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1.
Sci Rep ; 11(1): 16193, 2021 08 10.
Article in English | MEDLINE | ID: covidwho-1351975

ABSTRACT

We have optimised a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the detection of SARS-CoV-2 from extracted RNA for clinical application. We improved the stability and reliability of the RT-LAMP assay by the addition of a temperature-dependent switch oligonucleotide to reduce self- or off-target amplification. We then developed freeze-dried master mix for single step RT-LAMP reaction, simplifying the operation for end users and improving long-term storage and transportation. The assay can detect as low as 13 copies of SARS-CoV2 RNA per reaction (25-µL). Cross reactivity with other human coronaviruses was not observed. We have applied the new RT-LAMP assay for testing clinical extracted RNA samples extracted from swabs of 72 patients in the UK and 126 samples from Greece and demonstrated the overall sensitivity of 90.2% (95% CI 83.8-94.7%) and specificity of 92.4% (95% CI 83.2-97.5%). Among 115 positive samples which Ct values were less than 34, the RT-LAMP assay was able to detect 110 of them with 95.6% sensitivity. The specificity was 100% when RNA elution used RNase-free water. The outcome of RT-LAMP can be reported by both colorimetric detection and quantifiable fluorescent reading. Objective measures with a digitized reading data flow would allow for the sharing of results for local or national surveillance.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , COVID-19 Nucleic Acid Testing/standards , Humans , Molecular Diagnostic Techniques/standards , Nucleic Acid Amplification Techniques/standards , Sensitivity and Specificity
2.
Environ Microbiol ; 22(10): 4085-4092, 2020 10.
Article in English | MEDLINE | ID: covidwho-739051
3.
Biodes Manuf ; : 1-4, 2020 May 11.
Article in English | MEDLINE | ID: covidwho-232677

ABSTRACT

We present an example of applying 'need-driven' product design principle to the development of a rapid test kit to detect SARS-COV-2 (COVID-19). The tests are intended for use in the field and, longer term, for home use. They detect whether a subject is currently infected with the virus and is infectious. The urgent need for large numbers of tests in field setting imposes constraints such as short test time and lack of access to specialist equipment, laboratories and skilled technicians to perform the test and interpret results. To meet these needs, an antigen test based on RT-LAMP with colorimetric readout was chosen. Direct use of swab sample with no RNA extraction was explored. After extensive experimental study (reported elsewhere), a rapid test kit has been fabricated to satisfy all design criteria.

4.
Microb Biotechnol ; 13(4): 950-961, 2020 07.
Article in English | MEDLINE | ID: covidwho-116666

ABSTRACT

The pandemic coronavirus SARS-CoV-2 in the world has caused a large infected population suffering from COVID-19. To curb the spreading of the virus, WHO urgently demanded an extension of screening and testing; thus, a rapid and simple diagnostic method is needed. We applied a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) to achieve the detection of SARS-CoV-2 in 30 min. We designed four sets of LAMP primers (6 primers in each set), targeting the viral RNA of SARS-CoV-2 in the regions of orf1ab, S gene and N gene. A colorimetric change was used to report the results, which enables the outcome of viral RNA amplification to be read by the naked eye without the need of expensive or dedicated instrument. The sensitivity can be 80 copies of viral RNA per ml in a sample. We validated the RT-LAMP method in a hospital in China, employing 16 clinic samples with 8 positives and 8 negatives. The testing results are consistent with the conventional RT-qPCR. In addition, we also show that one-step process without RNA extraction is feasible to achieve RNA amplification directly from a sample. This rapid, simple and sensitive RT-LAMP method paves a way for a large screening at public domain and hospitals, particularly regional hospitals and medical centres in rural areas.


Subject(s)
Betacoronavirus/isolation & purification , Coronavirus Infections/diagnosis , Nucleic Acid Amplification Techniques/methods , Pneumonia, Viral/diagnosis , Betacoronavirus/classification , Betacoronavirus/genetics , COVID-19 , China , Coronavirus Infections/virology , DNA Primers/genetics , Humans , Pandemics , Pneumonia, Viral/virology , SARS-CoV-2 , Sensitivity and Specificity , Viral Proteins/genetics
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