). As the name suggests, SARS-CoV-2 is closely related to a group of severe acute respiratory syndromeerelated coronaviruses, namely subgenus Sarbecovirus, showing 96% identity to a bat coronavirus. 1, 2 Diagnosis of coronavirus disease 2019 can be done through computed tomographic scan of suspicious patients, and a confirmatory laboratory test is performed using published quantitative RT-PCR Q11 (RT-qPCR) methods 3e6 and recommendations from The World Health Organization (https://www.who.int/emergencies/diseases/ novel-coronavirus-2019/technical-guidance/laboratoryguidance, last accessed April 2, 2020). Although RT-qPCR methods are used as the gold standard for detection of pathogens because of their high sensitivity and specificity, there are still some caveats. Briefly, laboratory-level facilities, with reliable supply of electricity, expensive instruments, and trained personnel, are required to properly perform RT-qPCR tests. These restrictions hinder use of RT-qPCR methods for various point-of-care situations, where pathogen detection might be required. 7 To overcome such cost restriction of RT-qPCR and still detect pathogens' nucleic acids, isothermal amplification methods have been developed. 8 Among such methods, the loop-mediated isothermal amplification (LAMP) method has some advantages to be applied for point-of-care testing. 9 Well-optimized LAMP assay shows sensitivity comparable to that of PCR, <10 copies per reaction. 10 Intercalating fluorescent dyes are compatible with LAMP reaction so that amplification can be observed in real-time. 11 Because amplification efficiency of LAMP reaction is high, changes 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 in reaction mixture components made it possible to detect the result with colorimetric detection methods. 12e14 Moreover, unpurified sample can be directly used for LAMP. 15e17 This indicates that high-throughput test is possible when use of unpurified specimen is combined with noninstrumental (eg, colorimetric) detection. In this study, one-step reverse transcription LAMP (RT-LAMP) methods to detect SARS-CoV-2 were designed and evaluated. A pair of LAMP primer sets specific to SARS-CoV-2 and accompanying optimized reaction conditions was provided. The leuco crystal violet method was applied to achieve colorimetric detection of LAMP reaction. 13 SARS-CoV-2 viral RNA was prepared, as previously described. 18 In vitro transcribed standard RNA for SARS-CoV-2 was prepared, as previously described. 18 To prepare standard RNA for hCoV-229E, first amplicon of PCR (forward primer, 5 0 -GCTAGTGGATGATCATGCTTTG-3 0 ; reverse primer, 5 0 -TGGGGCCATAAACTGTTCTATTAC-3 0 ) was cloned to pBluescript II KS (þ) plasmid with BamHI and XhoI. Then, in vitro transcription template was prepared by restriction enzyme cut with BglI and XhoI and purification after agarose gel electrophoresis. For hCoV-OC43 and MERS-CoV, amplicons of PCR (OC43 forward primer, 5 0 -AGCAACCAGGCTGATGTCAATACC-3 0 ; OC43 reverse primer, 5 0 -AGCAGACCTTCCTGAGCCTTCAAT-3 0 ; MERS-CoV, UpE region 19 ) were synthesized and cloned to pBIC-A plasmid (Bioneer, Daejeon, Republic of Korea). In vitro transcription template for hCoV-OC43 and MERS-CoV was prepared by restriction enzyme cut with BamHI-XhoI or SspI-XhoI, respectively. In vitro transcriptions were done with EZ T7 High Yield In Vitro Transcription kit (Enzynomics, Daejeon, Republic of Korea), per manufacturer's instructions. RNA products were then purified using Agencourt RNAClean XP (Beckman Coulter, Brea, CA). Standard RNA copy numbers were calculated from concentration measured by NanoDrop Lite (Thermo Scientific, Waltham, MA). All restriction enzymes were purchased from Enzynomics. To evaluate genomic copy number of viral RNAs, dilutions of standard RNAs and viral RNAs in TE buffer (10 mmol/L Tris-Cl, pH 8.0, and 1 mmol/L EDTA) are subjected to one-step RT-qPCR. RT-qPCRs were performed using cDNA of SARS-CoV-2 was made using SuperScript IV Reverse Transcriptase (Invitrogen, Waltham, MA) following manufacturer's instructions with modifications. Briefly, 10 pmol of random hexamer was used as reverse transcription primer, and reaction was performed as follows: 20 minutes at 25 C, 30 minutes at 55 C, and 10 minutes at 80 C. Louis, MO; C0775), 60 mmol/L sodium sulfite (Sigma; S0505), and 5 mmol/L b-cyclodextrin (Sigma; C4767) was directly added to LAMP reaction mixture to 1Â concentration. When using WarmStart Colorimetric LAMP 2X Master Mix (NEB), same concentrations of primers and 0.4 mmol/L SYTO-9 were added. Isothermal incubation and fluorescence signal measurement were performed using LightCycler 96 instrument at 69 C with additional heat inactivation (5 minutes at 95 C) and melting curve analysis steps. Fluorescence signals were measured for every minute during 60 (screening and optimization) or 30 [limit of detection (LoD) and cross-reactivity] minutes of incubation. Any changed conditions are specified for each experiment. 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 AY313906, AY559094, AY502924, AY278491, and AY502927) were aligned by MEGA7 software Q15 . 20 SARS-CoV-2 specific regions for LAMP primer design are manually selected: two regions from Nsp3 (3055 to 3591 and 6172 to 7273), two regions from Spike (21540 to 22549 and 22890 to 23779), and one region from Orf8 (27824 to 28396). Whole Nucleocapsid gene region is also included as Nucleocapsid is usual target of molecular diagnosis because of the abundance of its mRNA. 21 Two to five basic LAMP primer sets are designed and selected with PrimerExplorer V5 (http://primerexplorer. jp/lampv5e/index.html, last accessed February 22, 2020) for each target region. Loop primers are designed by PrimerExplorer V5 or manually selected. Sixteen LAMP primer sets with proper both of loop forward primer and loop backward primer are selected and subjected to further screening. First round of screening was done using WarmStart Colorimetric LAMP 2X Master Mix with cDNA, of which corresponding RNA concentration is 8.3 Â 10 4 copies/ reaction. Of 16 primer sets, nine were selected by threshold time from the results of 40 minutes' incubation at 65 C and subjected to further screening. Second round of screening was done using Bst 3.0. The same amount of cDNA as the first round of screening is used. Two primer sets that showed relatively early nonspecific amplification are discarded, and the remaining seven primer sets are subjected to further screening. Third and fourth rounds of screening were done by checking sensitivity to dilutions of cDNA and RNA, respectively. Five of seven primer sets showed specific amplification for at least one replicate of duplicate, with cDNA concentration corresponding to 1.7 Â 10 1 copies of input RNA (Supplemental Figure S1 ). Next, sensitivity of the five primer sets to RNA dilutions was evaluated in RT-LAMP using Bst 3.0 and SuperScript IV Reverse Transcriptase. Two primer sets, both targeting Nsp3, showed best sensitivity that showed specific amplification at 10 À6 dilution of RNA ( Figure 1 ). Two additional primer sets Q16 , one targeting Spike and the other targeting Nucleocapsid, were added for reaction optimization experiments as they showed fast threshold time for cDNA and to keep ranges of target genes. Primer sequences are represented in Table 1 ½T1 ½T1 . print & web 4C=FPO The Journal of Molecular Diagnosticsjmd.amjpathol. org 3 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 ). Next, whether amplification is improved at lower temperature (65 C) was evaluated, because optimal reaction temperature of SuperScript IV Reverse Transcriptase is 50 C to 55 C and the manufacturer recommends 65 C to 72 C for optimal performance of Bst 3.0. Notably, Nsp3_1- 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 ). The LoD of optimized RT-LAMP assays was assessed through 5 to 1000 RNA copies/reaction in triplicate. Among four primer sets subjected to reaction optimization, S_1-2-2 and N_21 sets showed relatively poor sensitivity. For Nsp3_1-61 and Nsp3_2-24 primer sets, LoD was additionally evaluated with twofold SuperScript IV Reverse Transcriptase (20 U/reaction). LoD and Tt_av were improved by increasing reverse transcriptase amount ( Table 2 ½T2 ½T2 and Supplemental Figure S2 ). As a result, both Nsp3_1-61 and Nsp3_2-24 RT-LAMP assays could detect as low RNA concentration as 100 copies per reaction. The cross-reactivity of two SARS-CoV-2 RT-LAMP assays targeting Nsp3 to other human coronaviruses was not found for hCoV-229E, hCoV-OC43, and MERS-CoV ( Figure 2 ). In this study, LAMP primer sets targeting SARS-CoV-2 were designed and screened. Reaction optimization was also performed for selected primer sets. In summary, a pair of RT-LAMP assays for detection of SARS-CoV-2 with LoD of 100 copies per reaction was designed and evaluated. These RT-LAMP assays showed specificity to SARS-CoV-2 versus alphacoronavirus (hCoV-229E), betacoronavirus (hCoV-OC43), and MERS-CoV. Although specificity to SARS-CoV could not be tested because a proper sample was not in hand, specificity of the RT-LAMP assays is easily expectable from the mismatching bases in primer binding sites (Figure 1) . Especially, both of F1/B1 sites of Nsp3_1-61 primer set are in SARS-CoV-2 specific region, of which aligning SARS-CoV sequence does not exist. About the sensitivity of LAMP assays, note that average threshold time is not well correlated with LoD. In fact, average threshold times of S_1-2-2 and N_21 primer sets for 1000 copies were faster than that of Nsp3_1-61: 7.33 (ratio of positive replicates, 2:3) and 5.06 (ratio of positive replicates, 1:3) minutes, respectively. This disrelation is previously reported. 22 One peculiar observation is that both S_1-2-2 and N_21 showed good sensitivity to cDNA template (Supplemental Figure S1 ), unlike to RNA. Observed difference of sensitivity to RNA and cDNA seems significant even when accounting for slight amplification that might happened during reverse transcription. The reason may include stochastic nature of forming proper The Journal of Molecular Diagnosticsjmd.amjpathol. org 5 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 LAMP amplification intermediate-dumbbell structure, 22 higher stability of DNA/RNA double strand than DNA/ DNA double strand, and more. The LoD of RT-LAMP assays suggested in this study, 100 copies per reaction, may not be enough for sensitive screening of suspicious patients. This relatively high LoD would be from the target sequences used for primer design that are selected by specificity versus SARS-CoV. Indeed, target GC percentage and Tm Q19 of primers had to be adjusted to get enough number of starting sets for primer screening during LAMP primer design to give less-optimal primer sets. Therefore, there might be better target sequences for LAMP assay of SARS-CoV-2 in the viewpoint of sensitivity. However, expected high specificity of RT-LAMP assays suggested in this study would be a good feature for a confirmatory test. In addition, considering high viral load of SARS-CoV-2 at early stage after symptom onset 23 suggested RT-LAMP assays may still be useable for screening tests. In conclusion highly specific RT-LAMP assays for detection of SARS-CoV-2 were developed. The results of these RT-LAMP assays can be detected within 30 minutes after amplification reaction begins. In addition, optimized reaction conditions were provided, to which leuco crystal violet colorimetric detection method is applied that can be used for point-of-care tests. 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Supplemental material for this article can be found at https://doi.org/10.1016/j.jmoldx.2020.03.006.