Validation of a single-step, single-tube reverse transcription-loop-mediated isothermal amplification assay for rapid detection of SARS-CoV-2 RNA

Lee, Jean; Best, Nickala; McAuley, Julie; Porter, Jessica L.; Seemann, Torsten; Schultz, Mark B.; Sait, Michelle; Orlando, Nicole; Mercoulia, Karolina; Ballard, Susan A; Druce, Julian; Tran, Thomas; Catton, Mike; Pryor, Melinda J.; Cui, Huanhuan; Luttick, Angela; McDonald, Sean; Greenhalgh, Arran; Kwong, Jason C; Sherry, Norelle L; Graham, Maryza; Hoang, Tuyet; Hérisse, Marion; Pidot, Sacha J.; Williamson, Deborah A; Howden, Benjamin P; Monk, Ian R.; Stinear, Timothy P.

doi:10.1101/2020.04.28.067363

Cited by 19 publications

(23 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…RT-PCR remains the most sensitive nucleic acid amplification method for COVID-19 diagnosis compared to MCDA and LAMP. This result is in agreement with previous LAMP COVID-19 assays which showed RT-PCR having greater sensitivity 9-11 . The limit of detection for our MCDA N gene assay was 1000 copies/reaction or an equivalent N gene average Ct value of 32.4 (Table 1).…”

Section: Discussionsupporting

confidence: 93%

Development and comparison of a novel multiple cross displacement amplification (MCDA) assay with other nucleic acid amplification methods for SARS-CoV-2 detection

Luu

Payne

Zhang

et al. 2020

Preprint

View full text Add to dashboard Cite

Objectives: To develop a novel multiple cross displacement amplification (MCDA) assay for COVID-19 and compare its speed and sensitivity to existing loop-mediated isothermal amplification (LAMP) and real-time PCR (RT-PCR) methods. Methods: Two MCDA assays targeting the SARS-CoV-2 N gene and ORF1ab was designed. The fastest time to detection and sensitivity of MCDA was compared to LAMP and RT-PCR using 7 DNA standards and transcribed RNA. Results: For the N gene, MCDA was consistently faster than LAMP and RT-PCR by 10 and 20 minutes, respectively with a fastest time to detection of 5.2 minutes. RT-PCR had the highest sensitivity with a limit of detection of 100 copies/reaction compared with MCDA (1000 copies/reaction) and LAMP (5000/reaction). For ORF1ab, MCDA and LAMP had similar speed with fastest time to detection at 9.7 and 8.4 minutes, respectively. LAMP was more sensitive for ORF1ab detection with 500 copies/reaction compared to MCDA (5000 copies/reaction). Conclusions: Different nucleic acid amplification methods provide different advantages. MCDA is the fastest nucleic acid amplification method for COVID-19 while RT-PCR is still the most sensitive. These advantages should be considered when determining the most suitable nucleic acid amplification methods for different applications.

show abstract

Section: Discussionsupporting

confidence: 93%

Development and comparison of a novel multiple cross displacement amplification (MCDA) assay with other nucleic acid amplification methods for SARS-CoV-2 detection

Luu

Payne

Zhang

et al. 2020

Preprint

View full text Add to dashboard Cite

show abstract

“…Thus, the great advantage of having a rapid and accurate CLEIA is the ability for primary screening to establish a "gray zone" reserving definitive diagnosis by confirmatory NAAT testing. Reverse transcriptase loop-mediated isothermal amplification (LAMP) [7] has become the second most common NAAT after PCR with several advantages over PCR: rapid turn-around time within 30 minutes, ease of implementation, and potential utility at point of care using a simple device [6,9,[21][22][23][24][25]. We recently reported that LAMP had equivalent efficacy to PCR using saliva in both All rights reserved.…”

Section: Discussionmentioning

confidence: 99%

Performance of qualitative and quantitative antigen tests for SARS-CoV-2 in early symptomatic patients using saliva

Yokota

Sakurazawa

Sato

et al. 2020

Preprint

View full text Add to dashboard Cite

BackgroundThe rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an urgent need for the prevention and containment of disease outbreaks in communities. Although the gold standard is polymerase chain reaction (PCR), antigen tests such as immunochromatographic assay (ICA) and chemiluminescent enzyme immunoassay (CLEIA) that can yield results within 30 minutes.MethodsWe evaluated performance of ICA and CLEIA using 34 frozen PCR-positive specimens (17 saliva and 17 nasopharyngeal swab) and 307 PCR-negative samples.ResultsICA detected SARS-CoV-2 in only 14 (41%) samples, with positivity of 24% in saliva and 59% in NPS. Notably, ICA detected SARS-CoV-2 in 5 (83%) of 6 samples collected within 4 days after symptom onset. CLEIA detected SARS-CoV-2 in 31 (91%) samples, with positivity of 82% in saliva and 100% in NPS. CLEIA was negative in 3 samples with low viral load by PCR.ConclusionsThese results suggest that use of ICA should be limited to earlier time after symptom onset and CLEIA is more sensitive and can be used in situations where quick results are required.

show abstract

“…All SARS‐CoV‐2 infection cultures were conducted within the High Containment Facilities in a PC3 laboratory at the Doherty Institute. Stocks of SARS‐CoV‐2 isolate hCoV‐19/Australia/VIC01/2020 39 were produced as previously described 40 . To assess the potential for SARS‐CoV‐2 to infect and replicate virus genome, cells were pelleted by centrifugation, washed with PBS and pelleted again.…”

Section: Methodsmentioning

confidence: 99%

“…Stocks of SARS-CoV-2 isolate hCoV-19/ Australia/VIC01/2020 39 were produced as previously described. 40 To assess the potential for SARS-CoV-2 to infect and replicate virus genome, cells were pelleted by centrifugation, washed with PBS and pelleted again. The cells were then lysed and viral RNA extracted using the QiaAmp Viral RNA extraction kit (Qiagen, Australia) as per the manufacturer's instructions and stored at À80°C.…”

Section: Rna Extraction and Sars-cov2 Qrt-pcrmentioning

confidence: 99%

Influenza, but not SARS‐CoV‐2, infection induces a rapid interferon response that wanes with age and diminished tissue‐resident memory CD8⁺ T cells

et al. 2021

View full text Add to dashboard Cite

Older individuals exhibit a diminished ability to respond to and clear respiratory pathogens and, as such, experience a higher rate of lung infections with a higher mortality rate. It is unclear why respiratory pathogens impact older people disproportionately. Using human lung tissue from donors aged 22-68 years, we assessed how the immune cell landscape in lungs changes throughout life and investigated how these immune cells respond following in vitro exposure to influenza virus and SARS-CoV-2, two clinically relevant respiratory viruses. While the frequency of most immune cell subsets profiled in the human lung remained stable with age, memory CD8 + T cells declined, with the tissueresident memory (Trm) CD8 + T-cell subset being most susceptible to age-associated attrition. Infection of lung tissue with influenza virus resulted in an age-associated attenuation in the antiviral immune response, with aged donors producing less type I interferon (IFN), GM-CSF and IFNc, the latter correlated with a reduction of IFNc-producing memory CD8 + T cells. In contrast, irrespective of donor age, exposure of human lung cells to SARS-CoV-2, a pathogen for which all donors were immunologically na€ ıve, did not trigger activation of local immune cells and did not result in the induction of an early IFN response. Our findings show that the attrition of tissue-bound pathogen-specific Trm in the lung that occurs with advanced age, or their absence in immunologically na€ ıve individuals, results in a diminished early antiviral immune response which creates a window of opportunity for respiratory pathogens to gain a greater foothold.

show abstract

Validation of a single-step, single-tube reverse transcription-loop-mediated isothermal amplification assay for rapid detection of SARS-CoV-2 RNA

Cited by 19 publications

References 27 publications

Development and comparison of a novel multiple cross displacement amplification (MCDA) assay with other nucleic acid amplification methods for SARS-CoV-2 detection

Development and comparison of a novel multiple cross displacement amplification (MCDA) assay with other nucleic acid amplification methods for SARS-CoV-2 detection

Performance of qualitative and quantitative antigen tests for SARS-CoV-2 in early symptomatic patients using saliva

Influenza, but not SARS‐CoV‐2, infection induces a rapid interferon response that wanes with age and diminished tissue‐resident memory CD8⁺ T cells

Contact Info

Product

Resources

About

Validation of a single-step, single-tube reverse transcription-loop-mediated isothermal amplification assay for rapid detection of SARS-CoV-2 RNA

Cited by 19 publications

References 27 publications

Development and comparison of a novel multiple cross displacement amplification (MCDA) assay with other nucleic acid amplification methods for SARS-CoV-2 detection

Development and comparison of a novel multiple cross displacement amplification (MCDA) assay with other nucleic acid amplification methods for SARS-CoV-2 detection

Performance of qualitative and quantitative antigen tests for SARS-CoV-2 in early symptomatic patients using saliva

Influenza, but not SARS‐CoV‐2, infection induces a rapid interferon response that wanes with age and diminished tissue‐resident memory CD8+ T cells

Contact Info

Product

Resources

About

Influenza, but not SARS‐CoV‐2, infection induces a rapid interferon response that wanes with age and diminished tissue‐resident memory CD8⁺ T cells