Semi-nested RT-PCR enables sensitive and high-throughput detection of SARS-CoV-2 based on melting analysis.

BACKGROUND: Asymptomatic transmission was found to be the Achilles' heel of the symptom-based screening strategy, necessitating the implementation of mass testing to efficiently contain the transmission of COVID-19 pandemic. However, the global shortage of molecular reagents and the low throughput of available realtime PCR facilities were major limiting factors. METHODS: A novel semi-nested and heptaplex (7-plex) RT-PCR assay with melting analysis for detection of SARS-CoV-2 RNA has been established for either individual testing or 96-sample pooled testing. The complex melting spectrum collected from the heptaplex RT-PCR amplicons was interpreted with the support of an artificial intelligence algorithm for the detection of SARS-CoV-2 RNA. The analytical and clinical performance of the semi-nested RT-PCR assay was evaluated using RNAs synthesized in-vitro and those isolated from nasopharyngeal samples. RESULTS: The LOD of the assay for individual testing was estimated to be 7.2 copies/reaction. Clinical performance evaluation indicated a sensitivity of 100% (95% CI: 97.83-100) and a specificity of 99.87% (95% CI: 99.55-99.98). More importantly, the assay supports a breakthrough sample pooling method, which makes possible parallel screening of up to 96 samples in one real-time PCR well without loss of sensitivity. As a result, up to 8,820 individual pre-amplified samples could be screened for SARS-CoV-2 within each 96-well plate of realtime PCR using the pooled testing procedure. CONCLUSION: The novel semi-nested RT-PCR assay provides a solution for highly multiplex (7-plex) detection of SARS-CoV-2 and enables 96-sample pooled detection for increase of testing capacity. .

Sero-Prevalence of SARS-CoV-2 Antibodies in High-Risk Populations in Vietnam.

As a response to the coronavirus disease 2019 (COVID-19) pandemic, Vietnam enforced strict quarantine, contact tracing and physical distancing policies resulting in one of the lowest numbers of individuals infected with severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) globally. This study aimed to determine the prevalence of SARS-CoV-2 antibody positivity among high-risk populations in Vietnam. A prevalence survey was undertaken within four communities in Vietnam, where at least two COVID-19 cases had been confirmed. Participants were classified according to the location of exposure: household contacts, close contacts, community members, and healthcare workers (HCWs) responsible for treating COVID-19 cases. Participants completed a baseline questionnaire and SARS-CoV-2 IgG antibodies were quantified using a commercial assay. A total of 3049 community members and 149 health care workers consented to the study. Among 13 individuals who were seropositive (0.4%), five household contacts (5/27, 18.5%), one close contact (1/53, 1.9%), and seven community members (7/2954, 0.2%) had detectable SARS-CoV-2 antibodies. All HCWs were negative for SARS-CoV-2 antibodies. Participants were tested a median of 15.1 (interquartile range from 14.9 to 15.2) weeks after exposure. Our study found a low prevalence of SARS-CoV-2 antibodies in high-risk communities and healthcare workers in communities in Vietnam with known COVID-19 cases.