Dry loop–mediated isothermal amplification assay for detection of SARS-CoV-2 from clinical specimens

TLDR: The dry LAMP method for detection of SARS-CoV-2 RNA was fast and easy to use, solves the cold chain problem, and therefore represents a promising tool for diagnosis of COVID-19 in developing countries.

Abstract: Coronavirus disease 2019 (COVID-19) has had a major disease burden on many countries around the world. The spread of COVID-19 is anticipated to have a major impact on developing countries including African nations. To establish a point-of-care test for COVID-19, we developed a dry loop mediated isothermal amplification (LAMP) method to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. We carried out reverse transcription (RT)-LAMP using the Loopamp SARS-CoV-2 Detection kit (Eiken Chemical, Tokyo, Japan). The entire mixture except for the primers is dried and immobilized inside the tube lid. To determine the specificity of the kit, 22 viral genomes associated with respiratory infections, including the SARS coronavirus, were tested. No LAMP product was detected in reactions performed with RNA from these pathogens. The sensitivity of this assay, determined by either a real-time turbidity assay or colorimetric change of the reaction mixture, as evaluated by the naked eye or under illumination with ultraviolet light, was 10 copies/reaction. After the initial validation analysis, we analyzed 24 nasopharyngeal swab specimens collected from patients suspected to have COVID-19. Nineteen (79.2%) of the 24 samples were positive for SARS-CoV-2 RNA, as determined by real-time RT-PCR analysis. Using the Loopamp SARS-CoV-2 Detection kit, we detected SARS-CoV-2 RNA in 15 (62.5%) of the 24 samples. Thus, the sensitivity, specificity, positive predictive value, and negative predictive value of the Loopamp 2019-CoV-2 detection reagent kit were 94.0%, 96.0%, 95.9%, and 94.1%, respectively. The dry LAMP method for detection of SARS-CoV-2 RNA was fast and easy to use, solves the cold chain problem, and therefore represents a promising tool for diagnosis of COVID-19 in developing countries.

Figures

Table 2. Comparison of real-time RT-PCR and dry loop–mediated isothermal amplification assay results of SARS-CoV-2 using clinical specimens.

Table 1. Specificity of dry loop-mediated isothermal amplification assay for SARS-CoV-2.

Full text

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Research article
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Dry loop–mediated isothermal amplification assay for detection of SARS-CoV-2 from
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clinical specimens
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Yuki Higashimoto
1*
, Masaru Ihira
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, Yoshiki Kawamura
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, Masato Inaba
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, Kazuya Shirato
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,
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Tadaki Suzuki
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, Hideki Hasegawa
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, Tsutomu Kageyama
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, Yohei Doi
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, Tadayoshi Hata
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, and
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Tetsushi Yoshikawa
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Faculty of Medical Technology, Fujita Health University School of Health Sciences,
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Toyoake, Aichi, Japan
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Faculty of Clinical Engineering, Fujita Health University School of Health Sciences,
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Toyoake, Aichi, Japan
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Department of Pediatrics, Fujita Health University School of Medicine, Toyoake, Aichi,
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Japan
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Department of Infectious Diseases, Fujita Health University School of Medicine, Toyoake,
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Aichi, Japan
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Department of Virology III, National Institute of Infectious Diseases, Musashimurayama,
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Tokyo, Japan
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Department of Pathology, National Institute of Infectious Diseases, Musashimurayama,
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. CC-BY-ND 4.0 International licenseIt is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.(which was not certified by peer review)preprint
The copyright holder for thisthis version posted September 30, 2020. ; https://doi.org/10.1101/2020.09.29.20204297doi: medRxiv preprint
NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.

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Tokyo, Japan
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Influenza Virus Research Center, National Institute of Infectious Diseases,
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Musashimurayama, Tokyo, Japan
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Department of Clinical Laboratory, Fujita Health University Hospital, Toyoake, Japan
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* Corresponding Author
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E-mail: yhigashi@fujita-hu.ac.jp (YH)
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. CC-BY-ND 4.0 International licenseIt is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.(which was not certified by peer review)preprint
The copyright holder for thisthis version posted September 30, 2020. ; https://doi.org/10.1101/2020.09.29.20204297doi: medRxiv preprint

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Abstract
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Coronavirus disease 2019 (COVID-19) has had a major disease burden on many countries around
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the world. The spread of COVID-19 is anticipated to have a major impact on developing countries
31
including African nations. To establish a point-of-care test for COVID-19, we developed a dry
32
loop–mediated isothermal amplification (LAMP) method to detect severe acute respiratory
33
syndrome coronavirus 2 (SARS-CoV-2) RNA. We carried out reverse transcription (RT)-LAMP
34
using the Loopamp SARS-CoV-2 Detection kit (Eiken Chemical, Tokyo, Japan). The entire mixture
35
except for the primers is dried and immobilized inside the tube lid. To determine the specificity of
36
the kit, 22 viral genomes associated with respiratory infections, including the SARS coronavirus,
37
were tested. No LAMP product was detected in reactions performed with RNA from these
38
pathogens. The sensitivity of this assay, determined by either a real-time turbidity assay or
39
colorimetric change of the reaction mixture, as evaluated by the naked eye or under illumination
40
with ultraviolet light, was 10 copies/reaction. After the initial validation analysis, we analyzed 24
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nasopharyngeal swab specimens collected from patients suspected to have COVID-19. Nineteen
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(79.2%) of the 24 samples were positive for SARS-CoV-2 RNA, as determined by real-time
43
RT-PCR analysis. Using the Loopamp SARS-CoV-2 Detection kit, we detected SARS-CoV-2 RNA
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in 15 (62.5%) of the 24 samples. Thus, the sensitivity, specificity, positive predictive value, and
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negative predictive value of the Loopamp 2019-CoV-2 detection reagent kit were 94.0%, 96.0%,
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95.9%, and 94.1%, respectively. The dry LAMP method for detection of SARS-CoV-2 RNA was
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fast and easy to use, solves the cold chain problem, and therefore represents a promising tool for
48
. CC-BY-ND 4.0 International licenseIt is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.(which was not certified by peer review)preprint
The copyright holder for thisthis version posted September 30, 2020. ; https://doi.org/10.1101/2020.09.29.20204297doi: medRxiv preprint

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diagnosis of COVID-19 in developing countries.
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50
. CC-BY-ND 4.0 International licenseIt is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.(which was not certified by peer review)preprint
The copyright holder for thisthis version posted September 30, 2020. ; https://doi.org/10.1101/2020.09.29.20204297doi: medRxiv preprint

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Author summary
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Coronavirus disease 2019 (COVID-19) is a major public health problem around the world. A
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reliable point-of-care (POC) test for severe acute respiratory syndrome coronavirus 2 (SARS
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CoV-2) is urgently needed, especially in developing countries. The loop-mediated isothermal
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amplification (LAMP) method amplifies template nucleotides under isothermal conditions with
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high efficiency and specificity, both of which are major advantages for a POC test. In addition,
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because dry LAMP reagents can be stored at 4°C, it is suitable for use in developing countries.
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We evaluated the specificity and sensitivity of the Loopamp SARS-CoV-2 Detection kit (Eiken
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Chemical, Tokyo, Japan), a dry LAMP method for amplifying viral RNA. The initial validation
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study revealed that the method was highly specific and sensitive (lower detection limit: 10
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copies/reaction). We then analyzed 24 nasopharyngeal swab specimens from patients suspected to
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have COVID-19. Using the Loopamp SARS-CoV-2 Detection kit, SARS-CoV-2 RNA was detected
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in 15 (62.5%) of the 24 samples. Compared with the standard real-time reverse transcription PCR,
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the sensitivity, specificity, positive predictive value, and negative predictive value of the Loopamp
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SARS-CoV-2 Detection kit were 78.9%, 100%, 100%, and 55.6%, respectively.
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. CC-BY-ND 4.0 International licenseIt is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.(which was not certified by peer review)preprint
The copyright holder for thisthis version posted September 30, 2020. ; https://doi.org/10.1101/2020.09.29.20204297doi: medRxiv preprint

Frequently asked questions

Q1. What are the contributions in this paper?

It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

Q2. What is the advantage of the LAMP assay?

Ease of use, speed, and low cost of amplifying target nucleotides is a significant advantage of the 179 LAMP assay as a POC test. 

Q3. Why is LAMP widely used for POC testing?

Due to its speed, ease of use, and cost-effectiveness, LAMP has been 88 widely used for POC testing for various infectious diseases, including COVID-19 [14-17]. 

Q4. How many RNAs were used for the initial validation analysis?

96978Materials and methods 9899Viruses and RNA for initial validation analysis 100 A total of 22 respiratory pathogens were used for the initial validation analysis of specificity of the 101 primers in this study. 

Q5. How many false negative samples were collected?

The four false-negative samples contained low copies of viral 171 RNA (2.6, 2.3, 2.2, and 0.5 copies/reaction) and were collected during the convalescent phase of 172 illness (days 7 to 20). 

Q6. What is the importance of a reliable point-of-care test for severe acute respiratory syndrome?

A 52 reliable point-of-care (POC) test for severe acute respiratory syndrome coronavirus 2 (SARS 53 CoV-2) is urgently needed, especially in developing countries. 

Q7. Why is there an urgent need to develop rapid diagnostic tests for COVID-19?

82 In addition, because expansion of COVID-19 to developing countries is a major public health 83 concern, there is an urgent need to develop rapid diagnostic tests for COVID-19. 

Q8. What is the newest version of the RNA-based detection method?

Rapid and visual detection of 338232019 novel coronavirus (SARS-CoV-2) by a reverse transcription loop-mediated isothermal 339amplification assay. 

Q9. How many samples were collected from patients suspected of having COVID-19?

In order to evaluate the performance of SARS-CoV-2 dry LAMP for analyzing clinical 200 samples, the authors analyzed 24 nasopharyngeal swab specimens collected from patients suspected of 201 having COVID-19. 

Q10. What was the sensitivity of the RT-LAMP kit?

To determine the sensitivity of the Loopamp 159 SARS-CoV-2 Detection kit, in vitro transcribed RNAs were serially diluted in 10 mM Tris buffer 160 containing 0.1 mM EDTA and 50 ng/mL of carrier RNA were used to define the detection limit. 

Q11. How many large hospitals are using the method?

The method is very useful for testing large numbers of samples at 80 large hospitals, diagnostic companies, and local health facilities. 

Q12. What is the importance of a point-of-care test for COVID-19?

a point-of-care (POC) 81 test for COVID-19 is also important for management of suspected patients in less resourced settings. 

Q13. How was the sensitivity of the LAMP kit?

Compared with the standard real-time reverse transcription PCR, 63 the sensitivity, specificity, positive predictive value, and negative predictive value of the Loopamp 64 SARS-CoV-2 Detection kit were 78.9%, 100%, 100%, and 55.6%, respectively. 

Q14. What is the pmcid of the PMCPMC?

3182020/04/22. doi: 10.1093/clinchem/hvaa102. PubMed PMID: 32315390; PubMed Central 319PMCID: PMCPMC7188121. 3202222. Shirato K, Kawase M, Matsuyama S. Wild-type human coronaviruses prefer 321cell-surface TMPRSS2 to endosomal cathepsins for cell entry. 

Q15. how is the sars-cov-2 dry lamp method used?

In summary, this study demonstrated that the SARS-CoV-2 dry LAMP method is a reliable 210 method for rapid diagnosis of COVID-19.