Reverse Transcription Recombinase-Aided Amplification Assay With Lateral Flow Dipstick Assay for Rapid Detection of 2019 Novel Coronavirus.

Background: The 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. Methods: Targeting 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. Results: The 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. Conclusion: This work provides a convenient POCT tool for rapid screening, diagnosis, and monitoring of suspected patients in SARS-CoV-2 endemic areas.

Quorum sensing inhibition and tobramycin acceleration in Chromobacterium violaceum by two natural cinnamic acid derivatives.

Chromobacterium violaceum, one free-living Gram-negative bacterium, is abundantly presented in tropics and sub-tropics soil and aquatic environment; it is also an opportunistic human pathogen. Here, two cinnamic acid derivatives, i.e., 4-dimethylaminocinnamic acid (DCA) and 4-methoxycinnamic acid (MCA), were identified as potential quorum sensing (QS) and biofilm inhibitors in C. violaceum ATCC12472. Both DCA (100 µg/mL) and MCA (200 µg/mL) inhibited the levels of N-decanoyl-homoserine lactone (C10-HSL) and reduced the production of certain virulence factors in C. violaceum, including violacein, hemolysin, and chitinase. Metabolomics analysis indicated that QS-related metabolites, such as ethanolamine and L-methionine, were down-regulated after treatment with DCA and MCA. Quantitative real-time polymerase chain reaction (qRT-PCR) demonstrated that DCA and MCA markedly suppressed the expression of two QS-related genes (cviI and cviR). In addition, DCA and MCA also inhibited biofilm formation and enhanced the susceptibility of biofilms to tobramycin, which was evidenced by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Our results indicated that DCA and MCA can serve as QS-based agent for controlling pathogens.Key Points • DCA and MCA inhibited QS and biofilm formation in C. violaceum.• The combination of DCA or MCA and tobramycin removed the preformed biofilm of C. violaceum. • DCA or MCA inhibited virulence factors and expressions of cviI and cviR of C. violaceum.• DCA or MCA are potential antibiotic accelerants for treating C. violaceum infection.