A highly susceptible hACE2-transgenic mouse model for SARS-CoV-2 research.

Several animal models have been used to assist the development of vaccines and therapeutics since the COVID-19 outbreak. Due to the lack of binding affinity of mouse angiotensin-converting enzyme II (ACE2) to the S protein of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), increasing the susceptibility of mice to SARS-CoV-2 infection was considered in several ways. Here, we generated a COVID-19 mouse model expressing human ACE2 (hACE2) under the control of the CAG promoter. Overexpression of hACE2 did not pose a significant effect on weight growth. After SARS-CoV-2 inoculation, mice showed obvious viral replication and production of inflammation within 7 days, with a gradual decrease in body weight until death. Virological testing found that the virus can replicate in the respiratory system, small intestine, and brain. Additionally, this mouse model was applied to compare two antibody drug candidates, the anti-RBD antibody (MW06) and the mouse CD24-conjugated anti-RBD antibody (mCD24-MW06). Differences in antiviral effects between these two antibodies can be demonstrated in this mouse model when a challenge dose that invalidates the anti-RBD antibody treatment was used. This study provided a new mouse model for studying SARS-CoV-2 pathogenesis and evaluating potential interventions.

Preparation of polyclonal antibody against a universal bacterial antigen OmpA deduced by bioinformatic analysis and preliminary evaluation of concentration effects on foodborne pathogens.

Rapid and ultrasensitive microbial detection in actual samples have challenges because of target pathogen diversity and low abundance. In this study, we attempted to capture and concentrate multiple pathogens by combining magnetic beads with polyclonal antibodies against a universal antigen of ompA, LAMOA-1, before further detection. A protein sequence consisting of 241 amino acids with spatial conformation similar to E. coli ompA was identified and expressed as a recombinant protein in prokaryotes according to the results of sequence alignment among 432 sequences of ompA belonging to intestinal bacteria from gram-negative bacteria. Purified from immunized rabbits, the anti-LAMOA-1 antibody was shown to effectively recognize 12 foodborne bacterial species. Antibody-conjugated beads were used to concentrate the bacteria when the bacterial concentration in artificially contaminated samples is between 10 and 100 CFU/mL, which shortens detection duration by 8-24 h. The enrichment strategy is potentially beneficial for detection of foodborne pathogens.

Novel insight into the underlying dysregulation mechanisms of immune cell-to-cell communication by analyzing multitissue single-cell atlas of two COVID-19 patients.

How does SARS-CoV-2 cause lung microenvironment disturbance and inflammatory storm is still obscure. We here performed the single-cell transcriptome sequencing from lung, blood, and bone marrow of two dead COVID-19 patients and detected the cellular communication among them. Our results demonstrated that SARS-CoV-2 infection increase the frequency of cellular communication between alveolar type I cells (AT1) or alveolar type II cells (AT2) and myeloid cells triggering immune activation and inflammation microenvironment and then induce the disorder of fibroblasts, club, and ciliated cells, which may cause increased pulmonary fibrosis and mucus accumulation. Further study showed that the increase of T cells in the lungs may be mainly recruited by myeloid cells through ligands/receptors (e.g., ANXA1/FPR1, C5AR1/RPS19, and CCL5/CCR1). Interestingly, we also found that certain ligands/receptors (e.g., ANXA1/FPR1, CD74/COPA, CXCLs/CXCRs, ALOX5/ALOX5AP, CCL5/CCR1) are significantly activated and shared among lungs, blood and bone marrow of COVID-19 patients, implying that the dysregulation of ligands/receptors may lead to immune cell's activation, migration, and the inflammatory storm in different tissues of COVID-19 patients. Collectively, our study revealed a possible mechanism by which the disorder of cell communication caused by SARS-CoV-2 infection results in the lung inflammatory microenvironment and systemic immune responses across tissues in COVID-19 patients.

Amplification-Free Detection of SARS-CoV-2 Down to Single Virus Level by Portable Carbon Nanotube Biosensors.

The rapid and sensitive detection of trace-level viruses in a simple and reliable way is of great importance for epidemic prevention and control. Here, a multi-functionalized floating gate carbon nanotube field effect transistor (FG-CNT FET) based biosensor is reported for the single virus level detection of SARS-CoV-2 virus antigen and RNA rapidly with a portable sensing platform. The aptamers functionalized sensors can detect SARS-CoV-2 antigens from unprocessed nasopharyngeal swab samples within 1 min. Meanwhile, enhanced by a multi-probe strategy, the FG-CNT FET-based biosensor can detect the long chain RNA directly without amplification down to single virus level within 1 min. The device, constructed with packaged sensor chips and a portable sensing terminal, can distinguish 10 COVID-19 patients from 10 healthy individuals in clinical tests both by the RNAs and antigens by a combination detection strategy with an combined overall percent agreement (OPA) close to 100%. The results provide a general and simple method to enhance the sensitivity of FET-based biochemical sensors for the detection of nucleic acid molecules and demonstrate that the CNT FG FET biosensor is a versatile and reliable integrated platform for ultrasensitive multibiomarker detection without amplification and has great potential for point-of-care (POC) clinical tests.

Single-cell transcriptomics of immune cells in lymph nodes reveals their composition and alterations in functional dynamics during the early stages of bubonic plague.

Bubonic plague caused by Yersinia pestis is highly infectious and often fatal. Characterization of the host immune response and its subsequent suppression by Y. pestis is critical to understanding the pathogenesis of Y. pestis. Here, we utilized single-cell RNA sequencing to systematically profile the transcriptomes of immune cells in draining lymph nodes (dLNs) during the early stage of Y. pestis infection. Dendritic cells responded to Y. pestis within 2 h post-infection (hpi), followed by the activation of macrophages/monocytes (Mφs/Mons) and recruitment of polymorphonuclear neutrophils (PMNs) to dLNs at 24 hpi. Analysis of cell-to-cell communication suggests that PMNs may be recruited to lymph nodes following the secretion of CCL9 by Mφs/Mons stimulated through CCR1-CCL9 interaction. Significant functional suppression of all the three innate immune cell types occurred during the early stage of infection. In summary, we present a dynamic immune landscape, at single-cell resolution, of murine dLNs involved in the response to Y. pestis infection, which may facilitate the understanding of the plague pathogenesis of during the early stage of infection.

Targeted screening of genetic associations with COVID-19 susceptibility and severity.

The COVID-19 pandemic has resulted in great morbidity and mortality worldwide and human genetic factors have been implicated in the susceptibility and severity of COVID-19. However, few replicate researches have been performed, and studies on associated genes mainly focused on genic regions while regulatory regions were a lack of in-depth dissection. Here, based on previously reported associated variants and genes, we designed a capture panel covering 1,238 candidate variants and 25 regulatory regions of 19 candidate genes and targeted-sequenced 96 mild and 145 severe COVID-19 patients. Genetic association analysis was conducted between mild and severe COVID-19 patients, between all COVID-19 patients and general population, or between severe COVID-19 patients and general population. A total of 49 variants were confirmed to be associated with susceptibility or severity of COVID-19 (p < 0.05), corresponding to 18 independent loci. Specifically, rs1799964 in the promoter of inflammation-related gene TNF, rs9975538 in the intron of interferon receptor gene IFNAR2, rs429358 in the exon of APOE, rs1886814 in the intron of FOXP4-AS1 and a list of variants in the widely reported 3p21.31 and ABO gene were confirmed. It is worth noting that, for the confirmed variants, the phenotypes of the cases and controls were highly consistent between our study and previous reports, and the confirmed variants identified between mild and severe patients were quite different from those identified between patients and general population, suggesting the genetic basis of susceptibility and severity of SARS-CoV-2 infection might be quite different. Moreover, we newly identified 67 significant associated variants in the 12 regulatory regions of 11 candidate genes (p < 0.05). Further annotation by RegulomeDB database and GTEx eQTL data filtered out two variants (rs11246060 and rs28655829) in the enhancer of broad-spectrum antiviral gene IFITM3 that might affect disease severity by regulating the gene expression. Collectively, we confirmed a list of previously reported variants and identified novel regulatory variants associated with susceptibility and severity of COVID-19, which might provide biological and clinical insights into COVID-19 pathogenesis and treatment.

Comparison of the respiratory tract microbiome in hospitalized COVID-19 patients with different disease severity.

Little is known about the characteristics of respiratory tract microbiome in Coronavirus disease 2019 (COVID-19) inpatients with different severity. We conducted a study that expected to clarify these characteristics as much as possible. A cross-sectional study was conducted to characterize respiratory tract microbial communities of 69 COVID-19 inpatients from 64 nasopharyngeal swabs and 5 sputum specimens using 16S ribosomal RNA gene V3-V4 region sequencing. The bacterial profiles were analyzed to find potential biomarkers by the two-step method, the combination of random forest model and the linear discriminant analysis effect size, and explore the connections with clinical characteristics by Spearman's rank test. Compared with mild COVID-19 patients, severe patients had significantly decreased bacterial diversity (p-values were less than 0.05 in the alpha and beta diversity) and relative lower abundance of opportunistic pathogens, including Actinomyces, Prevotella, Rothia, Streptococcus, Veillonella. Eight potential biomarkers including Treponema, Leptotrichia, Lachnoanaerobaculum, Parvimonas, Alloprevotella, Porphyromonas, Gemella, and Streptococcus were found to distinguish the mild COVID-19 patients from the severe COVID-19 patients. The genera of Actinomyces and Prevotella were negatively correlated with age in two groups. Intensive care unit admission, neutrophil count, and lymphocyte count were significantly correlated with different genera in the two groups. In addition, there was a positive correlation between Klebsiella and white blood cell count in two groups. The respiratory tract microbiome had significant differences in COVID-19 patients with different severity. The value of the respiratory tract microbiome as predictive biomarkers for COVID-19 severity deserves further exploration.

Comparison of reverse-transcription qPCR and droplet digital PCR for the detection of SARS-CoV-2 in clinical specimens of hospitalized patients.

Accurate detection of severe acute respiratory syndrome coronavirus 2 is not only necessary for viral load monitoring to optimize treatment in hospitalized coronavirus disease 2019 patients, but also critical for deciding whether the patient could be discharged without any risk of viral shedding. Digital droplet PCR (ddPCR) is more sensitive than reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and is usually considered the superior choice. In the current study, we compared the clinical performance of RT-qPCR and ddPCR using oropharyngeal swab samples from patients hospitalized in the temporary Huoshenshan Hospital, Wuhan, Hubei, China. Results demonstrated that ddPCR was indeed more sensitive than RT-qPCR. Negative results might be caused by poor sampling technique or recovered patients, as the range of viral load in these patients varied significantly. In addition, both methods were highly correlated in terms of their ability to detect all three target genes as well as the ratio of copies of viral genes to that of the IC gene. Furthermore, our results evidenced that both methods detected the N gene more easily than the ORF gene. Taken together, these findings imply that the use of ddPCR, as an alternative to RT-qPCR, is necessary for the accurate diagnosis of hospitalized coronavirus disease 2019 patients.

Global transcriptomic characterization of T cells in individuals with chronic HIV-1 infection.

To obtain a comprehensive scenario of T cell profiles and synergistic immune responses, we performed single-cell RNA sequencing (scRNA-seq) on the peripheral T cells of 14 individuals with chronic human immunodeficiency virus 1 (HIV-1) infection, including nine treatment-naive (TP) and eight antiretroviral therapy (ART) participants (of whom three were paired with TP cases), and compared the results with four healthy donors (HD). Through analyzing the transcriptional profiles of CD4+ and CD8+ T cells, coupled with assembled T cell receptor sequences, we observed the significant loss of naive T cells, prolonged inflammation, and increased response to interferon-α in TP individuals, which could be partially restored by ART. Interestingly, we revealed that CD4+ and CD8+ Effector-GNLY clusters were expanded in TP cases, and persistently increased in ART individuals where they were typically correlated with poor immune restoration. This transcriptional dataset enables a deeper understanding of the pathogenesis of HIV-1 infection and is also a rich resource for developing novel immune targeted therapeutic strategies.

GP73 is a glucogenic hormone contributing to SARS-CoV-2-induced hyperglycemia.

Severe cases of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are associated with elevated blood glucose levels and metabolic complications. However, the molecular mechanisms for how SARS-CoV-2 infection alters glycometabolic control are incompletely understood. Here, we connect the circulating protein GP73 with enhanced hepatic gluconeogenesis during SARS-CoV-2 infection. We first demonstrate that GP73 secretion is induced in multiple tissues upon fasting and that GP73 stimulates hepatic gluconeogenesis through the cAMP/PKA signaling pathway. We further show that GP73 secretion is increased in cultured cells infected with SARS-CoV-2, after overexpression of SARS-CoV-2 nucleocapsid and spike proteins and in lungs and livers of mice infected with a mouse-adapted SARS-CoV-2 strain. GP73 blockade with an antibody inhibits excessive glucogenesis stimulated by SARS-CoV-2 in vitro and lowers elevated fasting blood glucose levels in infected mice. In patients with COVID-19, plasma GP73 levels are elevated and positively correlate with blood glucose levels. Our data suggest that GP73 is a glucogenic hormone that likely contributes to SARS-CoV-2-induced abnormalities in systemic glucose metabolism.

Genomic characteristics and pathogenicity of a new bat adenoviruses strains that was isolated in at sites along the southeastern coasts of the P. R. of China from 2015 to 2019.

Bats are important reservoirs for many kinds of emerging zoonotic viruses. In order to explore potential pathogens carried by bats and trace the source of adenovirus outbreaks on the southeastern coast of China, we took pharyngeal and anal swabs from a total of 552 bats (Rhinolophus pusillus) collected from various areas of Chinese southeastern coast. Adenoviruses were identified in 36 out of the 552 samples (6.5%) . Complete genome sequences of two adenovirus isolations from Vero E6 cells were obtained, which were further validated as identical strains via next-generation sequencing and were named Bat-Advcxc6. The cell culture inoculated with the two samples exhibited remarkable cytopathic changes. The full genome has 37,315 bp and owns 29 open reading frames. Phylogenetic analyses confirmed that Bat-Advcxc6 represented a novel bat adenovirus species in the genus Mastadenovirus. Transmission electron microgram showed clear virus particles. Bat-Advcxc6 shared similar characteristics of G + C contents with Bat mastadenovirus WIV11 (Bat mastadenovirus C) found in China in 2016, but differed from this serotype due to a <75% similarity with DNA polymerase amino acid sequences in WIV11. As it is a newly found adenovirus strain according to the international classification criteria, further analyses of virus dynamics, epithelial invasion, and immunization assays are required to explore its potential threats of cross-species transmission.

Rapid Identification of Pseudomonas fluorescens Harboring Thermostable Alkaline Protease by Real-Time Loop-Mediated Isothermal Amplification.

ABSTRACT: Thermostable alkaline protease (TAP) harbored by Pseudomonas fluorescens decomposes protein in milk and dairy products, leading to milk and dairy product spoilage during storage. Thus, a specific, sensitive, rapid, and simple method is required to detect TAP-harboring P. fluorescens. Two sets of primers targeting the aprX and gyrB genes of P. fluorescens were designed. The detection system and conditions were optimized, and a real-time loop-mediated isothermal amplification (real-time LAMP) method was developed for the simultaneous detection of TAP-harboring P. fluorescens in two separate reaction tubes. The phylogenetic tree targeting aprX showed that P. fluorescens and Pseudomonas lurida clustered on the same branch. The phylogenetic tree targeting gyrB showed that P. fluorescens clustered on the same branch with 95% confidence value, whereas P. lurida clustered on different branches. DNA of 16 strains of P. fluorescens and 34 strains of non-P. fluorescens was detected by real-time LAMP. TAP-harboring P. fluorescens can only be identified when the real-time LAMP detection results of both aprX and gyrB are positive. The dissociation temperatures of aprX and gyrB in the real-time LAMP-amplified products were approximately 90.0 and 88.0°C, respectively. The detection limits of the real-time LAMP targeting aprX and gyrB were 4.9 CFU per reaction in pure culture and 2.2 CFU per reaction in skimmed milk. The coefficient of variation of the repeatability test was less than 2%, indicating that the established real-time LAMP of P. fluorescens targeting gyrB and aprX has good stability and repeatability. Real-time LAMP was used to test 200 raw milk samples for the presence of TAP-harboring P. fluorescens in 3 h, and the coincidence rate of the results with those obtained using the traditional method, which takes at least 5 to 7 days, was 100%. Real-time LAMP will be a practical and effective method for accurate and rapid identification of TAP-harboring P. fluorescens in raw milk.

Genome-wide association study of COVID-19 severity among the Chinese population.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes a broad clinical spectrum of coronavirus disease 2019 (COVID-19). The development of COVID-19 may be the result of a complex interaction between the microbial, environmental, and host genetic components. To reveal genetic determinants of susceptibility to COVID-19 severity in the Chinese population, we performed a genome-wide association study on 885 severe or critical COVID-19 patients (cases) and 546 mild or moderate patients (controls) from two hospitals, Huoshenshan and Union hospitals at Wuhan city in China. We identified two loci on chromosome 11q23.3 and 11q14.2, which are significantly associated with the COVID-19 severity in the meta-analyses of the two cohorts (index rs1712779: odds ratio [OR] = 0.49; 95% confidence interval [CI], 0.38-0.63 for T allele; P = 1.38 × 10-8; and index rs10831496: OR = 1.66; 95% CI, 1.38-1.98 for A allele; P = 4.04 × 10-8, respectively). The results for rs1712779 were validated in other two small COVID-19 cohorts in the Asian populations (P = 0.029 and 0.031, respectively). Furthermore, we identified significant eQTL associations for REXO2, C11orf71, NNMT, and CADM1 at 11q23.3, and CTSC at 11q14.2, respectively. In conclusion, our findings highlight two loci at 11q23.3 and 11q14.2 conferring susceptibility to the severity of COVID-19, which might provide novel insights into the pathogenesis and clinical treatment of this disease.

Environmental contamination with SARS-CoV-2 in COVID-19 hospitals in Wuhan, China, 2020.

Coronavirus disease 2019 (COVID-19) pandemic has caused high number of infections and deaths of healthcare workers globally. Distribution and possible transmission route of SARS-CoV-2 in hospital environment should be clarified. We herein collected 431 environmental (391 surface and 40 air) samples in the intensive care unit (ICU) and general wards (GWs) of three hospitals in Wuhan, China from February 21 to March 4, 2020, and detected SARS-CoV-2 RNA by real-time quantitative PCR. The viral positive rate in the contaminated areas was 17.8% (28/157), whereas there was no virus detected in the clean areas. Higher positive rate (22/59, 37.3%) was found in ICU than that in GWs (3/63, 4.8%). The surfaces of computer keyboards and mouse in the ICU were the most contaminated (8/10, 80.0%), followed by the ground (6/9, 66.7%) and outer glove (2/5, 40.0%). From 17 air samples in the contaminated areas, only one sample collected at a distance of around 30 cm from the patient was positive. Enhanced surface disinfection and hand hygiene effectively decontaminated the virus from the environment. This finding might help understand the transmission route and contamination risk of SARS-CoV-2 and evaluate the effectiveness of infection prevention and control measures in healthcare facilities.

Development and evaluation of a loop-mediated isothermal amplification assay for clinical diagnosis of respiratory human adenoviruses emergent in China.

Three human adenovirus (HAdV) genotypes, HAdV-7, HAdV-14, and HAdV-55, emerged as the most prevalent variants in China over the past decade and caused both sporadic, fatal cases and frequent, large outbreaks. Early diagnosis is essential to control infections and endemics. Here, we established a loop-mediated isothermal amplification (LAMP) assay coupled with an instrument-free nucleic acid extraction device recently developed by our group; the assay could detect all the 3 prevalent HAdV genotypes. Specificity analysis showed no cross-reactivity with other common respiratory pathogens and the analytical sensitivity was as low as 10 copies/µL. All detection steps could be completed within 1 hour. The assay's performance was evaluated using clinical samples and compared with the gold standard RT-PCR method, showing highly consistent results. The LAMP assay developed here could be readily used in basic laboratory facilities and with minimal DNA extraction equipment, and as a reliable screening test in a resource-limited setting.

Clinical features and antibody response of patients from a COVID-19 treatment hospital in Wuhan, China.

Coronavirus disease 2019 (COVID-19) has rapidly evolved into a global pandemic. A total of 1578 patients admitted into a newly built hospital specialized for COVID-19 treatment in Wuhan, China, were enrolled. Clinical features and the levels of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoglobulin (Ig)M and IgG were analyzed. In total, 1532 patients (97.2%) were identified as laboratory-confirmed cases. Seventy-seven patients were identified as asymptomatic carriers (n = 64) or SARS-CoV-2 RNA positive before symptom onset (n = 13). The positive rates of SARS-CoV-2 IgM and IgG were 80.4% and 96.8%, respectively. The median of IgM and IgG titers were 37.0A U/ml (interquartile range [IQR]: 13.4-81.1 AU/ml) and 156.9 AU/ml (IQR: 102.8-183.3 AU/ml), respectively. The IgM and IgG levels of asymptomatic patients (median titers, 8.3 AU/ml and 100.3 AU/ml) were much lower than those in symptomatic patients (median titers, 38.0 AU/ml and 158.2 AU/ml). A much lower IgG level was observed in critically ill patients 42-60 days after symptom onset. There were 153 patients with viral RNA shedding after IgG detection. These patients had a higher proportion of critical illness during hospitalization (p < .001) and a longer hospital stay (p < .001) compared to patients with viral clearance after IgG detection. Coronary heart disease (odds ratio [OR], 1.89 [95% confidence interval [CI], 1.11-3.24]; p = .020), and intensive care unit admission (OR, 2.47 [95% CI, 1.31-4.66]; p = .005) were independent risk factors associated with viral RNA shedding after IgG detection. Symptomatic patients produced more antibodies than asymptomatic patients. The patients who had SARS-CoV-2 RNA shedding after developing IgG were more likely to be sicker patients.

Development and evaluation of a serological test for diagnosis of COVID-19 with selected recombinant spike proteins.

Serological test is a valuable diagnostic tool for coronavirus disease 2019 (COVID-19). However, considerable improvements to these tests are needed, especially in the detection sensitivity. In this study, six recombinant nucleocapsid and spike proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were prepared and evaluated, including three prokaryotic expression nucleocapsid proteins (rN, rN1, rN2) and three eukaryotic expression spike proteins (rS1, rS-RBD, rS-RBD-mFc). The recombinant proteins with the highest ELISA titers (rS1 and rS-RBD-mFc) were selected to develop a double-antigen sandwich colloidal gold immunochromatography assay (GICA) to detect total antibodies against SARS-CoV-2. The clinical evaluation results showed that the sensitivity and specificity of GICA were 92.09% (419/455) and 99.44% (706/710), respectively. Moreover, a significant number (65.63%, 21/32) of COVID-19 patients with undetectable viral RNA were correctly diagnosed by the GICA method. In conclusion, the eukaryotic expression spike proteins (rS1 and rS-RBD-mFc) are more suitable than the prokaryotic expression nucleocapsid proteins for serological diagnosis of SARS-CoV-2. The proposed GICA for detection of total antibodies could be a powerful complement to the current RNA tests for COVID-19.

Pretreatment with Retro-2 protects cells from death caused by ricin toxin by retaining the capacity of protein synthesis.

The current study explores the detoxification effect of Retro-2 on ricin toxin (RT) cytotoxicity, as well as the mechanisms underlying such effects, to provide a basis for follow-up clinical applications of Retro-2. The mouse-derived mononuclear/macrophage cell line, RAW264.7, was used to evaluate the detoxification effect of Retro-2 on RT by detecting cell viability, capacity for protein synthesis and the expression of cytokines, as well as endoplasmic reticulum stress (ERS)-related mRNA. The results indicated that many cells died when challenged with concentrations of RT ≥50ng/mL. The protein synthesis capacity of cells decreased when challenged with 200ng/mL RT for 2hours. Furthermore, the synthesis and release of many cytokines decreased, while the expression of cytokines or ERS-related mRNA increased when challenged with 200ng/mL of RT for 12 or more hours. However, cell viability, capacity for protein synthesis and release levels of many cytokines were higher, while the expression levels of cytokine, or ERS-related mRNA, were lower in cells pretreated with 20µm Retro-2 and challenged with RT, compared with those that had not been pretreated with Retro-2. In conclusion, Retro-2 retained the capacity for protein synthesis inhibited by RT, alleviated ERS induced by RT and increased the viability of cells challenged with RT. Retro-2 shows the potential for clinical applications.

Multiplex real-time SYBR Green I PCR assays for simultaneous detection of 15 common enteric pathogens in stool samples.

Diarrheal diseases account for more than 50% of foodborne diseases worldwide, the majority of which occur in infants and young children. The traditional bacterial detection method is complex and time-consuming; therefore, it is necessary to establish a rapid and convenient detection method that can detect multiple pathogens simultaneously. In this study, we developed a set of five multiplex real-time SYBR Green I PCR assays to simultaneously detect 15 common enteric pathogens based on the Homo-Tag Assisted Non-Dimer system. These assays effectively reduced primer-dimer formation and improved the stability, uniformity, and amplification efficiency of multiplex PCR. The detection limit of the multiplex SYBR Green I PCR system was approximately 104-106 CFU/mL for stool specimens. Furthermore, we vitrified heat-unstable components on the cap of a reaction tube, showing that Taq DNA polymerase, dNTPs, primers, and SYBR Green I remained stable at 25 °C. In summary, we developed multiplex SYBR Green I PCR assays that can simultaneously detect 15 enteric pathogens. This method is comprehensive, rapid, inexpensive, accurate, and simple and displays high specificity.

A small non-coding RNA facilitates Brucella melitensis intracellular survival by regulating the expression of virulence factor.

Brucella species are the causative agents of brucellosis, a worldwide zoonotic disease that affects a broad range of mammals and causes great economic losses. Small regulatory RNAs (sRNAs) are post-transcriptional regulatory molecules that participate in the stress adaptation and pathogenesis of Brucella. In this study, we characterized the role of a novel sRNA, BSR1141, in the intracellular survival and virulence of Brucella melitensis. The results show that BSR1141 was highly induced during host infections and under in vitro stress situations that simulated the conditions encountered within host phagocytes. In addition, a BSR1141 mutant showed reduced survival both under in vitro stress conditions and in mice, confirming the role of BSR1141 in Brucella intracellular survival. Bioinformatic and experimental approaches revealed that BSR1141 affects the expression of many target genes, including the Brucella virulence component virB2. These data indicate that BSR1141 could influence the expression of virB2, which is important for B. melitensis pathogenesis and intracellular survival. This work provides new insight into the mechanism of adaptation to environmental stress and into the pathogenesis of intracellular pathogens.