Dry loop mediated isothermal amplification assay for detection of SARS-CoV-2 from clinical specimens

Coronavirus disease 2019 (COVID-19) has had a major disease burden on many countries around the world. The spread of COVID-19 is anticipated to have a major impact on developing countries including African nations. To establish a point-of-care test for COVID-19, we developed a dry loop-mediated isothermal amplification (LAMP) method to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. We carried out reverse transcription (RT)-LAMP using the Loopamp SARS-CoV-2 Detection kit (Eiken Chemical, Tokyo, Japan). The entire mixture except for the primers is dried and immobilized inside the tube lid. To determine the specificity of the kit, 22 viral genomes associated with respiratory infections, including the SARS coronavirus, were tested. No LAMP product was detected in reactions performed with RNA from these pathogens. The sensitivity of this assay, determined by either a real-time turbidity assay or colorimetric change of the reaction mixture, as evaluated by the naked eye or under illumination with ultraviolet light, was 10 copies/reaction. After the initial validation analysis, we analyzed 24 nasopharyngeal swab specimens collected from patients suspected to have COVID-19. Nineteen (79.2%) of the 24 samples were positive for SARS-CoV-2 RNA, as determined by real-time RT-PCR analysis. Using the Loopamp SARS-CoV-2 Detection kit, we detected SARS-CoV-2 RNA in 15 (62.5%) of the 24 samples. Thus, the sensitivity, specificity, positive predictive value, and negative predictive value of the Loopamp 2019-CoV-2 detection reagent kit were 94.0%, 96.0%, 95.9%, and 94.1%, respectively. The dry LAMP method for detection of SARS-CoV-2 RNA was fast and easy to use, solves the cold chain problem, and therefore represents a promising tool for diagnosis of COVID-19 in developing countries. Author summaryCoronavirus disease 2019 (COVID-19) is a major public health problem around the world. A reliable point-of-care (POC) test for severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) is urgently needed, especially in developing countries. The loop-mediated isothermal amplification (LAMP) method amplifies template nucleotides under isothermal conditions with high efficiency and specificity, both of which are major advantages for a POC test. In addition, because dry LAMP reagents can be stored at 4{degrees}C, it is suitable for use in developing countries. We evaluated the specificity and sensitivity of the Loopamp SARS-CoV-2 Detection kit (Eiken Chemical, Tokyo, Japan), a dry LAMP method for amplifying viral RNA. The initial validation study revealed that the method was highly specific and sensitive (lower detection limit: 10 copies/reaction). We then analyzed 24 nasopharyngeal swab specimens from patients suspected to have COVID-19. Using the Loopamp SARS-CoV-2 Detection kit, SARS-CoV-2 RNA was detected in 15 (62.5%) of the 24 samples. Compared with the standard real-time reverse transcription PCR, the sensitivity, specificity, positive predictive value, and negative predictive value of the Loopamp SARS-CoV-2 Detection kit were 78.9%, 100%, 100%, and 55.6%, respectively.

Haplotype networks of SARS-CoV-2 infections in the Diamond Princess cruise ship outbreak

The Diamond Princess (DP) cruise ship was put under quarantine offshore Yokohama, Japan, after a passenger who disembarked in Hong Kong was confirmed as a COVID-19 case. We performed whole genome sequencing of SARS-CoV-2 directly from PCR-positive clinical specimens and conducted a haplotype network analysis of the outbreak. All tested isolates exhibited a transversion at G11083T, suggesting that SARS-CoV-2 dissemination on the DP originated from a single introduction event before the quarantine started. Although further spreading might have been prevented by quarantine, some progeny clusters were linked to transmission through mass-gathering events in the recreational areas and direct transmission among passengers who shared cabins during the quarantine. This study demonstrates the usefulness of haplotype network analysis in identifying potential infection routes. One Sentence SummaryGenome-based tracing of SARS-CoV-2 infections among passengers and crews in Diamond Princess cruise ship during the quarantine

A clinic-based direct real-time fluorescent reverse transcription loop-mediated isothermal amplification assay for influenza virus.

Seasonal influenza virus causes acute respiratory tract infections, which can be severe in children and the elderly. At present, rapid influenza diagnostic tests (RIDTs) are popular at clinical sites because they enable early diagnosis and avoid unnecessary use of antibiotics; in addition, high risk patients with underlying disease can be given antiviral drugs. However, the sensitivity and specificity of some of those tests are relatively poor. To overcome these problems, nucleic acid-based molecular point-of-care tests have been developed; however, they are significantly more expensive than RIDTs. Previously, the authors developed real-time reverse transcription loop-mediated isothermal amplification (rRT-LAMP) assays using a quenching primer to detect influenza viruses. However, the assay is limited to laboratory use because it requires a nucleic acid purification step and preparation of reaction mixtures on ice. Therefore, the authors developed and validated direct rRT-LAMP assays that require no nucleic acid purification steps using commercial RNA isolation kits, and no storage and handling of reagents on ice. These assays can be performed within 10-30 min and require only mixing a clinical specimen with extraction reagent followed by addition of a lyophilized detection reagent. The established assay showed high sensitivity and specificity when validated using 310 clinical specimens. Thus, the assay is a powerful tool for molecular diagnosis of seasonal influenza virus infection in the clinic.