ToxR represses the synthesis of c-di-GMP in Vibrio parahaemolyticus / 生物工程学报

Vibrio parahaemolyticus, the main pathogen causing seafood related food poisoning worldwide, has strong biofilm formation ability. ToxR is a membrane binding regulatory protein, which has regulatory effect on biofilm formation of V. parahaemolyticus, but the specific mechanism has not been reported. c-di-GMP is an important second messenger in bacteria and is involved in regulating a variety of bacterial behaviors including biofilm formation. In this study, we investigated the regulation of ToxR on c-di-GMP metabolism in V. parahaemolyticus. Intracellular c-di-GMP in the wild type (WT) and toxR mutant (ΔtoxR) strains were extracted by ultrasonication, and the concentrations of c-di-GMP were then determined by enzyme linked immunosorbent assay (ELISA). Three c-di-GMP metabolism-related genes scrA, scrG and vpa0198 were selected as the target genes. Quantitative real-time PCR (q-PCR) was employed to calculate the transcriptional variation of each target gene between WT and ΔtoxR strains. The regulatory DNA region of each target gene was cloned into the pHR309 plasmid harboring a promoterless lacZ gene. The recombinant plasmid was subsequently transferred into WT and ΔtoxR strains to detect the β-galactosidase activity in the cellular extracts. The recombinant lacZ plasmid containing each of the target gene was also transferred into E. coli 100λpir strain harboring the pBAD33 plasmid or the recombinant pBAD33-toxR to test whether ToxR could regulate the expression of the target gene in a heterologous host. The regulatory DNA region of each target gene was amplified by PCR, and the over-expressed His-ToxR was purified. The electrophoretic mobility shift assay (EMSA) was applied to verify whether His-ToxR directly bound to the target promoter region. ELISA results showed that the intracellular c-di-GMP level significantly enhanced in ΔtoxR strain relative to that in WT strain, suggesting that ToxR inhibited the production of c-di-GMP in V. parahaemolyticus. qPCR results showed that the mRNA levels of scrA, scrG and vpa0198 significantly increased in ΔtoxR strain relative to those in WT strain, suggesting that ToxR repressed the transcription of scrA, scrG and vpa0198. lacZ fusion assay showed that ToxR was able to repress the promoter activities of scrA, scrG and vpa0198 in both V. parahaemolyticus and E. coli 100λpir. EMSA results showed that His-ToxR was able to bind to the regulatory DNA regions of scrA and scrG, but not to the regulatory DNA region of vpa0198. In conclusion, ToxR inhibited the production of c-di-GMP in V. parahaemolyticus via directly regulating the transcription of enzyme genes associated with c-di-GMP metabolism, which would be beneficial for V. parahaemolyticus to precisely control bacterial behaviors including biofilm formation.

Transcriptional regulation of pilABCD by OpaR in Vibrio parahaemolyticus / 中华微生物学和免疫学杂志

Objective:To study the transcriptional regulation of pilABCD by the master quorum sensing (QS) regulator OpaR in Vibrio parahaemolyticus. Methods:Total RNAs were extracted from the wild type (WT) and opaR mutant (Δ opaR) strain. Quantitative real-time PCR (qPCR) was employed to calculate the transcriptional variation of pilA (the first gene of pilABCD operon) between WT and Δ opaR. The regulatory DNA region of pilABCD was cloned into the corresponding restriction endonuclease sites of pHRP309 harboring a promoterless lacZ reporter gene. The recombinant pHRP309 plasmid was then transferred into WT and Δ opaR, respectively, to detect the β-galactosidase activity in cellular extracts using a β-Galactosidase Enzyme Assay System (Promega). The primer extension assay was applied to map the transcription start site of pilABCD using the total RNAs extracted from the WT strain as the template. The regulatory DNA region of pilABCD was amplified by PCR, and the over-expressed His-OpaR was purified under native conditions with nickel loaded HiTrap Chelating Sepharose columns (Amersham). Thereafter, the electrophoretic mobility shift assay (EMSA) was applied to analyze the DNA-binding activity of His-OpaR to the target DNA in vitro, and the DNase I footprinting assay was further employed to detect the DNA-binding sites of His-OpaR within the target DNA. Results:The results of qPCR and LacZ fusion assays showed that OpaR activated the transcription of pilABCD, leading to a gradual increase in the expression level of pilA with the extension of culture time. The primer extension assay detected only one transcription start site located at 155 bp upstream of pilA. The results of EMSA and DNase Ⅰ footprinting assays showed that His-OpaR protected two DNA regions located from -246 to -197 bp and -181 to -131 bp upstream of pilA. Conclusions:Vibrio parahaemolyticus OpaR activated the transcription of pilABCD in a direct manner.

Comparative Evaluation of 19 Reverse Transcription Loop-Mediated Isothermal Amplification Assays for Detection of SARS-CoV-2

Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has caused a global pandemics. To facilitate the detection of SARS-CoV-2 infection, various RT-LAMP assays using 19 sets of primers had been developed, but never been compared. We performed comparative evaluation of the 19 sets of primers using 4 RNA standards and 29 clinical samples from COVID-19 patients. Six of 15 sets of primers were firstly identified to have faster amplification when tested with four RNA standards, and were further subjected to parallel comparison with the remaining four primer sets using 29 clinical samples. Among these 10 primer sets, Set-4 had the highest positive detection rate of SARS-CoV-2 (82.8%), followed by Set-10, Set-11, Set-13 and Set-14 (75.9%), and Set-14 showed the fastest amplification speed (< 8.5 minutes), followed by Set-17 (< 12.5 minutes). Based on the overall detection performance, Set-4, Set-10, Set-11, Set-13, Set-14 and Set-17 that target Nsp3, S, S, E, N and N gene regions of SARS-CoV-2, respectively, are determined to be better than the other primer sets. Two RT-LAMP assays with the Set-14 primers in combination with any one of four other primer sets (Set-4, Set-10, Set-11, and Set-13) are recommended to be used in the COVID-19 surveillance.

Temporal dynamics of human respiratory and gut microbiomes during the course of COVID-19 in adults

SARS-CoV-2 infects multiple organs including the respiratory tract and gut. Whether regional microbiomes are disturbed significantly to affect the disease progression of COVID-19 is largely unknown. To address this question, we performed cross-sectional and longitudinal analyses of throat and anal swabs from 35 COVID-19 adults and 15 controls by 16S rRNA gene sequencing. The results allowed a partitioning of patients into 3-4 categories (I-IV) with distinct microbial community types in both sites. Lower-diversity community types often appeared in the early phase of COVID-19, and synchronous fast restoration of both the respiratory and gut microbiomes from early dysbiosis towards late near-normal was observed in 6/8 mild COVID-19 adult patients despite they had a relatively slow clinical recovery. The synchronous shift of the community types was associated with significantly positive bacterial interactions between the respiratory tract and gut, possibly along the airway-gut axis. These findings reveal previously unknown interactions between respiratory and gut microbiomes, and suggest that modulations of regional microbiota might help to improve the recovery from COVID-19 in adult patients.

SARS-CoV-2 detection using digital PCR for COVID-19 diagnosis, treatment monitoring and criteria for discharge

BackgroundSARS-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. MethodsDigital 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. ResultsThe 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. ConclusionsDigital 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.

Construction of infectious clone of HBV adefovir-resistant strain RT A181T and its expression in Huh7 cells / 国际检验医学杂志

Objective To rapidly construct hepatitis B virus(HBV)adefovir(ADV)‐resistant strain(RT A181T)infectious clone by using SOE‐PCR ,to observe the expression of recombinant plasmids in Huh7 cells and to establish the in vitro cell model of HBV ADV‐resistant strain(RT A181T) .Methods The conservative primers were designed ,the full‐length HBV genome was amplified from serum in the patients with ADV‐resistant chronic hepatitis B .Then ,the 1 .3‐fold genome‐length infectious clone pHBV1 .3 was constructed by using the SOE‐PCR technique ,which was transfected into human hepatoma cells Huh7 cell line .ELISA ,Western Blot and Real‐time PCR were adopted to detect infectious cloning replication and expression ,meanwhile the antiviral drugs lamivu‐dine(LAM ) and ADV were used to verify the infectious clone copy and inhibition of expression .Results The HBV ADV‐resistant infectious cloning plasmids pHBV1 .3 was successfully constructed .This plasmid could be effectively replicated ,transcripted and ex‐pressed in Huh7 cell lines .LAM could effectively inhibit the replication and expression of the infectious clone ,while ADV could not inhibit the replication and expression of the infectious clone .Conclusion The constructed infectious clone plasmids pHBV1 .3(RT A181T ) of HBV ADV‐resistant strain can efficiently replicate and express protein in vitro ,its transfected cells can be used in the study of HBV replication mechanism and antiviral study .

Enterovirus 71 molecular epidemiology research in Nantong,2014 / 国际检验医学杂志

Objective To analyze enterovirus 71 (EV71) molecular epidemiology characteristics in Nantong region of Jiangsu , 2014 .Methods The pharyngeal swab samples were selected from 52 children with hand ,foot and mouth disease .After extracting the virus nucleic acid ,the EV71 VP1 gene of the virus were amplified by RT‐PCR amplification .The phylogenetic tree was con‐structed between isolated and EV71 reference strains from other parts of the country .Results 12 strains EV71 VP1 gene were iso‐lated from 52 cases of clinical specimens .The nucleotide and amino acid homology of 12 strains EV71 VP1 gene was 93 .4% -99 .1% ,the phylogenetic tree analysis showed that 12 strains of VP1 gene all belong to C4 genotype C4a subgenotypes .Conclusion The isolated strains in Nantong in 2014 are all the EV71 C4a subgenotypes of C4 genotype ,the same with the most of isolates in re‐cent years .

Construction of recombinant eukaryotic expression plasmid containing 1.3-fold-overlength genome of HBV and its expression in Huh7 cells / 实用医学杂志

Objective To construct 1.3-fold-overlength infectious clone of hepatitis B virus isolated from Chinese patients , observe the expression of plasmid in Huh7 of liver cancer cells and establish genome of HBV in vitro. Methods HBV DNA in serum was extracted from HBV patient. SOE-PCR was performed to produce a 1.3-fold-overlength genome of HBV. The plasmid was named pHBV1.3 (C). After that,pHBV1.3 (C) was transfected into Huh7 cells, HBV related viral antigens and DNA were detected by ELISA,Western Blot and Fluorescence quantitative PCR. Furthermore, adefovir dipivoxil, a clinic anti-viral drug, was utilized to test the sensitivity of the new infectious clone. Results An infectious clone of pHBV1.3 (C) was successfully constructed. HBV gene carried in pHBV1.3 (C) could be efficiently replicated and expressed in Huh7 cells. Adefovir could inhibit HBV replication in this HBV cell model. Conclusions A recombinant plasmid containing 1.3-fold-overlength of HBV genotype C was successfully constructed. This construct is competent to support viral transcription and replication in vitro , suggesting that infectious cells are expected to be a new model of HBV infection in vitro.

Pathogen detection and clinical characteristics of hand, foot and mouth disease children in Nantong / 实用医学杂志

Objective To analyze the epidemiological characteristics of pathogens of children with hand , foot and mouth disease (HFMD) in Nantong area, in order to provide the basis for the treatment and prevention of HFMD. Methods Multiplex RT-PCR (fluorescence PCR method) was used to detect EV71, CoxA16 and non-infectious cases of EV71, CoxA16 of children with HFMD in Nantong. VP1 gene of strains isolated from 12 cases of EV71 was amplified and underwent phylogenetic tree analysis. 208 cases clinical data of children with HFMD were analyzed and summarized. Results In 208 cases of children with HFMD swab samples, EV71 of 64 cases were positive (30.8%); CoxA16 of 28 cases were positive (13.5%); 40 cases of other enterovirus were positive (19.2%). Phylogenetic tree analysis showed that EV71 epidemic strains are C4 subtypes in Nantong region. The clinical manifestations of enterovirus infections in EV71 , CoxA16 and other enterovirus are caused by rash, fever and other symptoms, the difference of which was not statistically significant (P > 0.05). In laboratory examination, EV71 infection made white blood cell count higher than CoxA16 and other enterovirus, but the difference was not statistically significant (P>0.05). Conclusions EV71, CoxA16 and other enterovirus intestinal virus are main pathogens of HFMD in Nantong. EV71 C4 subtype is the main popular subtype of EV71 in Nantong. The elevation of white blood cell count of HFMD is the main clinical manifestations and characteristics , but can not serve as the identification of children infected with EV71, CoxA16 and other enterovirus.