key: cord-292347-d7xq7x5g authors: Carter, Linda J.; Garner, Linda V.; Smoot, Jeffrey W.; Li, Yingzhu; Zhou, Qiongqiong; Saveson, Catherine J.; Sasso, Janet M.; Gregg, Anne C.; Soares, Divya J.; Beskid, Tiffany R.; Jervey, Susan R.; Liu, Cynthia title: Assay Techniques and Test Development for COVID-19 Diagnosis date: 2020-04-30 journal: ACS Cent Sci DOI: 10.1021/acscentsci.0c00501 sha: doc_id: 292347 cord_uid: d7xq7x5g nan One of the many challenges for containing the spread of 17 COVID-19 is the ability to identify asymptomatic cases that 18 result in spreading of the virus to close contacts. A study of the 19 passengers on a Diamond Princess Cruise ship forced into 20 temporary quarantine from an early outbreak of COVID-19, 21 estimated the asymptomatic proportion (among all infected 22 cases) at 17.9% (95%CrI: 15.5−20.2%). 1 Therefore, the actual 23 number of SARS-CoV-2-infected individuals may be much 24 higher than currently accounted for based on positive test 25 results. 2 Having accurate, convenient, and rapid testing for wide-26 spread deployment can aid in eliminating the silent spread of 27 COVID-19 by asymptomatic viral carriers. Table 1 in section 2.5 130 and Supporting Information Table S1 ). 12 Table 1 in section 2.5 and 139 Supporting Information I. 140 RT-PCR tests are constantly evolving with improved detec-141 tion methods and more automated procedures. For example, the 142 ePlex SARS-CoV-2 test developed by GenMark Diagnostics, 143 Inc. 21 uses "The True Sample-to-Answer Solution" ePlex instru-144 ment to detect SARS-CoV-2 in nasopharyngeal swabs. Each test 145 cartridge contains reagents for magnetic solid-phase extraction The RT-PCR creates a cDNA copy of a specific segment of the viral RNA, which is converted to dsDNA that is exponentially amplified. Although RT-PCR is the most widely used method for 155 detecting SARS-CoV-2 infections, it has the disadvantage of 156 requiring expensive laboratory instrumentation highly skilled 157 laboratory personnel, and can take days to generate results. 158 As a result, a number of companies and laboratories around 159 the globe are working to further improve the efficiency and 160 timeliness of the RT-PCR technologies and develop various 161 other techniques. 162 2.2. Isothermal Nucleic Acid Amplification. RT-PCR 163 requires multiple temperature changes for each cycle, involving 164 sophisticated thermal cycling equipment. 22 Isothermal nucleic 165 acid amplification is an alternative strategy that allows ampli-166 fication at a constant temperature and eliminates the need for a 167 thermal cycler. Therefore, several methods based on this prin-168 ciple have been developed. As shown in Table 1 Step 1: At the 3′-end of the viral RNA, reverse transcriptase and BIP primer initiate conversion of RNA to cDNA. Step 2: At the same end, DNA polymerase and B3 primer continue to generate the second cDNA strand to displace and release the first cDNA strand. Step 3: The FIP primer binds to the released cDNA strand and DNA polymerase generates the complementary strand. Step 4: F3 primer binds to the 3′ end, and DNA polymerase then generates a new strand while displacing the old strand. LAMP cycling produces various sized double-stranded looped DNA structures containing alternately inverted repeats of the target sequence as detected by a DNA indicator dye. Reagents*: Primers and master mix containing reverse transcriptase, DNA polymerase with strand displacing activity, dNTPs, and buffers. 212 platform is distinctive because of its high testing throughput 213 (up to 1000 tests in 24 h) and its capability to simultaneously 214 screen for other common respiratory viruses whose symptoms 215 overlap with COVID-19 using the same patient sample and 216 collection vial. 217 The initial step involves hybridization of the viral RNA 218 target to a specific capture probe and an additional oligonu-219 cleotide containing a T7 promoter primer, which are captured 220 onto magnetic microparticles by application of a magnetic 221 field. Then, the captured RNA target hybridized to the T7 pro-222 moter primer is reverse transcribed into a complementary 223 cDNA. The RNase H activity of the reverse transcriptase sub-224 sequently degrades the target RNA strand from the hybrid 225 RNA−cDNA duplex, leaving a single-stranded cDNA, which 226 includes the T7 promoter. An additional primer is used to gen-227 erate a double-stranded DNA, which is subsequently tran-228 scribed into RNA amplicons by T7 RNA polymerase. These 229 new RNA amplicons then reenter the TMA process allowing 230 this exponential amplification process to generate billions of 231 RNA amplicons in less than 1 h. The detection process 232 involves the use of single-stranded nucleic acid torches that 233 hybridize specifically to the RNA amplicon in real time. Each 234 torch is conjugated to a fluorophore and a quencher. When the 235 torch hybridizes to the RNA amplicon, the fluorophore is able 236 to emit a signal upon excitation. 237 2.2.3. CRISPR-Based Assays. Clustered Regularly Interspaced 238 Short Palindromic Repeats (CRISPR) represents a family of 239 nucleic acid sequences found in prokaryotic organisms, such as 240 bacteria. These sequences can be recognized and cut by a set of 241 bacterial enzymes, called CRISPR-associated enzymes, exem-242 plified by Cas9, Cas12, and Cas13. Certain enzymes in the 243 Cas12 and Cas13 families can be programmed to target and 244 cut viral RNA sequences. 29 245 Two companies, Mammoth Biosciences and Sherlock 246 Biosciences, established by the CRISPR pioneer scientists, 247 are independently exploring the possibility of using the gene-248 editing CRISPR methodology for detection of SARS-CoV-2. 249 The SHERLOCK method developed by Sherlock Biosciences 250 uses Cas13 that is capable of excising reporter RNA sequences 251 in response to activation by SARS-CoV-2-specific guide RNA. 30 252 The DETECTR assay by Mammoth Biosciences relies on the 253 cleavage of reporter RNA by Cas12a to specifically detect 254 viral RNA sequences of the E and N genes, followed by iso-255 thermal amplification of the target, resulting in a visual readout 256 with a fluorophore. 31 These CRISPR-based methods, as depicted 257 in Figure 3 , do not require complex instrumentation and 258 can be read using paper strips to detect the presence of the 259 SARS-CoV-2 virus without loss of sensitivity or specificity. 311 of SARS-CoV-2 allows for potential contact tracing, molecular 312 epidemiology, and studies of viral evolution. Metagenomics 313 approaches such as sequence-independent single primer 314 amplification (SISPA) provide additional checks on sequence 315 divergence. This dual technique is particularly relevant to 316 SARS-CoV-2 in assessment of its rate of mutation and to detect 317 its possible recombination with other human coronaviruses, 318 both of which have implications for vaccine development and 319 antiviral efficacy. 320 Amplicon and metagenomics MinION based sequencing 321 were used by Moore et al. (2020) to rapidly (within 8 h) 322 sequence the genome of SARS-CoV-2 and the other micro-323 biome in nasopharyngeal swabs obtained from patients with 324 COVID-19 by the ISARIC 4C consortium. 39 For the amplicon-325 based system, the group chose 16 primer binding sites from con-326 served regions in the SARS-CoV-2 genome to sequentially 327 amplify roughly 1000 bp fragments with an approximately 200 bp 328 overlapping region. These primer sets were then used to gen-329 erate 30 amplicons from the cDNA, which were subsequently 330 sequenced using MinION. 331 A next-generation shotgun metagenomics sequencing plat-332 form has been developed by Illumina with the ability not only 333 to detect the presence of multiple strains of coronaviruses but 334 also to comprehensively examine multiple pathogenic organi-335 sms present in a complex sample. The Illumina metagenomics 336 workflow involves sample preparation using their TruSeq Ribo-337 Zero Gold rRNA depletion kit, library preparation using TruSeq 338 stranded total RNA, sequencing using the Illumina benchtop 339 375 While RT-PCR-based viral RNA detection has been widely 376 used in diagnosis of COVID-19, it cannot be used to monitor 377 the progress of the disease stages and cannot be applied to 378 broad identification of past infection and immunity. 379 Serological testing is defined as an analysis of blood serum or 380 plasma and has been operationally expanded to include testing 381 of saliva, sputum, and other biological fluids for the presence 382 of immunoglobulin M (IgM) and immunoglobulin G (IgG) 383 antibodies. This test plays an important role in epidemiology and 384 vaccine development, providing an assessment of both short-term 385 (days to weeks) and long-term (years or permanence) trajectories 386 of antibody response, as well as antibody abundance and 387 diversity. IgM first becomes detectable in serum after a few days 388 and lasts a couple of weeks upon infection and is followed by a 389 switch to IgG. Thus, IgM can be an indicator of early stage infec-390 tion, and IgG can be an indicator of current or prior infection. 391 IgG may also be used to suggest the presence of post-infection 392 immunity. In recent years, the sophistication and sensitivity of 393 immunological assays have increased not only for the detection of 394 antibodies themselves but also for the application of antibodies 395 (primarily monoclonal antibodies) to the detection of pathogen-396 derived antigens. These tests have a huge potential for the epide-397 miology of COVID-19, 32,42−45 but test results can be impacted by 398 at least three situations: (1) a subset of subjects with a positive 399 result from molecular genetic assays for SARS-CoV-2 infection 400 are seronegative due to the lag in antibody production following 401 infection, (2) the subjects may be seropositive yet negative for 402 molecular genetic assay results reflecting clearance of an earlier, 403 milder infection, and (3) limitation in sensitivity and specificity of 404 the assays. The last issue is particularly important because even a 405 small percentage of false positive results due to low specificity 406 (cross reaction) may lead to misleading predictive antibody 407 prevalence among a given population, which may have unde-408 sirable impact on the socioeconomic decisions and overall 409 public confidence in the results. 46,47 410 The determination of SARS-CoV-2 exposure relies largely 411 on the detection of either IgM or IgG antibodies that are 412 specific for various viral antigens including, but not exclusively, 413 the spike glycoprotein (S1 and S2 subunits, receptor-binding 414 domain) and nucleocapsid protein. The methodology for these 415 determinations includes the traditional enzyme-linked immu-416 nosorbent assay (ELISA), immunochromatographic lateral flow 417 assay, neutralization bioassay, and specific chemosensors. Each 418 of these formats brings advantages (speed, multiplexing, 419 automation) and disadvantages (trained personnel, dedicated 420 laboratories). Complementary to these antibody-detecting 421 methods are the rapid antigen tests wherein antibodies are 422 used to detect the presence of viral antigen(s) in serological 423 samples. Development of high-throughput serology tests is a 424 current focus of major diagnostic companies. 45 The FDA 425 granted EUA status to the first serology test, qSARS-CoV-2 426 IgG/IgM Rapid Test, manufactured by Cellex Inc., on April 1, 427 2020, 48 but continues to allow clinical laboratories and com-428 mercial manufacturers to launch serology tests without an 429 EUA. 431 ELISA is a microwell, plate-based assay technique designed for 432 detecting and quantifying substances such as peptides, pro-433 teins, antibodies, and hormones. The test can be qualitative or 434 quantitative, and the time to results is typically 1−5 h. In the 435 case of SARS-CoV-2 as shown in Figure 5A , the plate wells are Neutralization assays deter-460 mine the ability of an antibody to inhibit virus infection of cul-461 tured cells and the resulting cytopathic effects of viral replica-462 tion. For this assay, patient samples of whole blood, serum, or 463 plasma are diluted and added at decreasing concentrations to 464 the cell cultures. If neutralizing antibodies are present, their 465 levels can be measured by determining the threshold at which 466 they are able to prevent viral replication in the infected cell 467 cultures. The time to results for neutralization assays is 468 typically 3−5 days, but recent advances have reduced this to 469 hours. 49,50 This type of testing requires cell culture facilities, 470 and in the case of SARS coronavirus, Biosafety Level 3 (BSL3) 471 laboratories. Despite these limitations, determination of neu-472 tralizing antibodies is important in the short term for the 473 therapeutic application of convalescent plasma and, in the long 474 term, for vaccine development. Biosensor tests rely on converting the 486 specific interaction of biomolecules into a measurable readout 487 via optical, electrical, enzymatic, and other methods. Surface 488 plasmon resonance (SPR) is a technique that measures inter-489 ference with incident light at a solid boundary due to local 490 disturbances such as the adsorption of antibody or antigen. 491 An SPR-based biosensor was developed for the diagnosis of 492 SARS using coronaviral surface antigen (SCVme) anchored 493 onto a gold substrate. 53 The SPR chip had a lower limit of detec-494 tion of 200 ng/mL for anti-SCVme antibodies within 10 min. 495 Most recently, PathSensors Inc. announced a CANARY bio-496 sensor to detect the novel SARS coronavirus. This platform 497 utilizes a cell-based immunosensor that couples capture of the 498 virus with signal amplification to provide a result in 3−5 min. 499 The biosensor is slated to be available for research purposes in 500 May 2020. 54 501 3.6. Rapid Antigen Test. Complementary to molecular 502 genetic assays are the rapid antigen tests that allow detection of 503 viral antigens. 55, 56 As shown in Figure 5B , these tests rely on 504 specific monoclonal antibodies to provide a mechanism for the 505 capture of viral antigens from an analytical sample. These assays 506 are not restricted to a particular format. Examples include a 507 colorimetric enzyme immunoassay for SARS-CoV in 2004, 57 an 508 enhanced chemiluminescent immunoassay for SARS-CoV in 509 2005, 58 and more recently a fluorescence lateral flow assay 59 for 510 the detection of SARS-CoV-2 nucleocapsid protein. Table 2 513 provides a collection of current available serological and immuno-514 logical assays for COVID-19 diagnosis, and a complete list of simi-515 lar assays is provided in Supporting Information Table S2. 564 While the past few months have witnessed rapid progress in 565 diagnostic kit development for COVID-19, the race continues 566 to develop even more efficient laboratory techniques and cost-567 effective, point-of-care test kits that can be deployed in mass 568 quantities. This report provides a broad survey of molecular 569 genetic assays, and serological and immunological tests for 570 identification of COVID-19 infection. While RT-PCR has been 571 the dominant technique for detection of viral RNA, other 572 nucleic acid assays including isothermal amplification assays, 573 hybridization microarray assays, amplicon-based metagenomics 574 sequencing, and the cutting-edge CRISPR-related technologies 575 are also under development or have resulted in approved 576 tests. 60 The efficiency of such testing has also been significantly 577 improved. This report also provides trend analysis of journal 578 articles related to COVID-19 diagnostic techniques. The 579 number of FDA EUA-approved tests available for COVID-19 580 diagnosis keeps growing, but the many new tests are still in 581 various stages of development. Ultrarapid test kits and point-582 of-care tests are a major focus of development in order to speed 583 up the response time for treatment and eliminate the need for 584 elaborate laboratory equipment and waiting time involved with 585 testing in approved laboratories. 586 The urgent need for accurate and rapid diagnosis of SARS-t4 587 CoV-2 infection remains critical as global healthcare systems 588 continue to operate during the course of the COVID-19 pan-589 demic. In particular, serological and immunological testing of 590 infected asymptomatic and symptomatic individuals, and their 591 close contacts, is expected to be in high demand. In addition to 592 its role complementary to molecular genetic testing to confirm 593 suspected cases, this type of testing would provide valuable 594 information about the course and degree of immune response 595 as well as the durability of immunity in both infected indi-596 viduals and participants in vaccine clinical trials. The results 597 from these tests may assist epidemiological assessment and can 598 be used to manage the return to normal activities. However, 599 many questions regarding serological tests remain to be addressed, 600 including their degree of sensitivity and specificity. Finally, it 601 remains to be confirmed that the presence of antibodies against 602 SARS-CoV-2 indeed correlates with immunity to the virus. 603 In summary, significant progress has been made in the devel- Albaiu for their encouragement and support 663 for this work, especially Dana's effort in critical review Estimating 669 the asymptomatic proportion of coronavirus disease 2019 (COVID-670 19) cases on board the Diamond Princess cruise ship The Importance of diagnostic testing for COVID-19 Infectious Diseases Hub Evaluation of 684 coronavirus in tears and conjunctival secretions of patients with 685 SARS-CoV-2 infection American College of Physicians. COVID-19 found in sputum 687 and feces samples after pharyngeal specimens no longer positive Clinical and 691 virologic characteristics of the first 12 patients with coronavirus 692 disease 2019 (COVID-19) in the United States Rutgers University; New Rutgers Saliva Test for Coronavirus 695 Gets FDA Approval: Emergency Use Authorization Granted for New 696 /news/new-rutgers-saliva-test-coronavirus-gets-fda Accelerated emergency use 700 authorization (EUA) summary SARS-CoV-2 ASSAY Real-time PCR for mRNA 707 quantitation Diagnostic Tests. 360Dx coronavirus-test-tracker-launched-covid-19-tests COVID-19 test kits included on the ARTG for legal Australian Government, Department of Health covid-19-test-kits-included-artg-legal-supply-australia Emergency Use Authorization: Emergency Use Authorization 716 EUA) information, and list of all current EUAs mcm-legal-regulatory-and-policy-framework/emergency-use-719 Find Evaluation Update: SARS-CoV-2 Molecular Diagnostics COVID-10 Diagnostics Resource Centre; Foundation for Innovative 722 New Diagnostics (FIND) LabCorp launches test for coronavirus disease 2019 Laboratory Corporation of America, News release Emergency use authorization for the Real-Time RT-PCR Diagnostic Panel and Prevention). U.S. Food & Drug Administration TaqPath COVID-19 Multiplex Diagnostic Solution AllplexTM 2019-nCoV Assay cobas SARS-CoV-2 Test (for the COVID-19 Coronavirus) GenMark Responds to the SARS-CoV-2 Outbreak Loop-mediated isothermal 747 amplification of DNA An Update on Abbott's Work on COVID-19 Testing Rapid molecular detection of SARS-CoV-2 COVID-19) virus RNA using colorimeteric LAMP Kits for nucleic acid sequence 764 amplification methods Hologic's Molecular Test for the Novel Coronavirus CoV-2, Receives FDA Emergency Use Authorization. Coronavirus 770 Update What is CRISPR? Ask the Brain Massachusetts Institute of 773 Technology A protocol for 775 detection of COVID-19 using CRISPR diagnostics CRISPR−Cas12-based detection of 779 SARS-CoV-2 Serology-based tests for COVID-19 Questions: How COVID-19 tests work and why 786 they're in short supply Massachusetts Institute of Technology. news.mit.edu/ 788 2020/how-covid-19-tests-work-why-they-are-in-short-supply-0410 Detection of severe acute respiratory syndrome 792 coronavirus RNA in plasma during the course of infection Comprehensive detection and identification of seven animal 796 coronaviruses and human respiratory coronavirus 229E with a 797 microarray hybridization assay Development of a 799 single nucleotide polymorphism DNA microarray for the detection 800 and genotyping of the SARS coronavirus Generic detection of coronaviruses and 805 differentiation at the prototype strain level by reverse-transcription-806 PCR and nonfluorescent low-density microarray Initial performance evaluation 810 of a spotted array Mobile Analysis Platform (MAP) for the detection 811 of influenza A/B, RSV, and MERS coronavirus Amplicon based MinION sequencing of 820 SARS-CoV-2 and metagenomics characterisation of nasopharyngeal 821 swabs from patients with Comprehensive Workflow for Detecting Coronavirus Using Illumina Benchtop Systems − A Shotgun Metagenomics Sequencing 825 Workflow for Effective Detection and Characterization of Coronavirus 826 Strains. Infectious Disease Surveillance Diagnosing COVID-19: The Disease and Tools for 839 Detection SARS-CoV-2 Viral Load in Upper Respiratory 843 Specimens of Infected Patients Serology testing for COVID-19 COVID-19/COVID-19-fact-sheets/200228-Serology-testing-COVID FDA Fact Sheet: Serological testing for antibodies to SARS-850 CoV-2 infection. U.S. Food & Drug Administration Screening tests: a review 853 with examples Emergency Use Authorization for qSARS IgG/IgM Rapid Test Scalable, semi-automated fluorescence reduction neutralization assay 861 samples Diagnosis of Corona Virus Disease Announced the Development of a SARS-886 Analysis of serologic cross-reactivity between common human 889 -2 using coronavirus antigen microarray Sensitive and specific monoclonal antibody-based capture enzyme 897 Diagnosis of Acute Respiratory Syndrome Coronavirus 2 903 Infection by Detection of Nucleocapsid Protein Chemiluminescent immunometric detection 906 of SARS-CoV in sera as an early marker for the diagnosis of SARS Standard coronavirus test, if available, works 910 well�but can new diagnostics help in this pandemic? Science