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Single- and Two-Stage, Closed-Tube, Point-of-Care, Molecular Detection of SARS-CoV-2.
Song, Jinzhao; El-Tholoth, Mohamed; Li, Yize; Graham-Wooten, Jevon; Liang, Yining; Li, Juan; Li, Weijian; Weiss, Susan R; Collman, Ronald G; Bau, Haim H.
  • Song J; The Cancer Hospital of the University of Chinese Academy of Sciences (Zhejiang Cancer Hospital), Institute of Basic Medicine and Cancer (IBMC), Chinese Academy of Sciences, Hangzhou, Zhejiang 310022, China.
  • El-Tholoth M; Department of Mechanical Engineering and Applied Mechanics, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States.
  • Li Y; Department of Mechanical Engineering and Applied Mechanics, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States.
  • Graham-Wooten J; Department of Virology, Faculty of Veterinary Medicine, Mansoura University, Mansoura 35516, Egypt.
  • Liang Y; Health Sciences Division, Veterinary Sciences Program, Al Ain Men's Campus, Higher Colleges of Technology, Al Ain 17155, UAE.
  • Li J; Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States.
  • Li W; Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States.
  • Weiss SR; Department of Mechanical Engineering and Applied Mechanics, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States.
  • Collman RG; The Cancer Hospital of the University of Chinese Academy of Sciences (Zhejiang Cancer Hospital), Institute of Basic Medicine and Cancer (IBMC), Chinese Academy of Sciences, Hangzhou, Zhejiang 310022, China.
  • Bau HH; The Cancer Hospital of the University of Chinese Academy of Sciences (Zhejiang Cancer Hospital), Institute of Basic Medicine and Cancer (IBMC), Chinese Academy of Sciences, Hangzhou, Zhejiang 310022, China.
Anal Chem ; 93(38): 13063-13071, 2021 09 28.
Article in English | MEDLINE | ID: covidwho-1428693
ABSTRACT
Short of a vaccine, frequent and rapid testing, preferably at home, is the most effective strategy to contain the COVID-19 pandemic. Herein, we report on single-stage and two-stage molecular diagnostic tests that can be carried out with simple or no instrumentation. Our single-stage amplification is reverse transcription-loop mediated isothermal amplification (RT-LAMP) with custom-designed primers targeting the ORF1ab and the N gene regions of the virus genome. Our new two-stage amplification, dubbed Penn-RAMP, comprises recombinase isothermal amplification (RT-RPA) as its first stage and LAMP as its second stage. We compared various sample preparation strategies aimed at deactivating the virus while preserving its RNA and tested contrived and patient samples, consisting of nasopharyngeal swabs, oropharyngeal swabs, and saliva. Amplicons were detected either in real time with fluorescent intercalating dye or after amplification with the intercalating colorimetric dye LCV, which is insensitive to sample's PH. Our single RT-LAMP tests can be carried out instrumentation-free. To enable concurrent testing of multiple samples, we developed an inexpensive heat block that supports both the single-stage and two-stage amplification. Our RT-LAMP and Penn-RAMP assays have, respectively, analytical sensitivities of 50 and 5 virions/reaction. Both our single- and two-stage assays have successfully detected SARS-CoV-2 in patients with viral loads corresponding to the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) threshold cycle smaller than 32 while operating with minimally processed samples, without nucleic acid isolation. Penn-RAMP provides a 10-fold better sensitivity than RT-LAMP and does not need thermal cycling like PCR assays. All reagents are amenable to dry, refrigeration-free storage. The SARS-CoV-2 test described herein is suitable for screening at home, at the point of need, and in resource-poor settings.
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Full text: Available Collection: International databases Database: MEDLINE Main subject: SARS-CoV-2 / COVID-19 Type of study: Diagnostic study Topics: Vaccines Limits: Humans Language: English Journal: Anal Chem Year: 2021 Document Type: Article Affiliation country: Acs.analchem.1c03016

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Full text: Available Collection: International databases Database: MEDLINE Main subject: SARS-CoV-2 / COVID-19 Type of study: Diagnostic study Topics: Vaccines Limits: Humans Language: English Journal: Anal Chem Year: 2021 Document Type: Article Affiliation country: Acs.analchem.1c03016