COVID-19 has become a major global public health burden, currently causing a rapidly growing number of infections and significant morbidity and mortality around the world. Early detection with fast and sensitive assays and timely intervention are crucial for interrupting the spread of the COVID-19 virus (SARS-CoV-2). Using a mismatch-tolerant amplification technique, we developed a simple, rapid, sensitive and visual reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for SARS-CoV-2 detection based on its N gene. The assay has a high specificity and sensitivity, and robust reproducibility, and its results can be monitored using a real-time PCR machine or visualized via colorimetric change from red to yellow. The limit of detection (LOD) of the assay is 118.6 copies of SARS-CoV-2 RNA per 25 μL reaction. The reaction can be completed within 30 min for real-time fluorescence monitoring, or 40 min for visual detection when the template input is more than 200 copies per 25 μL reaction. To evaluate the viability of the assay, a comparison between the RT-LAMP and a commercial RT-qPCR assay was made using 56 clinical samples. The SARS-CoV-2 RT-LAMP assay showed perfect agreement in detection with the RT-qPCR assay. The newly-developed SARS-CoV-2 RT-LAMP assay is a simple and rapid method for COVID-19 surveillance.

Vibrio parahaemolyticus, the leading cause of seafood-associated gastroenteritis worldwide, has a strong ability to form biofilms on surfaces. Quorum sensing (QS) is a process widely used by bacteria to communicate with each other and control gene expression via the secretion and detection of autoinducers. OpaR is the master QS regulator of V. parahaemolyticus operating under high cell density (HCD). OpaR regulation of V. parahaemolyticus biofilm formation has been reported, but the regulatory mechanisms are still not fully understood. bis-(3′-5′)-cyclic di-GMP (c-di-GMP) is an omnipresent intracellular second messenger that regulates diverse behaviors of bacteria including activation of biofilm formation. In this work, we showed that OpaR repressed biofilm formation and decreased the intracellular concentration of c-di-GMP in V. parahaemolyticus RIMD2210633. The OpaR box-like sequences were detected within the regulatory DNA regions of scrA, scrG, VP0117, VPA0198, VPA1176, VP0699, and VP2979, encoding a group of GGDEF and/or EAL-type proteins. The results of qPCR, LacZ fusion, EMSA, and DNase I footprinting assays demonstrated that OpaR bound to the upstream DNA regions of scrA, VP0117, VPA0198, VPA1176, and VP0699 to repress their transcription, whereas it positively and directly regulated the transcription of scrG and VP2979. Thus, transcriptional regulation of these genes by OpaR led directly to changes in the intracellular concentration of c-di-GMP. The direct association between QS and c-di-GMP metabolism in V. parahaemolyticus RIMD2210633 would be conducive to precise control of gene transcription and bacterial behaviors such as biofilm formation.

Summary: Background: SARS-CoV-2 nucleic acid detection by RT-PCR is one of the criteria approved by China FDA for diagnosis of COVID-19. However, inaccurate test results ( for example, high false negative rate and some false positive rate) were reported in both China and US CDC using RT-PCR method. Inaccurate results are caused by inadequate detection sensitivity of RT-PCR, low viral load in some patients, difficulty to collect samples from COVID-19 patients, insufficient sample loading during RT-PCR tests, and RNA degradation during sample handling process. False negative detection could subject patients to multiple tests before diagnosis can be made, which burdens health care system. Delayed diagnosis could cause infected patients to miss the best treatment time window. False negative detection could also lead to prematurely releasing infected patients who still carry residual SARS-CoV-2 virus. In this case, these patients could infect many others. A high sensitivity RNA detection method to resolve the existing issues of RT-PCR is in need for more accurate COVID-19 diagnosis. Methods: Digital PCR (dPCR) instrument DropX-2000 and assay kits were used to detect SARS-CoV-2 from 108 clinical specimens from 36 patients including pharyngeal swab, stool and blood from different days during hospitalization. Double-blinded experiment data of 108 clinical specimens by dPCR methods were compared with results from officially approved RT-PCR assay. A total of 109 samples including 108 clinical specimens and 1 negative control sample were tested in this study. All of 109 samples, 26 were from 21patients reported as positive by officially approved clinical RT-PCR detection in local CDC and then hospitalized in Nantong Third Hospital. Among the 109 samples, dPCR detected 30 positive samples on ORFA1ab gene, 47 samples with N gene positive, and 30 samples with double positive on ORFA1ab and N genes. Results: The lower limit of detection of the optimize dPCR is at least 10-fold lower than that of RT-PCR. The overall accuracy of dPCR for clinical detection is 96.3%. 4 out 4 of (100 %) negative pharyngeal swab samples checked by RT-PCR were positive judged by dPCR based on the follow-up investigation. 2 of 2 samples in the RT-PCR grey area (Ct value > 37) were confirmed by dPCR with positive results. 1 patient being tested positive by RT-PCR was confirmed to be negative by dPCR. The dPCR results show clear viral loading decrease in 12 patients as treatment proceed, which can be a useful tool for monitoring COVID-19 treatment. Conclusions: Digital PCR shows improved lower limit of detection, sensitivity and accuracy, enabling COVID-19 detection with less false negative and false positive results comparing with RT-PCR, especially for the tests with low viral load specimens. We showed evidences that dPCR is powerful in detecting asymptomatic patients and suspected patients. Digital PCR is capable of checking the negative results caused by insufficient sample loading by quantifying internal reference gene from human RNA in the PCR reactions. Multi-channel fluorescence dPCR system (FAM/HEX/CY5/ROX) is able to detect more target genes in a single multiplex assay, providing quantitative count of viral load in specimens, which is a powerful tool for monitoring COVID-19 treatment.

Mature biofilm formation by Vibrio parahaemolyticus requires exopolysaccharide (EPS), type IV pili, and capsular polysaccharide (CPS). Production of each is strictly regulated by various control pathways including quorum sensing (QS) and bis-(3′–5′)-cyclic di-GMP (c-di-GMP). QsvR, an AraC-type regulator, integrates into the QS regulatory cascade via direct control of the transcription of the master QS regulators, AphA and OpaR. Deletion of qsvR in wild-type or opaR mutant backgrounds altered the biofilm formation by V. parahaemolyticus, suggesting that QsvR may coordinate with OpaR to control biofilm formation. Herein, we demonstrated both QsvR and OpaR repressed biofilm-associated phenotypes, c-di-GMP metabolism, and the formation of V. parahaemolyticus translucent (TR) colonies. QsvR restored the biofilm-associated phenotypic changes caused by opaR mutation, and vice versa. In addition, QsvR and OpaR worked coordinately to regulate the transcription of EPS-associated genes, type IV pili genes, CPS genes and c-di-GMP metabolism-related genes. These results demonstrated how QsvR works with the QS system to regulate biofilm formation by precisely controlling the transcription of multiple biofilm formation-associated genes in V. parahaemolyticus.

We showed that Vibrio parahaemolyticus was able to naturally and reversibly switch between wrinkly and smooth phenotypes and disclosed the gene expression profiles related to wrinkly-smooth switching, showing that the significantly differentially expressed genes between the two colony morphology phenotypes were involved in various biological behaviors, including virulence factor production, biofilm formation, metabolism, adaptation, and colonization.