tetano
Editor, Senior Moderator
Microbiol Spectr
. 2022 Nov 29;e0196222.
doi: 10.1128/spectrum.01962-22. Online ahead of print.
Development and Validation of a Novel COVID-19 nsp8 One-Tube RT-LAMP-CRISPR Assay for SARS-CoV-2 Diagnosis
Cyril Chik-Yan Yip[SUP] #[/SUP][SUP] 1 [/SUP], Siddharth Sridhar[SUP] #[/SUP][SUP] 1 2 3 [/SUP], Wan-Mui Chan[SUP] 2 [/SUP], Jonathan Daniel Ip[SUP] 2 [/SUP], Allen Wing-Ho Chu[SUP] 2 [/SUP], Kit-Hang Leung[SUP] 2 [/SUP], Vincent Chi-Chung Cheng[SUP] 1 [/SUP], Kwok-Yung Yuen[SUP] 1 2 3 4 [/SUP], Kelvin Kai-Wang To[SUP] 1 2 3 4 [/SUP]
Affiliations
Abstract
Accurate and simple diagnostic tests for coronavirus disease 2019 (COVID-19) are essential components of the pandemic response. In this study, we evaluated a one-tube reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay coupled with clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein-mediated endpoint detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in clinical samples. RT-LAMP-CRISPR is fast and affordable, does not require bulky thermocyclers, and minimizes carryover contamination risk. Results can be read either visually or with a fluorometer. RT-LAMP-CRISPR assays using primers targeting a highly expressed nsp8 gene and previously described nucleocapsid (N) gene primers were designed. The analytical characteristics and diagnostic performance of RT-LAMP-CRISPR assays were compared to those of a commercial real-time RT-PCR E gene assay. The limits of detection (LODs) of the nsp8 and N RT-LAMP-CRISPR assays were 750 and 2,000 copies/mL, which were higher than that of the commercial real-time RT-PCR assay (31.3 copies/mL). Despite the higher LOD, RT-LAMP-CRISPR assays showed diagnostic sensitivity and specificity of 98.6% and 100%, respectively, equivalent to those of the real-time RT-PCR assay (P = 0.5). The median fluorescence reading from the nsp8 assay (378.3 raw fluorescence unit [RFU] [range, 215.6 to 592.6]) was significantly higher than that of the N gene assay (342.0 RFU [range, 143.0 to 576.6]) (P < 0.0001). In conclusion, we demonstrate that RT-LAMP-CRISPR assays using primers rationally designed from highly expressed gene targets are highly sensitive, specific, and easy to perform. Such assays are a valuable asset in resource-limited settings. IMPORTANCE Accurate tests for the diagnosis of SARS-CoV-2, the virus causing coronavirus disease 2019 (COVID-19), are important for timely treatment and infection control decisions. Conventional tests such as real-time reverse transcription-PCR (RT-PCR) require specialized equipment and are expensive. On the other hand, rapid antigen tests suffer from a lack of sensitivity. In this study, we describe a novel assay format for the diagnosis of COVID-19 that is based on principles of loop-mediated isothermal amplification (LAMP) and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas chemistry. A major advantage of this assay format is that it does not require expensive equipment to perform, and results can be read visually. This method proved to be fast, easy to perform, and inexpensive. The test compared well against an RT-PCR assay in terms of the ability to detect SARS-CoV-2 RNA in clinical samples. No false-positive test results were observed. The new assay format is ideal for SARS-CoV-2 diagnosis in resource-limited settings.
Keywords: CRISPR; RT-LAMP; SARS-CoV-2; diagnostic assay; nsp8.
. 2022 Nov 29;e0196222.
doi: 10.1128/spectrum.01962-22. Online ahead of print.
Development and Validation of a Novel COVID-19 nsp8 One-Tube RT-LAMP-CRISPR Assay for SARS-CoV-2 Diagnosis
Cyril Chik-Yan Yip[SUP] #[/SUP][SUP] 1 [/SUP], Siddharth Sridhar[SUP] #[/SUP][SUP] 1 2 3 [/SUP], Wan-Mui Chan[SUP] 2 [/SUP], Jonathan Daniel Ip[SUP] 2 [/SUP], Allen Wing-Ho Chu[SUP] 2 [/SUP], Kit-Hang Leung[SUP] 2 [/SUP], Vincent Chi-Chung Cheng[SUP] 1 [/SUP], Kwok-Yung Yuen[SUP] 1 2 3 4 [/SUP], Kelvin Kai-Wang To[SUP] 1 2 3 4 [/SUP]
Affiliations
- PMID: 36445095
- DOI: 10.1128/spectrum.01962-22
Abstract
Accurate and simple diagnostic tests for coronavirus disease 2019 (COVID-19) are essential components of the pandemic response. In this study, we evaluated a one-tube reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay coupled with clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein-mediated endpoint detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in clinical samples. RT-LAMP-CRISPR is fast and affordable, does not require bulky thermocyclers, and minimizes carryover contamination risk. Results can be read either visually or with a fluorometer. RT-LAMP-CRISPR assays using primers targeting a highly expressed nsp8 gene and previously described nucleocapsid (N) gene primers were designed. The analytical characteristics and diagnostic performance of RT-LAMP-CRISPR assays were compared to those of a commercial real-time RT-PCR E gene assay. The limits of detection (LODs) of the nsp8 and N RT-LAMP-CRISPR assays were 750 and 2,000 copies/mL, which were higher than that of the commercial real-time RT-PCR assay (31.3 copies/mL). Despite the higher LOD, RT-LAMP-CRISPR assays showed diagnostic sensitivity and specificity of 98.6% and 100%, respectively, equivalent to those of the real-time RT-PCR assay (P = 0.5). The median fluorescence reading from the nsp8 assay (378.3 raw fluorescence unit [RFU] [range, 215.6 to 592.6]) was significantly higher than that of the N gene assay (342.0 RFU [range, 143.0 to 576.6]) (P < 0.0001). In conclusion, we demonstrate that RT-LAMP-CRISPR assays using primers rationally designed from highly expressed gene targets are highly sensitive, specific, and easy to perform. Such assays are a valuable asset in resource-limited settings. IMPORTANCE Accurate tests for the diagnosis of SARS-CoV-2, the virus causing coronavirus disease 2019 (COVID-19), are important for timely treatment and infection control decisions. Conventional tests such as real-time reverse transcription-PCR (RT-PCR) require specialized equipment and are expensive. On the other hand, rapid antigen tests suffer from a lack of sensitivity. In this study, we describe a novel assay format for the diagnosis of COVID-19 that is based on principles of loop-mediated isothermal amplification (LAMP) and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas chemistry. A major advantage of this assay format is that it does not require expensive equipment to perform, and results can be read visually. This method proved to be fast, easy to perform, and inexpensive. The test compared well against an RT-PCR assay in terms of the ability to detect SARS-CoV-2 RNA in clinical samples. No false-positive test results were observed. The new assay format is ideal for SARS-CoV-2 diagnosis in resource-limited settings.
Keywords: CRISPR; RT-LAMP; SARS-CoV-2; diagnostic assay; nsp8.