One-Stop Extraction and In Situ RT-qPCR for Ultrasensitive Detection of Highly Diluted SARS-CoV-2 in Large-Volume Samples from Aquatic Environments.

Surveillance of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in aquatic environments attracted attention due to its considerable impacts on human health and ecology, especially in countries with poor sanitation standards. Based on a strategy of one-stop extraction and in situ amplification, we developed an ultrasensitive method that uses a polyacrylamide derivative-modified filter disc (PAD-FD), in which highly diluted RNA can be efficiently concentrated onto the filter disc and directly used for amplification. A newly designed spin column with a cup-like filter base facilitated the non-contact transfer of the affinity filter disc from the column to a PCR tube. The limit of detection of the PAD-FD coupled with RT-qPCR is 10 copies/mL. Using 32 suspected SARS-CoV-2 samples, we demonstrated that the detection rate of our method (62.5%, 20/32) was triple the rate of the commercial kit (18.8%, 6/32). Using a PAD-FD, 56.3% (18/32) and 40.6% (13/32) of the 10-fold-dilution samples with river and tap water, respectively, were detected. Even when diluted 100-fold, 28.1% (9/32) and 37.5% (12/32) were still detected in river and tap water, respectively. We believe that the PAD-FD method offers an accurate testing tool for monitoring viral RNA in aquatic environments, contributing to the forewarning of the SARS-CoV-2 outbreak and the breaking of the transmission chain.

A Combination of Novel Nucleic Acid Cross-Linking Dye and Recombinase-Aided Amplification for the Rapid Detection of Viable Salmonella in Milk.

Salmonella, as an important foodborne pathogen, can cause various diseases, such as severe enteritis. In recent years, various types of nucleicacid-intercalating dyes have been utilized to detect viable Salmonella. However, in principle, the performance of existing nucleic acid dyes is limited because they depend on the integrity of cell membrane. Herein, based on the metabolic activity of bacteria, a novel DNA dye called thiazole orange monoazide (TOMA) was introduced to block the DNA from dead bacteria. Recombinase-aided amplification (RAA) was then performed to detect viable Salmonella in samples. In this study, the permeability of TOMA to the cell membrane of Salmonella was evaluated via confocal laser scanning microscopy and fluorescence emission spectrometry. The limit of detection (LOD) of the TOMA-RAA method was 2.0 × 104 CFU/mL in pure culture. The feasibility of the TOMA-RAA method in detecting Salmonella was assessed in spiked milk. The LOD for Salmonella was 3.5 × 102 CFU/mL after 3 h of enrichment and 3.5 × 100 CFU/mL after 5 h of enrichment. The proposed TOMA-RAA assay has great potential to be applied to accurately detect and monitor foodborne pathogens in milk and its byproducts.

A novel photoreactive DNA-binding dye for detecting viable Klebsiella pneumoniae in powdered infant formula.

In addition to Cronobacter spp., Klebsiella pneumoniae is another opportunistic bacterial pathogen present in powdered infant formula (PIF) that can cause pneumonia, septicemia, and other diseases. In this study, a rapid and specific method based on a fluorescence probe was developed for detecting viable K. pneumoniae in PIF samples via the combination of recombinase-aided amplification (RAA) with thiazole orange monoazide (TOMA) dye (the TOMA-RAA assay hereafter). As a novel photosensitive DNA-intercalating dye, TOMA was used to penetrate bacterial cells, including both dead and viable cells, as verified by confocal laser scanning microscopy and fluorescent emission spectrometry. Importantly, the RAA assay exhibited good performance in detecting K. pneumoniae within 40 min at 39°C. Under optimal conditions, the TOMA-RAA assay can detect as low as 2.6 × 103 cfu/mL of K. pneumoniae in pure culture and 2.3 × 104 cfu/g of K. pneumoniae in spiked PIF sample. After 3 h of pre-enrichment, 3 × 100 cfu/g of K. pneumoniae can be detected. Furthermore, the TOMA-RAA assay displayed an excellent anti-interference ability to nontarget bacteria. In short, the proposed method has great potential application for the rapid and accurate detection of viable K. pneumoniae in PIF.

Real-time recombinase-aided amplification with improved propidium monoazide for the rapid detection of viable Escherichia coli O157:H7 in milk.

Escherichia coli O157:H7, the causative agent of thrombotic thrombocytopenic purpura and hemolytic uremic syndrome in humans, generates a effective harm to community health because of its high pathogenicity. A real-time recombinase-aided amplification (rRAA) is an emerging method for nucleic acid detection. However, genomic DNA of bacteria could exist in food and the environment for a long time after death and could be amplified by rRAA assay, resulting in false-positive signal; thus, developing a fast and sensitive method is necessary to detect viable foodborne pathogens in food products. In our research, rRAA assay coupled with an enhanced nucleic acid binding dye named improved propidium monoazide (PMAxx) was established and applied in viable E. coli O157:H7 identification in skim milk. The PMAxx could eliminate interference from dead bacteria by permeating impaired membranes and covalently linking to DNA to prevent DNA amplification. The PMAxx-rRAA assay was performed with high sensitivity and good specificity. The PMAxx-rRAA assay could detect as low as 5.4 × 100 cfu/mL of viable E. coli O157:H7 in pure culture, and 7.9 × 100 cfu/mL of viable E. coli O157:H7 in skim milk. In addition, the PMAxx-rRAA assay was performed in the presence of a high concentration of dead bacteria or nontarget bacteria in skim milk to verify the capacity to resist interference from dead bacteria and nontarget bacteria. Therefore, the established PMAxx-rRAA assay is a valuable tool for the identification of viable E. coli O157:H7 in complex food matrix.

Synergistic inactivation of Escherichia coli O157:H7 and Staphylococcus aureus by gallic acid and thymol and its potential application on fresh-cut tomatoes.

Antibacterial activity against Escherichia coli O157:H7 and Staphylococcus aureus of five typical plant-derived compounds [gallic acid (G.A), citral (Cit), thymol (Thy), salicylic acid (S.A), lauric acid (L.A)] were investigated by determining the minimum inhibitory concentration (MIC) and the fractional inhibitory concentration index (FICI). The results showed that only a combination of Thy and G.A (TGA), with a concentration of 0.1 and 1.25 mg/mL, respectively, had a synergistic effect (FICI = 0.5) on both E. coli O157:H7 and S. aureus. The amount of Thy and G.A in mixture were four-fold lower than the MICs of the individuals shown to cause the equivalent antimicrobial activity in trypticase soy broth (TSB). The microbial reduction obtained in TSB with addition of TGA were significantly higher (P < 0.05) than the reduction shown for the broth supplemented with the separated phenolics. TGA caused the changes of morphology and membrane integrity of bacteria. Additionally, the application of TGA on fresh-cut tomatoes are investigated. Fresh-cut tomatoes inoculated with E. coli O157:H7and S. aureus were washed for 2min, 5min, 10min at 4 °C, 25 °C, 40 °C in 0.3% NaOCl, or water containing TGA at various concentrations. Overall, the reduction of TGA achieved against S. aureus is higher than E. coli O157:H7. Same concentrations of combined antimicrobials at a temperature of 40 °C further increased the degree of microbial inactivation, with an additional 0.89-1.51 log CFU/g reduction compared to that at 25 °C. Moreover, 1/2MICThy+1/2MICG.A at 25 °C for 10min or 40 °C for 5min were generally acceptable with sensorial scores higher than 7. Our results showed that TGA could work synergistically on the inactivation of the tested bacteria and may be used as an alternative disinfectant of fresh produce.

The fluorescent probe-based recombinase-aided amplification for rapid detection of Escherichia coli O157:H7.

Escherichia coli O157:H7 (E. coli O157:H7) is a common foodborne morbigenous microorganism, which can spread through fecal-oral transmission. Humans can be infected by ingesting foods and water contaminated with E. coli O157:H7, which can cause various symptoms. In present study, we have successfully developed a quick and hypersensitive fluorescent probe-based Recombinase-aided amplification (RAA) method and applied in E. coli O157:H7 detection at 39 °C in 20 min. The sensitivity of the assay in pure E. coli O157:H7 suspension was 5.6 × 100 CFU/mL. The fluorescent probe-based RAA assay was further applied in three samples, and the limit of detection (LOD) in skimmed milk, lettuces and lake water was 5.4 × 101 CFU/mL, 7.9 × 101 CFU/mL and 5.2 × 101 CFU/mL, separately. This method showed a high sensitivity and short detection time, which has the feasible application in on-site test in real samples.

Foodborne TiO2 Nanoparticles Induced More Severe Hepatotoxicity in Fructose-Induced Metabolic Syndrome Mice via Exacerbating Oxidative Stress-Mediated Intestinal Barrier Damage.

The hazard of titanium dioxide nanoparticles (TiO2 NPs) in diseased population should be given focus due to the huge number of these NPs in foods and medicine. This study aimed to evaluate the stronger biological adverse effect of oral exposure to TiO2 NPs in a fructose-induced metabolic syndrome mouse model. Compared to the normal mice, low-dose (2 mg/kg) TiO2 NPs did not cause severe hepatotoxicity. However, high-dose (20 mg/kg) TiO2 NPs induced aggravated hepatic inflammation, fibrosis, and apoptosis, with substantial alteration of related biochemical parameters in the mouse model. Moreover, significantly increased Ti and lipopolysaccharide burden were observed in metabolic syndrome murine liver and serum, which possibly worsened the portend intestinal leakage. The expression of tight junction-related protein showed that TiO2 NPs induced further increase in serious intestinal permeability. The intestinal inflammatory and oxidative stress response in the model were also assessed. Results showed that TiO2 NPs caused more severe intestinal inflammatory injury by intensifying the oxidative stress in metabolic syndrome mice and then induced further liver injury. This work provides information on the insights into the toxic effect of TiO2 NPs in sub-healthy population.

The responses of Salmonella enterica serovar Typhimurium to vanillin in apple juice through global transcriptomics.

Salmonella enterica serovar Typhimurium can survive some extreme environment in food processing, and vanillin generally recognized as safe is bactericidal to pathogens. Thus, we need to explore the responses of S. Typhimurium to vanillin in order to apply this antimicrobial agent in food processing. In this study, we exposed S. Typhimurium to commercial apple juice with/without vanillin (3.2 mg/mL) at 45 °C for 75 min to determine the survival rate. Subsequently, the 10-min cultures were selected for transcriptomic analysis. Using high-throughput RNA sequencing, genes related to vanillin resistance and their expression changes of S. Typhimurium were identified. The survival curve showed that S. Typhimurium treated with vanillin were inactivated by 5.5 log after 75 min, while the control group only decreased by 2.3 log. Such a discrepancy showed the significant antibacterial effect of vanillin on S. Typhimurium. As a result, 265 differentially expressed genes (DEGs) were found when coping with vanillin, among which, 225 showed up-regulation and 40 DEGs were down-regulated. Treated with vanillin, S. Typhimurium significantly up-regulated genes involved in cell membrane, acid tolerance response (ATR) and oxidative stress response, cold shock cross-protection, DNA repair, virulence factors and some key regulators. Firstly, membrane-related genes, including outer membrane (bamE, mepS, ygdI, lolB), inner membrane (yaiY, yicS) and other proteins (yciC, yjcH), were significantly up-regulated because of the damaged cell membrane. Then, up-regulated proteins associated with arginine synthesis (ArgABCDIG) and inward transportation (ArtI, ArtJ, ArtP and HisP), participated in ATR to pump out the protons inside the cell in this scenario. Next, superoxide stress response triggered by vanillin was found to have a significant up-regulation as well, which was controlled by SoxRS regulon. Besides, NADH-associated (nuoA, nuoB, nuoK, nadE, fre and STM3021), thioredoxin (trxA, trxC, tpx and bcp) and glutaredoxin (grxC and grxD) DEGs led to the increase of the oxidative stress response. Cold shock proteins such as CspA and CspC showed an up-regulation, suggesting it might play a role in cross-protecting S. Typhimurium from vanillin stress. Furthermore, DEGs in DNA repair and virulence factors, including flagellar assembly, adhesins and type III secretion system were up-regulated. Some regulators like fur, rpoE and csrA played a pivotal role in response to the stress caused by vanillin. Therefore, this study sounds an alarm for the risks caused by stress tolerance of S. Typhimurium in food industry.

Simultaneous detection and differentiation of SARS-CoV-2, influenza A virus and influenza B virus by one-step quadruplex real-time RT-PCR in patients with clinical manifestations.

OBJECTIVE: To develop a novel quadruplex real-time reverse transcription polymerase chain reaction (rRT-PCR) assay for the diagnosis of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection, differential diagnosis and detection of co-infections. METHODS: A one-step quadruplex rRT-PCR assay was developed for simultaneous detection and differentiation of SARS-CoV-2 ORF1ab and N genes, influenza A virus (hIAV) and influenza B virus (hIBV). RESULTS: The quadruplex rRT-PCR assay had good sensitivity and specificity. Correlation coefficients and amplification efficiencies of all singleplex and quadruplex rRT-PCR reactions were within acceptable ranges. The 95% lower limits of detection for plasmid standards and positive nucleic acid extracts of the quadruplex rRT-PCR assay were 57.38-95.11 copies/µL and 114.65-154.25 copies/µL, respectively. Excellent results were attained for inter- and intra-assay reproducibility. Among these clinical samples, only four samples showed results inconsistent with the singleplex rRT-PCR assays. CONCLUSIONS: To the authors' knowledge, this is the first study to report a quadruplex rRT-PCR assay for the detection of two SARS-CoV-2 genes, hIAV and hIBV with perfect clinical performance.

Global transcriptomic analysis of Cronobacter sakazakii CICC 21544 by RNA-seq under inorganic acid and organic acid stresses.

Cronobacter sakazakii is a common foodborne pathogen that can tolerate various stress conditions. Acidic environment is a common stress condition encountered by bacteria in food processing and gastrointestinal digestion, including both inorganic and organic acids. In order to elucidate the Acid Tolerance Response (ATR) of C. sakazakii, we performed high-throughput RNA-seq to compare gene expression under hydrochloric acid and citric acid stresses. In this study, 107 differentially expressed genes (DEGs) were identified in both acids, of which 85 DEGs were functionally related to the regulation of acid tolerance. Multiple layers of mechanisms may be applied by C. sakazakii in response to acid stress: Firstly, in order to reduce excessive intracellular protons, C. sakazakii pumps them out through trans-membrane proteins or consumes them through metabolic reactions. Secondly, under acidic conditions, a large amount of reactive oxygen species and hydroxyl radicals accumulate in the cells, resulting in oxidative damage. C. sakazakii protects cells by up-regulating the antioxidant stress genes such as soxS and madB. Thirdly, C. sakazakii chooses energy efficient metabolic pathways to reduce energy consumption and maintain necessary processes. Finally, genes involved in chemotaxis and motility were differentially expressed to respond to different acidic conditions. This study systematically analyzed the acid-resistant mechanism of C. sakazakii under the stress of organic and inorganic acids, and provided a theoretical basis for better control of its contamination in food.

Construction of Lentivirus-Based Reference Material for RT-PCR Detection of Middle East Respiratory Syndrome Coronavirus and Its Application in External Quality Assessment.

Nucleic acid amplification technology (NAT) has been the most used one for rapid detection of Middle East Respiratory Syndrome coronavirus (MERS-CoV) since MERS-CoV was first detected in 2012. It is important to develop stable and safe reference materials for assessing the quality of NAT kits and performing an external quality assessment (EQA) in different laboratories. In this study, the MERS-CoV RNA fragments including upE, ORF1b, and N were packed within human immunodeficiency virus type 1 (HIV-1)-like particles. The lyophilized virus-like particles (VLPs) were found to be stable at 37 °C or below and safe to be used not only as the control material for PCR detection of MERS-CoV but also as the reference material for EQA. In an EQA organized by Ningbo International Travel Healthcare Center in China, 49 participating institutions achieved 100% agreement in detecting MERS-CoV using various commercial diagnosis kits and different extraction methods. However, different Ct values reported by different sites for the same sample implied that a need exists to standardize the RNA extraction method and/or the PCR detection conditions between the laboratories.

Actinomyces tangfeifanii sp. nov., isolated from the vulture Aegypius monachus.

A novel, Gram-stain-positive, catalase-positive, non-spore-forming, short rod-shaped strain (VUL4_3T) was isolated from rectal swabs of Old World vultures (Aegypius monachus) from the Tibet-Qinghai Plateau, China. Based on the results of biochemical tests and 16S rRNA gene sequence comparison, strain VUL4_3T was determined to be a member of the genus Actinomyces that is closely related to the type strains of Actinomyces liubingyangii (97.7 % 16S rRNA gene sequence similarity) and Actinomyces marimammalium (96.5 %). Optimal growth occurred at 37 °C, pH 6-7 and with 1 % (w/v) NaCl. The typical major cellular fatty acids of strain VUL4_3T were C18 : 1ω9c, C16 : 0 and C18 : 0. The VUL4_3T genome contained 2 207 832 bp with an average G+C content of 51.9 mol%. DNA-DNA hybridization values between strain VUL4_3T and the above two species of the genus Actinomyces showed less than 32 % DNA-DNA relatedness, supporting a novel species status of strain VUL4_3T. Based on the phenotypic data and phylogenetic inference, the novel species Actinomycestangfeifanii sp. nov. is proposed. The type strain is VUL4_3T (=CGMCC 4.7369T=DSM 103436T).

Long Noncoding RNA GAPLINC Promotes Cells Migration and Invasion in Colorectal Cancer Cell by Regulating miR-34a/c-MET Signal Pathway.

BACKGROUND: Gastric adenocarcinoma predictive long intergenic noncoding RNA (GAPLINC) has been detected in colorectal cancer (CRC) cells and reportedly performs many functions related to tumor proliferation and metastasis. Aim The present study aimed to comprehensively explore the biological functions of GAPLINC and their underlying mechanism in CRC cell. METHODS: The human cancer LncRNA PCR array was used to detect the differentially expressed long noncoding RNAs in human CRC samples. Real-time PCR, dual-luciferase assay, RNA pull-down assay, Transwell assay, and western blot analysis were performed to explore the molecular mechanism underlying GAPLINC functions related to migration and invasion of a human CRC cell line (HCT116). RESULTS: Compared to the non-cancerous tissues, GAPLINC expression was obviously increased in CRC tissues. In HCT116, silencing of GAPLINC weakened cell migration and invasion, while overexpression of GAPLINC significantly promoted cell migration and invasion. Through dual-luciferase, RNA pull-down, and Transwell assays, we verified that miR-34a was the downstream molecule of GAPLINC and that miR-34a negatively regulated the migration and invasion of HCT116 cell. Furthermore, we found that GAPLINC positively regulated the miR-34a target gene c-MET in CRC tissues. CONCLUSIONS: Our findings revealed that GAPLINC was up-regulated in CRC tissues and was involved in the migration and invasion of CRC cells by regulating miR-34a/c-MET signaling pathway.

Development of Neutralization Assay Using an eGFP Chikungunya Virus.

Chikungunya virus (CHIKV), a member of the Alphavirus genus, is an important human emerging/re-emerging pathogen. Currently, there are no effective antiviral drugs or vaccines against CHIKV infection. Herein, we construct an infectious clone of CHIKV and an eGFP reporter CHIKV (eGFP-CHIKV) with an isolated strain (assigned to Asian lineage) from CHIKV-infected patients. The eGFP-CHIKV reporter virus allows for direct visualization of viral replication through the levels of eGFP expression. Using a known CHIKV inhibitor, ribavirin, we confirmed that the eGFP-CHIKV reporter virus could be used to identify inhibitors against CHIKV. Importantly, we developed a novel and reliable eGFP-CHIKV reporter virus-based neutralization assay that could be used for rapid screening neutralizing antibodies against CHIKV.

Genome-wide computational analyses of microRNAs and their targets from Canis familiaris.

By computational analyses, we identified 357 miRNA candidates from Canis familiaris genome, among which 300 are homology of characterized human miRNAs, the remains are not reported in any other animal. Of the 357 miRNA genes, 142 are organized into 53 clusters, and two clusters locate in the paternally imprinted region. These dog miRNAs may regulate more than 800 possible targets, which are involved in a wide range of cellular processes. Remarkably, miR-186 resides in the eighth intron of its target gene in the same orientation, suggesting a feedback regulation of miRNA on its host gene.