tetano
Editor, Senior Moderator
J Clin Virol
. 2021 Feb 11;136:104760.
doi: 10.1016/j.jcv.2021.104760. Online ahead of print.
Development of a point-of-care test to detect SARS-CoV-2 from saliva which combines a simple RNA extraction method with colorimetric reverse transcription loop-mediated isothermal amplification detection
Wataru Yamazaki[SUP] 1 [/SUP], Yasufumi Matsumura[SUP] 2 [/SUP], Uraiwan Thongchankaew-Seo[SUP] 3 [/SUP], Yasuko Yamazaki[SUP] 3 [/SUP], Miki Nagao[SUP] 2 [/SUP]
Affiliations
Abstract
The new coronavirus infection (COVID-19) is a major public health concern, with a high burden and risk for infection among patients and healthcare workers. Saliva droplets containing SARS-COV-2 are a major vector for COVID-19 infection, making saliva a promising alternative for COVID-19 testing using nasopharyngeal swab samples. To diagnose COVID-19 patients in the field, a point-of-care test (POCT) using saliva was conceptualized. We have developed a simple method for extracting RNA from saliva samples using semi-alkaline proteinase, a sputum homogenizer typically used for preparing samples for tuberculosis testing, and a subsequent simple heating step with no need for centrifugation or RNA extraction. Further, we newly developed a triplex reverse transcription loop-mediated isothermal amplification approach (RT-LAMP) which utilizes colorimetric readout using a heat block, with results evaluated with the unaided eye. In 44 clinical patients suspected of having COVID-19 infection, the test took 45 min, and resulted in a diagnostic sensitivity of 82.6% (19/23) and diagnostic specificity of 100% (21/21), compared to the reference standard. The limit of detection was 250 copies/reaction (25,000 copies/mL). Our newly developed POCT approach achieved simple RNA extraction and constant RT-LAMP detection. This POCT has the potential to be used for simple inspection stations in a field setting, helping reduce the risk of infection by simplifying and accelerating testing for COVID-19.
Keywords: COVID-19; LAMP; POCT; Point-of-care test; SARS-CoV-2, semi alkaline proteinase.
. 2021 Feb 11;136:104760.
doi: 10.1016/j.jcv.2021.104760. Online ahead of print.
Development of a point-of-care test to detect SARS-CoV-2 from saliva which combines a simple RNA extraction method with colorimetric reverse transcription loop-mediated isothermal amplification detection
Wataru Yamazaki[SUP] 1 [/SUP], Yasufumi Matsumura[SUP] 2 [/SUP], Uraiwan Thongchankaew-Seo[SUP] 3 [/SUP], Yasuko Yamazaki[SUP] 3 [/SUP], Miki Nagao[SUP] 2 [/SUP]
Affiliations
- PMID: 33610926
- DOI: 10.1016/j.jcv.2021.104760
Abstract
The new coronavirus infection (COVID-19) is a major public health concern, with a high burden and risk for infection among patients and healthcare workers. Saliva droplets containing SARS-COV-2 are a major vector for COVID-19 infection, making saliva a promising alternative for COVID-19 testing using nasopharyngeal swab samples. To diagnose COVID-19 patients in the field, a point-of-care test (POCT) using saliva was conceptualized. We have developed a simple method for extracting RNA from saliva samples using semi-alkaline proteinase, a sputum homogenizer typically used for preparing samples for tuberculosis testing, and a subsequent simple heating step with no need for centrifugation or RNA extraction. Further, we newly developed a triplex reverse transcription loop-mediated isothermal amplification approach (RT-LAMP) which utilizes colorimetric readout using a heat block, with results evaluated with the unaided eye. In 44 clinical patients suspected of having COVID-19 infection, the test took 45 min, and resulted in a diagnostic sensitivity of 82.6% (19/23) and diagnostic specificity of 100% (21/21), compared to the reference standard. The limit of detection was 250 copies/reaction (25,000 copies/mL). Our newly developed POCT approach achieved simple RNA extraction and constant RT-LAMP detection. This POCT has the potential to be used for simple inspection stations in a field setting, helping reduce the risk of infection by simplifying and accelerating testing for COVID-19.
Keywords: COVID-19; LAMP; POCT; Point-of-care test; SARS-CoV-2, semi alkaline proteinase.