BackgroundThe emerging Coronavirus Disease-2019 (COVID-19) has challenged the public health globally. With the increasing requirement of detection for SARS-CoV-2 outside of the laboratory setting, a rapid and precise Point of Care Test (POCT) is urgently needed.MethodsTargeting the nucleocapsid (N) gene of SARS-CoV-2, specific primers, and probes for reverse transcription recombinase-aided amplification coupled with lateral flow dipstick (RT-RAA/LFD) platform were designed. For specificity evaluation, it was tested with human coronaviruses, human influenza A virus, influenza B viruses, respiratory syncytial virus, and hepatitis B virus, respectively. For sensitivity assay, it was estimated by templates of recombinant plasmid and pseudovirus of SARS-CoV-2 RNA. For clinical assessment, 100 clinical samples (13 positive and 87 negatives for SARS-CoV-2) were tested via quantitative reverse transcription PCR (RT-qPCR) and RT-RAA/LFD, respectively.ResultsThe limit of detection was 1 copies/μl in RT-RAA/LFD assay, which could be conducted within 30 min at 39°C, without any cross-reaction with other human coronaviruses and clinical respiratory pathogens. Compared with RT-qPCR, the established POCT assay offered 100% specificity and 100% sensitivity in the detection of clinical samples.ConclusionThis work provides a convenient POCT tool for rapid screening, diagnosis, and monitoring of suspected patients in SARS-CoV-2 endemic areas.
To detect Zika virus more rapidly and accurately, we developed a novel method that utilized a real-time fluorescence reverse transcription loop-mediated isothermal amplification (LAMP) technique. The NS5 gene was amplified by a set of six specific primers that recognized six distinct sequences. The amplification process, including 60 min of thermostatic reaction with Bst DNA polymerase following real-time fluorescence reverse transcriptase using genomic Zika virus standard strain (MR766), was conducted through fluorescent signaling. Among the six pairs of primers that we designate here, NS5 was the most efficient with a high sensitivity of up to 3.3 ng/μl and reproducible specificity on eight pathogen samples that were used as negative controls. The real-time fluorescence reverse transcription LAMP detection process can be completed within 35 min. Our study demonstrated that real-time fluorescence reverse transcription LAMP could be highly beneficial and convenient clinical application to detect Zika virus due to its high specificity and stability.
Streptococcus agalactiae is a major pathogenic bacterium causing perinatal infections in humans. In the present study, a novel real-time fluorescence loop-mediated isothermal amplification technology was successfully developed and evaluated for the detection of S. agalactiae in a single reaction. Six specific primers were designed to amplify the corresponding six regions of fbs B gene of S. agalactiae, using Bst DNA polymerase with DNA strand displacement activity at a constant temperature for 60 min. The presence of S. agalactiae was indicated by the fluorescence in real-time. Amplification of the targeted gene fragment was optimized with the primer 1 in the current setup. Positive result was only obtained for Sa by Real-LAMP among 10 tested relevant bacterial strains, with the detection sensitivity of 300 pg/µl. Real-LAMP was demonstrated to be a simple and rapid detection tool for S. agalactiae with high specificity and stability, which ensures its wide application and broad prospective utilization in clinical practice for the rapid detection of S. agalactiae.
The present study reported the epidemiological, clinical features, laboratory data and management of a family of 5 members with COVID-19. It was found that the initial clinical manifestations of the 3 adults were fever, cough and sore throat; they all exhibited a higher erythrocyte sedimentation rate (ESR) and higher serum glucose level. The 2 children only exhibited a mild dry cough. As regards the 2-month-old infant who was breast-fed, the possible infection source was close contact with family members or respiratory droplets. Her mother's breast milk was negative for SARS-CoV-2, as shown by RT-PCR. The two children persisted to be positive for SARS-CoV-2 by anal swab after being released from the hospital, although they were negative for SARS-CoV-2 by a throat swab.
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