PMC:7668588 / 23053-28622 JSONTXT

{"project":"LitCovid-PubTator","denotations":[{"id":"275","span":{"begin":1105,"end":1109},"obj":"Gene"},{"id":"276","span":{"begin":553,"end":563},"obj":"Species"},{"id":"277","span":{"begin":1380,"end":1391},"obj":"Species"},{"id":"278","span":{"begin":1601,"end":1611},"obj":"Species"},{"id":"279","span":{"begin":1786,"end":1796},"obj":"Species"},{"id":"280","span":{"begin":1393,"end":1397},"obj":"Species"},{"id":"281","span":{"begin":1522,"end":1526},"obj":"Species"},{"id":"282","span":{"begin":82,"end":103},"obj":"Disease"},{"id":"283","span":{"begin":300,"end":308},"obj":"Disease"},{"id":"294","span":{"begin":1971,"end":1981},"obj":"Species"},{"id":"295","span":{"begin":2636,"end":2644},"obj":"Species"},{"id":"296","span":{"begin":2754,"end":2762},"obj":"Species"},{"id":"297","span":{"begin":2912,"end":2920},"obj":"Species"},{"id":"298","span":{"begin":3034,"end":3045},"obj":"Species"},{"id":"299","span":{"begin":2012,"end":2032},"obj":"Disease"},{"id":"300","span":{"begin":2102,"end":2134},"obj":"Disease"},{"id":"301","span":{"begin":2136,"end":2140},"obj":"Disease"},{"id":"302","span":{"begin":2367,"end":2387},"obj":"Disease"},{"id":"303","span":{"begin":2809,"end":2813},"obj":"Disease"},{"id":"318","span":{"begin":3178,"end":3186},"obj":"Species"},{"id":"319","span":{"begin":3407,"end":3415},"obj":"Species"},{"id":"320","span":{"begin":3567,"end":3581},"obj":"Species"},{"id":"321","span":{"begin":3783,"end":3793},"obj":"Species"},{"id":"322","span":{"begin":3898,"end":3908},"obj":"Species"},{"id":"323","span":{"begin":3925,"end":3933},"obj":"Species"},{"id":"324","span":{"begin":4250,"end":4260},"obj":"Species"},{"id":"325","span":{"begin":4584,"end":4589},"obj":"Species"},{"id":"326","span":{"begin":4756,"end":4760},"obj":"Chemical"},{"id":"327","span":{"begin":4832,"end":4836},"obj":"Chemical"},{"id":"328","span":{"begin":4967,"end":4970},"obj":"Chemical"},{"id":"329","span":{"begin":3169,"end":3177},"obj":"Disease"},{"id":"330","span":{"begin":3543,"end":3563},"obj":"Disease"},{"id":"331","span":{"begin":4509,"end":4517},"obj":"Disease"},{"id":"335","span":{"begin":5251,"end":5259},"obj":"Disease"},{"id":"336","span":{"begin":5454,"end":5462},"obj":"Disease"},{"id":"337","span":{"begin":5552,"end":5568},"obj":"Disease"}],"attributes":[{"id":"A275","pred":"tao:has_database_id","subj":"275","obj":"Gene:27074"},{"id":"A276","pred":"tao:has_database_id","subj":"276","obj":"Tax:2697049"},{"id":"A277","pred":"tao:has_database_id","subj":"277","obj":"Tax:11620"},{"id":"A278","pred":"tao:has_database_id","subj":"278","obj":"Tax:64320"},{"id":"A279","pred":"tao:has_database_id","subj":"279","obj":"Tax:2697049"},{"id":"A280","pred":"tao:has_database_id","subj":"280","obj":"Tax:11620"},{"id":"A281","pred":"tao:has_database_id","subj":"281","obj":"Tax:11620"},{"id":"A282","pred":"tao:has_database_id","subj":"282","obj":"MESH:C000657245"},{"id":"A283","pred":"tao:has_database_id","subj":"283","obj":"MESH:C000657245"},{"id":"A294","pred":"tao:has_database_id","subj":"294","obj":"Tax:2697049"},{"id":"A295","pred":"tao:has_database_id","subj":"295","obj":"Tax:694009"},{"id":"A296","pred":"tao:has_database_id","subj":"296","obj":"Tax:1335626"},{"id":"A297","pred":"tao:has_database_id","subj":"297","obj":"Tax:1335626"},{"id":"A298","pred":"tao:has_database_id","subj":"298","obj":"Tax:11118"},{"id":"A299","pred":"tao:has_database_id","subj":"299","obj":"MESH:D018352"},{"id":"A300","pred":"tao:has_database_id","subj":"300","obj":"MESH:D018352"},{"id":"A301","pred":"tao:has_database_id","subj":"301","obj":"MESH:D018352"},{"id":"A302","pred":"tao:has_database_id","subj":"302","obj":"MESH:D018352"},{"id":"A303","pred":"tao:has_database_id","subj":"303","obj":"MESH:D018352"},{"id":"A318","pred":"tao:has_database_id","subj":"318","obj":"Tax:9606"},{"id":"A319","pred":"tao:has_database_id","subj":"319","obj":"Tax:9606"},{"id":"A320","pred":"tao:has_database_id","subj":"320","obj":"Tax:9544"},{"id":"A321","pred":"tao:has_database_id","subj":"321","obj":"Tax:2697049"},{"id":"A322","pred":"tao:has_database_id","subj":"322","obj":"Tax:2697049"},{"id":"A323","pred":"tao:has_database_id","subj":"323","obj":"Tax:9606"},{"id":"A324","pred":"tao:has_database_id","subj":"324","obj":"Tax:2697049"},{"id":"A325","pred":"tao:has_database_id","subj":"325","obj":"Tax:9606"},{"id":"A329","pred":"tao:has_database_id","subj":"329","obj":"MESH:C000657245"},{"id":"A330","pred":"tao:has_database_id","subj":"330","obj":"MESH:C000657245"},{"id":"A331","pred":"tao:has_database_id","subj":"331","obj":"MESH:C000657245"},{"id":"A335","pred":"tao:has_database_id","subj":"335","obj":"MESH:C000657245"},{"id":"A336","pred":"tao:has_database_id","subj":"336","obj":"MESH:C000657245"},{"id":"A337","pred":"tao:has_database_id","subj":"337","obj":"MESH:D001102"}],"namespaces":[{"prefix":"Tax","uri":"https://www.ncbi.nlm.nih.gov/taxonomy/"},{"prefix":"MESH","uri":"https://id.nlm.nih.gov/mesh/"},{"prefix":"Gene","uri":"https://www.ncbi.nlm.nih.gov/gene/"},{"pr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this study, we developed a rapid and sensitive diagnostic assay for SARS-CoV-2 infections, which is based on RT-LAMP. The assay could detect 23.7 copies of viral RNA within 15 min from clinical specimens (Fig 1A, Table 6, and S1 Table). Currently, RT-qPCR is the gold standard assay of COVID-19 diagnosis and takes at least 2–3 hours to complete all processes including RNA extraction followed by a quantitative PCR step using a real-time thermal cycler. To shorten the assay time, several research groups developed RT-LAMP assays against SARS-CoV-2 [16–21]; however, the detection sensitivity and assay time can be improved. Huang et al. reported a novel RT-LAMP assay that could detect 2 copies of viral RNA after an additional gel electrophoresis step, requiring more assay time than the normal RT-LAMP method [21]. In addition to RT-LAMP, we also developed a remarkable rapid assay, Direct RT-LAMP, that does not require the extraction of viral RNA from clinical specimens. This assay could detect 203 copies of viral RNA in the virus-spiked swab solution within 10.5 min (Table 5). The LAMP method has an excellent specificity accomplished by 6 to 8 different primer binding regions in the target DNA sequence. Nevertheless, several viruses require multiple sets of primers to detect whole lineages/genotypes due to genetic diversity of their genome sequences. Lassa virus (LASV) has 6 lineages and has highly diverse genome even within the same lineage. There is a single report of a RT-LAMP assay for LASV, and the assay requires 3 sets of primers to detect only lineage II [22]. Zika virus also shows diversity in genome sequences between Asian and African genotypes, requiring the mixture of 2 primer sets to detect all strains in one reaction [13]. Fortunately, SARS-CoV-2 shows lower genetic diversity worldwide, and therefore would be suitable to be diagnosed using the simple RT-LAMP assay with one primer set (S1 Fig).\nBefore the emergence of SARS-CoV-2, two large outbreaks of novel coronavirus diseases were reported: severe acute respiratory syndrome (SARS) in 2002, and Middle East respiratory syndrome (MERS) in 2012 [23,24]. For the molecular diagnosis for these diseases, RT-PCR and RT-qPCR were used as gold standard assays with an immediate announcement of recommended protocols by WHO. Several years since the emergence of these coronavirus diseases, RT-LAMP assays were developed for faster diagnosis [25,26]. For SARS, Thai et al. developed a RT-LAMP assay which is 100-fold more sensitive than RT-PCR [25] and showed clear linearity between viral titer and detection time, and that could detect SARS-CoV even in RT-PCR negative samples. Similar to SARS, several RT-LAMP assays were developed for the detection of MERS-CoV. Shirato et al. developed a RT-LAMP assay for MERS with a sensitivity comparable to standard RT-qPCR, and with sufficient specificity to distinguish MERS-CoV from 20 other respiratory viruses [26]. These past reports indicate advantages in using RT-LAMP assays to detect coronavirus from clinical specimens with sufficient sensitivity.\nA recent clinical study that evaluated respiratory tract specimens of COVID-19 patients with severe and mild symptoms showed the initial viral load of 6.17 and 5.11 log10 copies per mL, respectively [27]. Posterior oropharyngeal saliva, which might represent a non-invasive reasonable specimen acceptable by patients, contained 4 to 8 log10 copies of viral genome per mL in the first 5 days from the symptom onset [27]. Similarly, experimental SARS-CoV-2 infection of rhesus monkeys showed that the initial viral load in nasal or throat swabs also contained 4 to 8 log10 copies of viral genome per mL within the first 5 days [28]. Again, our RT-LAMP assay could detect 23.7 copies of SARS-CoV-2 viral RNA, approximately equivalent to 1,778 copies per mL in clinical specimens, effectively detecting SARS-CoV-2 in samples from patients with both severe and mild symptoms during the acute phase. Whereas, our Direct RT-LAMP is approximately 60-fold less sensitive than RT-LAMP in the assay for clinical specimens, indicating the reliable detection limit of approximately 5 log10 copies per mL (Table 6). Thus, although this Direct RT-LAMP assay detects SARS-CoV-2 in high viral titer samples, it should not be used as a sole molecular diagnostic method but used with another assay such as qPCR to avoid false negatives. Nevertheless, Direct RT-LAMP would significantly contribute to the rapid first screening of COVID-19 during an outbreak (1) in the high-risk population (e.g. pregnant women, individuals with a pre-existing illness) who needs an immediate care, and (2) in the resource-limited settings where a centrifuge is unavailable. We also found that HBSS affected the detection time of Direct RT-LAMP (Fig 2), indicating that HBSS contains inhibitors of RT-LAMP amplification. Therefore, in the Direct RT-LAMP assay, it is preferable to suspend swab samples in PBS.\nSince the RT-LAMP assay can be performed with a portable battery-driven device, it may be available at point-of-care, even in poorly-equipped settings. Taken together, our results suggest that this new RT-LAMP protocol can be useful as a rapid and sensitive diagnostic assay for COVID-19, and that the Direct RT-LAMP version could also be made available as a much simpler, faster, and low-cost assay. These new assays may contribute to public health control in countries undergoing COVID-19 epidemics, not only for early disease detection, but also to monitor future expansion of viral infections."}

{"project":"LitCovid-sentences","denotations":[{"id":"T262","span":{"begin":0,"end":10},"obj":"Sentence"},{"id":"T263","span":{"begin":11,"end":131},"obj":"Sentence"},{"id":"T264","span":{"begin":132,"end":250},"obj":"Sentence"},{"id":"T265","span":{"begin":251,"end":468},"obj":"Sentence"},{"id":"T266","span":{"begin":469,"end":639},"obj":"Sentence"},{"id":"T267","span":{"begin":640,"end":832},"obj":"Sentence"},{"id":"T268","span":{"begin":833,"end":991},"obj":"Sentence"},{"id":"T269","span":{"begin":992,"end":1100},"obj":"Sentence"},{"id":"T270","span":{"begin":1101,"end":1229},"obj":"Sentence"},{"id":"T271","span":{"begin":1230,"end":1379},"obj":"Sentence"},{"id":"T272","span":{"begin":1380,"end":1473},"obj":"Sentence"},{"id":"T273","span":{"begin":1474,"end":1600},"obj":"Sentence"},{"id":"T274","span":{"begin":1601,"end":1772},"obj":"Sentence"},{"id":"T275","span":{"begin":1773,"end":1946},"obj":"Sentence"},{"id":"T276","span":{"begin":1947,"end":2158},"obj":"Sentence"},{"id":"T277","span":{"begin":2159,"end":2323},"obj":"Sentence"},{"id":"T278","span":{"begin":2324,"end":2448},"obj":"Sentence"},{"id":"T279","span":{"begin":2449,"end":2677},"obj":"Sentence"},{"id":"T280","span":{"begin":2678,"end":2763},"obj":"Sentence"},{"id":"T281","span":{"begin":2764,"end":2960},"obj":"Sentence"},{"id":"T282","span":{"begin":2961,"end":3098},"obj":"Sentence"},{"id":"T283","span":{"begin":3099,"end":3303},"obj":"Sentence"},{"id":"T284","span":{"begin":3304,"end":3518},"obj":"Sentence"},{"id":"T285","span":{"begin":3519,"end":3729},"obj":"Sentence"},{"id":"T286","span":{"begin":3730,"end":3992},"obj":"Sentence"},{"id":"T287","span":{"begin":3993,"end":4200},"obj":"Sentence"},{"id":"T288","span":{"begin":4201,"end":4416},"obj":"Sentence"},{"id":"T289","span":{"begin":4417,"end":4736},"obj":"Sentence"},{"id":"T290","span":{"begin":4737,"end":4882},"obj":"Sentence"},{"id":"T291","span":{"begin":4883,"end":4971},"obj":"Sentence"},{"id":"T292","span":{"begin":4972,"end":5123},"obj":"Sentence"},{"id":"T293","span":{"begin":5124,"end":5372},"obj":"Sentence"},{"id":"T294","span":{"begin":5373,"end":5569},"obj":"Sentence"}],"namespaces":[{"prefix":"_base","uri":"http://pubannotation.org/ontology/tao.owl#"}],"text":"Discussion\nIn this study, we developed a rapid and sensitive diagnostic assay for SARS-CoV-2 infections, which is based on RT-LAMP. The assay could detect 23.7 copies of viral RNA within 15 min from clinical specimens (Fig 1A, Table 6, and S1 Table). Currently, RT-qPCR is the gold standard assay of COVID-19 diagnosis and takes at least 2–3 hours to complete all processes including RNA extraction followed by a quantitative PCR step using a real-time thermal cycler. To shorten the assay time, several research groups developed RT-LAMP assays against SARS-CoV-2 [16–21]; however, the detection sensitivity and assay time can be improved. Huang et al. reported a novel RT-LAMP assay that could detect 2 copies of viral RNA after an additional gel electrophoresis step, requiring more assay time than the normal RT-LAMP method [21]. In addition to RT-LAMP, we also developed a remarkable rapid assay, Direct RT-LAMP, that does not require the extraction of viral RNA from clinical specimens. This assay could detect 203 copies of viral RNA in the virus-spiked swab solution within 10.5 min (Table 5). The LAMP method has an excellent specificity accomplished by 6 to 8 different primer binding regions in the target DNA sequence. Nevertheless, several viruses require multiple sets of primers to detect whole lineages/genotypes due to genetic diversity of their genome sequences. Lassa virus (LASV) has 6 lineages and has highly diverse genome even within the same lineage. There is a single report of a RT-LAMP assay for LASV, and the assay requires 3 sets of primers to detect only lineage II [22]. Zika virus also shows diversity in genome sequences between Asian and African genotypes, requiring the mixture of 2 primer sets to detect all strains in one reaction [13]. Fortunately, SARS-CoV-2 shows lower genetic diversity worldwide, and therefore would be suitable to be diagnosed using the simple RT-LAMP assay with one primer set (S1 Fig).\nBefore the emergence of SARS-CoV-2, two large outbreaks of novel coronavirus diseases were reported: severe acute respiratory syndrome (SARS) in 2002, and Middle East respiratory syndrome (MERS) in 2012 [23,24]. For the molecular diagnosis for these diseases, RT-PCR and RT-qPCR were used as gold standard assays with an immediate announcement of recommended protocols by WHO. Several years since the emergence of these coronavirus diseases, RT-LAMP assays were developed for faster diagnosis [25,26]. For SARS, Thai et al. developed a RT-LAMP assay which is 100-fold more sensitive than RT-PCR [25] and showed clear linearity between viral titer and detection time, and that could detect SARS-CoV even in RT-PCR negative samples. Similar to SARS, several RT-LAMP assays were developed for the detection of MERS-CoV. Shirato et al. developed a RT-LAMP assay for MERS with a sensitivity comparable to standard RT-qPCR, and with sufficient specificity to distinguish MERS-CoV from 20 other respiratory viruses [26]. These past reports indicate advantages in using RT-LAMP assays to detect coronavirus from clinical specimens with sufficient sensitivity.\nA recent clinical study that evaluated respiratory tract specimens of COVID-19 patients with severe and mild symptoms showed the initial viral load of 6.17 and 5.11 log10 copies per mL, respectively [27]. Posterior oropharyngeal saliva, which might represent a non-invasive reasonable specimen acceptable by patients, contained 4 to 8 log10 copies of viral genome per mL in the first 5 days from the symptom onset [27]. Similarly, experimental SARS-CoV-2 infection of rhesus monkeys showed that the initial viral load in nasal or throat swabs also contained 4 to 8 log10 copies of viral genome per mL within the first 5 days [28]. Again, our RT-LAMP assay could detect 23.7 copies of SARS-CoV-2 viral RNA, approximately equivalent to 1,778 copies per mL in clinical specimens, effectively detecting SARS-CoV-2 in samples from patients with both severe and mild symptoms during the acute phase. Whereas, our Direct RT-LAMP is approximately 60-fold less sensitive than RT-LAMP in the assay for clinical specimens, indicating the reliable detection limit of approximately 5 log10 copies per mL (Table 6). Thus, although this Direct RT-LAMP assay detects SARS-CoV-2 in high viral titer samples, it should not be used as a sole molecular diagnostic method but used with another assay such as qPCR to avoid false negatives. Nevertheless, Direct RT-LAMP would significantly contribute to the rapid first screening of COVID-19 during an outbreak (1) in the high-risk population (e.g. pregnant women, individuals with a pre-existing illness) who needs an immediate care, and (2) in the resource-limited settings where a centrifuge is unavailable. We also found that HBSS affected the detection time of Direct RT-LAMP (Fig 2), indicating that HBSS contains inhibitors of RT-LAMP amplification. Therefore, in the Direct RT-LAMP assay, it is preferable to suspend swab samples in PBS.\nSince the RT-LAMP assay can be performed with a portable battery-driven device, it may be available at point-of-care, even in poorly-equipped settings. Taken together, our results suggest that this new RT-LAMP protocol can be useful as a rapid and sensitive diagnostic assay for COVID-19, and that the Direct RT-LAMP version could also be made available as a much simpler, faster, and low-cost assay. These new assays may contribute to public health control in countries undergoing COVID-19 epidemics, not only for early disease detection, but also to monitor future expansion of viral infections."}