Epidemiological and Genomic analysis of Vibrio parahaemolyticus isolated from imported travelers at the port of Shanghai, China (2017-2019).

BACKGROUND: Vibrio parahaemolyticus is the predominant etiological agent of seafood-associated foodborne illnesses on a global scale. It is essential to elucidate the mechanisms by which this pathogen disseminates. Given the existing research predominantly concentrates on localized outbreaks, there is a pressing necessity for a comprehensive investigation to capture strains of V. parahaemolyticus cross borders. RESULTS: This study examined the frequency and genetic attributes of imported V. parahaemolyticus strains among travelers entering Shanghai Port, China, between 2017 and 2019.Through the collection of 21 strains from diverse countries and regions, Southeast Asia was pinpointed as a significant source for the emergence of V. parahaemolyticus. Phylogenetic analysis revealed clear delineation between strains originating from human and environmental sources, emphasizing that underlying genome data of foodborne pathogens is essential for environmental monitoring, food safety and early diagnosis of diseases. Furthermore, our study identified the presence of virulence genes (tdh and tlh) and approximately 120 antibiotic resistance-related genes in the majority of isolates, highlighting their crucial involvement in the pathogenesis of V. parahaemolyticus. CONCLUSIONS: This research enhanced our comprehension of the worldwide transmission of V. parahaemolyticus and its antimicrobial resistance patterns. The findings have important implications for public health interventions and antimicrobial stewardship strategies, underscoring the necessity for epidemiological surveillance of pathogen at international travel hubs.

Ultra-Fast Single-Nucleotide-Variation Detection Enabled by Argonaute-Mediated Transistor Platform.

"Test-and-go" single-nucleotide variation (SNV) detection within several minutes remains challenging, especially in low-abundance samples, since existing methods face a trade-off between sensitivity and testing speed. Sensitive detection usually relies on complex and time-consuming nucleic acid amplification or sequencing. Here, a graphene field-effect transistor (GFET) platform mediated by Argonaute protein that enables rapid, sensitive, and specific SNV detection is developed. The Argonaute protein provides a nanoscale binding channel to preorganize the DNA probe, accelerating target binding and rapidly recognizing SNVs with single-nucleotide resolution in unamplified tumor-associated microRNA, circulating tumor DNA, virus RNA, and reverse transcribed cDNA when a mismatch occurs in the seed region. An integrated microchip simultaneously detects multiple SNVs in agreement with sequencing results within 5 min, achieving the fastest SNV detection in a "test-and-go" manner without the requirement of nucleic acid extraction, reverse transcription, and amplification.

Application of whole-genome tiling array at Shanghai port, China: An alternative method for SARS-CoV-2 surveillance.

The ongoing coronavirus disease 2019 (COVID-19) pandemic, driven by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), highlights the critical role of genomic surveillance in tracking rapidly spreading viruses and their evolving lineages. The emergence of the SARS-CoV-2 tiling array, a comprehensive tool capable of capturing the entire viral genome, has presented a promising avenue for variants. This study introduces the SARS-CoV-2 tiling array as a novel method for port inspection. Using next-generation sequencing as a benchmark, 35 positive samples underwent sequencing through both methodologies, including the Alpha variant (B.1.1.7), Delta variants (AY.120, AY.122, AY.23.1), and Omicron variants (BA.1, BA.2, BA.2.75, BA.4, BA.5, BE.1, BF.7, BN.1, BQ.1, XBB.1) within the sample set. The whole-genome tiling array demonstrated successful identification of various sublineages of SARS-CoV-2. The average sequencing coverage rates were 99.22% (96.82%-99.92%) for the whole-genome tiling array and 98.56% (92.81%-99.59%) for Illumina sequencing, respectively. The match rates of these two methods ranged from 92.81%-99.59%, with an average rate of 98.56%. Among the benefits of the whole-genome tiling array are its cost-effectiveness and equipment simplification, making it particularly suitable for identifying SARS-CoV-2 variants in the front-line inspection department. The aforementioned findings provide valuable insights into the surveillance of COVID-19 and present a pragmatic solution for improving quarantine measures at entry points.

Accurate quantification of DNA using on-site PCR (osPCR) by characterizing DNA amplification at single-molecule resolution.

Despite the need in various applications, accurate quantification of nucleic acids still remains a challenge. The widely-used qPCR has reduced accuracy at ultralow template concentration and is susceptible to nonspecific amplifications. The more recently developed dPCR is costly and cannot handle high-concentration samples. We combine the strengths of qPCR and dPCR by performing PCR in silicon-based microfluidic chips and demonstrate high quantification accuracy in a large concentration range. Importantly, at low template concentration, we observe on-site PCR (osPCR), where only certain sites of the channel show amplification. The sites have almost identical ct values, showing osPCR is a quasi-single molecule phenomenon. Using osPCR, we can measure both the ct values and the absolute concentration of templates in the same reaction. Additionally, osPCR enables identification of each template molecule, allowing removal of nonspecific amplification during quantification and greatly improving quantification accuracy. We develop sectioning algorithm that improves the signal amplitude and demonstrate improved detection of COVID in patient samples.

RT-RPA-Cas12a-based assay facilitates the discrimination of SARS-CoV-2 variants of concern.

Timely and accurate detection of SARS-CoV-2 variants of concern (VOCs) is urgently needed for pandemic surveillance and control. Great efforts have been made from a mass of scientists in increasing the detection sensitivity and operability, and reducing the turn-around time and cost. Here, we report a nucleic acid testing-based method aiming to detect and discriminate SARS-CoV-2 mutations by combining RT-RPA and CRISPR-Cas12a detecting assays (RRCd). With a detection limit of 10 copies RNA/reaction, RRCd was validated in 194 clinical samples, showing 89% positive predictive agreement and 100% negative predictive agreement, respectively. Critically, using specific crRNAs, representatives of single nucleotide polymorphisms and small deletions in SARS-CoV-2 VOCs including N501Y, T478K and ΔH69-V70 were discriminated by RRCd, demonstrating 100% specificity in clinical samples with C t < 33. The method completes within 65 min and could offer visible results without using any electrical devices, which probably facilitate point-of-care testing of SARS-CoV-2 variants and other epidemic viruses.

Internet of medical things-enabled CRISPR diagnostics for rapid detection of SARS-CoV-2 variants of concern.

Previous studies have highlighted CRISPR-based nucleic acid detection as rapid and sensitive diagnostic methods for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we reported an optimized CRISPR-Cas12a diagnostic platform for the safe and rapid detection of SARS-CoV-2 variants of concern (VOCs). This platform, which was referred to as CALIBURN-v2, could complete the diagnosis on extracted RNA samples within 25 min in a closed-lid reaction mode and had 100-fold increase in detection sensitivity in comparison with previous platforms. Most importantly, by integrating a portable device and smartphone user interface, CALIBURN-v2 allowed for cloud server-based data collection and management, thus transforming the point-of-care testing (POCT) platform to internet of medical things (IoMT) applications. It was found that IoMT-enabled CALIBURN-v2 could achieve 95.56% (172 out of 180) sensitivity for SARS-CoV-2 wild type and 94.38% (84 out of 89) overall sensitivity for SARS-CoV-2 variants including Delta and Omicron strains. Therefore, our study provides a feasible approach for IoMT-enabled CRISPR diagnostics for the detection of SARS-CoV-2 VOCs.

Research on safety and compliance of imported microbial inoculants using high-throughput sequencing.

Microbial inoculants are widely used in wastewater treatment, soil remediation, and biological control. Safety and compliance for active constituents are considered to be the most important measures of imported microbial inoculants. Microbial inoculants composition was commonly identified by phenotypic culture, which is time-consuming and labor intense with occasionally false negative results provided, and can only be tested for specific species. High-throughput sequencing (HTS), known for its non-targeted detection of unknown species composition in samples, is suitable for composition consistency identification and biosafety analysis of imported microbial inoculants. In this study, the application of HTS for microflora distribution and resistance gene was verified in microbial inoculants for environmental protection and then applicated in imported microbial inoculants. Both Illumina- and Nanopore-based HTS methods identified the same dominant bacterial species successfully in the imported microbial inoculants. The main component of bacterial species was Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, and Enterococcus faecium, and further confirmed with traditional methods. The antibiotic resistance genes Bacillus subtilis mprF, bcrA, blt, lmrB, rphB, tet(L), tmrB, vmlR, ykkC, and ykkD were detected in all samples. Our results indicated that HTS processes the application potential to identify the active ingredients of microbial inoculants. Therefore, rapid and accurate identification of the microbial compositions in microbial formulation products is of high importance for port biosafety supervision.

Fully Automated CRISPR-LAMP Platform for SARS-CoV-2 Delta and Omicron Variants.

Integrated clustered regularly interspaced short palindromic repeat (CRISPR)-loop-mediated amplification (LAMP) technology is of great importance in CRISPR-based diagnostic systems, which urgently needs to be developed to improve diagnostic accuracy. A labor-free, contamination-free, and fully automated droplet manipulation platform for the CRISPR-LAMP technology has not been developed before. Herein, we propose a fully automated CRISPR-LAMP platform, which can precisely manipulate the CRISPR-LAMP droplet and perform combined reactions with high sensitivity and specificity. SARS-CoV-2 Spike T478K, D614G, P681R, and P681H mutations, typical point mutations of B.1.617.2 (Delta) and Omicron variants, are monitored with this platform with a detection limit of 102 copies/µL. Allele discrimination between the mutants and wild type is significant with the designed one/two-mismatch CRISPR RNA (crRNA) at a limit of 102 copies/µL. Chemically synthesized and modified crRNAs greatly increase the CRISPR-LAMP signal, which advance the wide application. Combined with the previously developed RdRp CRISPR-LAMP assay, clinical results showed that Spike T478K and P681H can discriminate the mutant type form the wild type with 70% (49.66-85.50%, 95% confidence interval) and 78% (57.27-90.62%, 95% confidence interval) sensitivity, respectively, and 100% specificity (51.68-100%, 95% confidence interval), and the RdRp target can detect SARS-CoV-2 strains with 85% sensitivity (65.39-95.14%, 95% confidence interval) and 100% specificity (51.68-100%, 95% confidence interval). We believe that this automatic digital microfluid (DMF) system can advance the integrated CRISPR-LAMP technology with higher stability, sensitivity, and practicability, also for other CRISPR-associated diagnostic platforms.

Increased genetic variation of A(H3N2) virus from influenza surveillance at the end of the 2016/2017 season for Shanghai port, China.

Influenza A(H3N2) virus exhibited complex seasonal patterns to evade pre-existing antibodies, resulting in changes in the antigenicity of the viron surface protein hemagglutinin (HA). To monitor the currently imported influenza viruses as well as to assess the capacity of health emergencies at the Shanghai port, we collected respiratory specimens of passengers from different countries and regions including some of Europe with influenza-like illness at the Shanghai port during 2016/2017, examined amino acid substitutions, and calculated the perfect-match vaccine efficacy using the p epitope model. Phylogenetic analysis of the HA genes revealed that influenza A(H3N2) viruses belonging to eight subclades were detected, and three amino acid substitutions in the subclade 3C.2a.4 were also added. Besides, two epidemic influenza virus strains were found in the 2016/2017 winter and 2016 summer. The results of lower predicted vaccine effectiveness in summer suggest that the imported A(H3N2) strains were not a good match for the A/Hong Kong/4801/2014 vaccine strain since the summer of 2017. Therefore, the Shanghai Port might stop the risk of the international spread of influenza for the first time, and curb the entry of A(H3N2) from overseas at the earliest stage of a probable influenza pandemic.

Moxa Wool in Different Purities and Different Growing Years Measured by Terahertz Spectroscopy.

Moxa wool is a traditional Chinese herbal medicine, which can warm channels to dispel coldness. At present, there is no unified index to evaluate the purity and growing years of moxa wool in the market. Terpineol is one of the effective substances in the volatile oil of moxa wool. Here, we characterize the purity and growing years of moxa wool by studying terpineol. Gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC) are the methods for monitoring terpineol at present, all of which have defects of complicated procedures. We established linear fitting to distinguish the different purities of moxa wool through the intensities (areas) of terpineol, the characteristic peaks, and the consequence presented; the coefficient of determination (R 2) was higher than 0.90. Furthermore, based on the characteristic peak position of standard terpineol, the correlation model with the purity and growing year of moxa wool was set up, thereby differentiating the quality of moxa wool. We have built the partial least squares (PLS) model of the growing years of moxa wool with high accuracy, and the determination coefficient is greater than 0.98. In addition, we compare the quantitative accuracy of Raman spectroscopy with terahertz technology. Finally, a new method of terahertz spectroscopy to evaluate quality of moxa wool was found. It provides a new idea for the identification of inferior moxa wool in the market and a new method for identifying the quality of moxa wool in traditional Chinese medicine.

Measuring heavy metal ions in water using nature existed microalgae as medium based on terahertz technology.

Heavy metal pollution in water seriously affects human health. The disadvantages of traditional metal ion detection methods involve long and cumbersome chemical pretreatment in the early stage, and large volume of samples. In this study, microalgae were used as the medium, and terahertz spectroscopy technology was employed to collect the changes of material components in it, so as to deduce the types and concentrations of heavy metal pollution in water. Through the partial least square(PLS), we establish the prediction model of heavy metal concentration, and the results show that the best detection time for Pb2+ is 6 h and Ni2+ is 18 h. The principal component analysis(PCA) shows that ß-carotene is the most affected substance. Afterward we collect five real surface waters in East China and verify that the judgment accuracy of Pb2+ and Ni2+ are 100% and 93.2% respectively. The results indicate that the time is shorter than the traditional pretreatment time from more than 20-6 h, the sample volume is reduced from 50 mL to 10 mL, the detection accuracy is improved from 10 ng/mL to 1 ng/mL. In a word, we provide a new fast and real-time method for biological monitoring of heavy metal pollution in water.

Multiplex, Real-Time, Point-of-care RT-LAMP for SARS-CoV-2 Detection Using the HFman Probe.

Viral evolution impacts diagnostic test performance through the emergence of variants with sequences affecting the efficiency of primer binding. Such variants that evade detection by nucleic acid-based tests are subject to selective pressure, enabling them to spread more efficiently. Here, we report a variant-tolerant diagnostic test for SARS-CoV-2 using a loop-mediated isothermal nucleic acid-based amplification (LAMP) assay containing high-fidelity DNA polymerase and a high-fidelity DNA polymerase-medicated probe (HFman probe). In addition to demonstrating a high tolerance to variable SARS-CoV-2 viral sequences, the mechanism also overcomes frequently observed limitations of LAMP assays arising from non-specific amplification within multiplexed reactions performed in a single "pot". Results showed excellent clinical performance (sensitivity 94.5%, specificity 100%, n = 190) when compared directly to a commercial gold standard reverse transcription quantitative polymerase chain reaction assay for the extracted RNA from nasopharyngeal samples and the capability of detecting a wide range of sequences containing at least alpha and delta variants. To further validate the test with no sample processing, directly from nasopharyngeal swabs, we also detected SARS-CoV-2 in positive clinical samples (n = 49), opening up the possibility for the assay's use in decentralized testing.

Resveratrol attenuates inflammation and apoptosis through alleviating endoplasmic reticulum stress via Akt/mTOR pathway in fungus-induced allergic airways inflammation.

BACKGROUND: Resveratrol has shown pleiotropic effects against inflammation and oxidative response. The present study aimed to investigate the effects and mechanisms of resveratrol on fungus-induced allergic airway inflammation. METHODS: Female BALB/c mice were injected intraperitoneally with Aspergillus fumigatus (Af) extract emulsified with aluminum on day 0 and 7 and intranasally challenged with Af extracts on day 14 and 15. Resveratrol or dexamethasone or a vehicle was injected intraperitoneally 1 h before each challenge. Mice were sacrificed for serum, bronchoalveolar lavage fluid (BALF), and lungs 24 h after the last challenge. The control group was administered with saline. BEAS-2B was used for the experiments in vitro that Af-exposed airway epithelial cells. RESULTS: Resveratrol and dexamethasone attenuated the airway inflammation and eosinophilia, and reduced not only the production of IL-4, IL-5, and IL-13 in the BALF and lung tissues but also the mRNA levels of lung IL-6, TNF-α, and TGF-ß induced by Af challenge (P < 0.05). Furthermore, Af-induced lung endoplasmic reticulum (ER) stress-related proteins PERK, CHOP, and GRP78 and the apoptosis markers including cleaved caspase-3 and cleaved caspase-7 were both suppressed significantly by resveratrol (P < 0.05). In vitro, activation of ER stress and the Akt/mTOR pathway in Af-exposed BEAS-2B cells were effectively ameliorated by resveratrol. Inhibition of the Akt/mTOR pathway using LY294002 suppressed the ER stress while ER stress inhibitor 4-PBA decreased the apoptosis in Af-exposed BEAS-2B cells. CONCLUSIONS: Our findings collectively revealed that resveratrol alleviated the Af-exposed allergic inflammation and apoptosis through inhibiting ER stress via Akt/mTOR pathway, exerting therapeutic effects on the fungus-induced allergic lung disorder.

Complete genome sequence of GII.9 norovirus.

Norovirus is recognized as one of the leading causes of acute gastroenteritis outbreaks. Genotype GII.9 was first detected in Norfolk, VA, USA, in 1997. However, the complete genome sequence of this genotype has not yet been determined. In this study, a complete genome sequence of GII.9[P7] norovirus, SCD1878_GII.9[P7], from a patient was determined using high-throughput sequencing and rapid amplification of cDNA ends (RACE) technology. The complete genome sequence of SCD1878_GII.9[P7] is 7544 nucleotides (nt) in length with a 3' poly(A) tail and contains three open reading frames. Sequence comparisons indicated that SCD1878_GII.9[P7] shares 92.1%-92.3% nucleotide sequence identity with GII.P7 (AB258331 and AB039777) and 96.7%-97.4% identity with GII.9 (AY038599 and DQ379715). The results suggested that SCD1878_GII.9[P7] is a member of P genotype GII.P7 and G genotype GII.9. This viral sequence fills a gap at the whole-genome level for the GII.9 genotype.

Imported human norovirus in travelers, Shanghai port, China 2018: An epidemiological and whole genome sequencing study.

BACKGROUND: Global mobility of the population has accelerated spread of the Human Norovirus (HuNoV), with long-distance travel in enclosed spaces increasing the opportunity for viral outbreaks. However, surveillance of HuNoV transmission is still lacking, especially in cross-border transportation. METHOD: From 533 self-reported patients, 83 swab samples (15.6%) tested positive for HuNoV by RT-qPCR. Positive samples were sequenced using next-generation sequencing (NGS). Epidemiological investigation and whole genome analysis were then conducted. RESULTS: Most cases occurred in February and March, with large outbreaks involving more than 34 people. A total of 74 HuNoV sequences that could be genotyped were obtained, with near-complete genomes (>7 kb) accounting for most sequences (57/74). A total of 19 different genotypes of viral whole genome sequences were included. The first whole genome sequence of GII.9[P7] was obtained. Rarely reported genotypes including GI.3[P10], GI.3[P13], GII.7[P7], GII.8[P8], and GIX.1[GII.P15] were sequenced and assembled successfully. Four possible sources of virus outbreaks in China were traced. Beyond HuNoV, whole genome sequences of food-borne viruses including Salivirus, Kobuvirus, and Enterovirus were obtained in further assembly. CONCLUSIONS: Surveillance of the etiology and epidemiology of HuNoV global spread through travelers will improve pre-travel health advice, empirical treatment, and estimates of vaccine-preventable diseases.

Study on the mechanism of production of γ-PGA and nattokinase in Bacillus subtilis natto based on RNA-seq analysis.

Poly-γ-glutamic acid (γ-PGA) and nattokinase (NK) are the main substances produced by Bacillus subtilis natto in solid-state fermentation and have wide application prospects. We found that our strains had higher activity of nattokinase when soybeans were used as substrate to increase the yield of γ-PGA. Commercial production of γ-PGA and nattokinase requires an understanding of the mechanism of co-production. Here, we obtained the maximum γ-PGA yield (358.5 g/kg, w/w) and highest activity of NK during fermentation and analyzed the transcriptome of Bacillus subtilis natto during co-production of γ-PGA and NK. By comparing changes in expression of genes encoding key enzymes and the metabolic pathways associated with the products in genetic engineering, the mechanism of co-production of γ-PGA and nattokinase can be summarized based on RNA-seq analysis. This study firstly provides new insights into the mechanism of co-production of γ-PGA and nattokinase by Bacillus subtilis natto and reveals potential molecular targets to promote the co-production of γ-PGA and nattokinase.

Toxoplasma gondii RPL40 is a circulating antigen with immune protection effect.

Screening and identification of protective antigens are essential for the prevention of infections with Toxoplasma gondii (Nicolle et Manceaux, 1908). In our previous study, T. gondii ribosomal-ubiquitin protein L40 (TgRPL40) was identified as a circulating antigen. However, the function and protective value of TgRPL40 was unknown. In the current study, recombinant TgRPL40 was expressed in Escherichia coli BL21 and antibody was prepared. Western blotting analysis indicated that TgRPL40 was present in circulating antigens and excretory/secretary antigens (ESA). Immunofluorescence and immunoelectron microscopy analysis revealed that TgRPL40 protein is widely distributed in the tachyzoites. Immunisation with recombinant TgRPL40 prolonged the survival of mice infected with tachyzoites. Quantitative real-time polymerase chain reaction analysis showed that immunisation with recombinant TgRPL40 reduced the parasite burden in blood, liver, spleen and brain of mice infected with tachyzoites. These observations indicate that TgRPL40 is a circulating antigen and is an effector of immune protection against acute T. gondii infection.

A novel wide-range microfluidic dilution device for drug screening.

Microfluidic dilution chip is a crucial approach to perform gradient dilution of experimental samples in many biological investigations. In this study, we developed two serial wide-range dilution chips with dilution rates of 1:1 and 1:4 on the basis of the microfluidic oscillator by designing a series chamber, which was similar to a series circuit. The size of this chamber was adjusted and mixed with the neighboring air chamber to form dilution rates by oscillatory methods. We applied this microfluidic oscillator to estimate cellular kinetics and perform an acute oxidative stress test on Caenorhabditis elegans (C. elegans) in order to further validate their effectiveness. We estimated the kinetic parameters of ß-galactosidase, the biocatalyst responsible for the hydrolysis of lactose, and found out that K m was 602 ± 73 µM and k cat was 72 ± 12/s. In addition, our result of the study on acute oxidative stress of C. elegans using this novel chip was consistent with the result using 96-well plates. Overall, we believe that this novel chip can be applied to enzymatic reaction kinetics to evaluate accurately drug screening in bio-nematode models such as C. elegans. In summary, we have provided a novel microfluidic dilution chip that can form a wide range of sample concentration gradients. Our chip may facilitate drug screening, drug toxicology, and environmental toxicology.

In Situ Capture RT-qPCR: A New Simple and Sensitive Method to Detect Human Norovirus in Oysters.

Human noroviruses (HuNoVs) are the major cause worldwide for non-bacterial acute gastroenteritis. In this study, we applied a novel viral receptor mediated in situ capture RT-qPCR (ISC-RT-qPCR) to detect HuNoVs in oysters and compared with the traditional RT-qPCR method. Ten HuNoVs RT-PCR positive and 5 negative clinical samples from gastroenteritis patients were used to compare specificity and sensitivity of ISC-RT-qPCR against that of the RT-qPCR assay. ISC-RT-qPCR had at a one-log and a two-log increase in sensitivity over that of the RT-qPCR assay for genotype I (GI) and GII, respectively. Distributions of HuNoVs in oyster tissues were investigated in artificially inoculated oysters. GI HuNoVs could be detected in all tissues in inoculated oysters by both ISC-RT-qPCR and RT-qPCR. GII HuNoVs could only be detected in gills and digestive glands by both methods. The number of viral genomic copies (vgc) measured by ISC-RT-qPCR was comparable with RT-qPCR in the detection of GI and GII HuNoVs in inoculated oysters. Thirty-six oyster samples from local market were assayed for HuNoVs by both assays. More HuNoVs could be detected by ISC-RT-qPCR in retail oysters. The detection rates of GI HuNoVs in gills, digestive glands, and residual tissues were 33.3, 25.0, and 19.4% by ISC-RT-qPCR; and 5.6, 11.1, and 11.1% by RT-qPCR, respectively. The detection rates of GII HuNoVs in gills were 2.8% by ISC-RT-qPCR; no GII HuNoV was detected in these oysters by RT-qPCR. Overall, all results demonstrated that ISC-RT-qPCR is a promising method for detecting HuNoVs in oyster samples.