Advances in detection methods for viable Salmonella spp.: current applications and challenges.

Salmonella is a common intestinal pathogen that can cause food poisoning and intestinal disease. The high prevalence of Salmonella necessitates efficient and sensitive methods for its identification, detection, and monitoring, especially of viable Salmonella. Conventional culture methods need to be more laborious and time-consuming. And they are relatively limited in their ability to detect Salmonella in the viable but non-culturable status if present in the sample to be tested. As a result, there is an increasing need for rapid and accurate techniques to detect viable Salmonella spp. This paper reviewed the status and progress of various methods reported in recent years that can be used to detect viable Salmonella, such as culture-based methods, molecular methods targeting RNAs and DNAs, phage-based methods, biosensors, and some techniques that have the potential for future application. This review can provide researchers with a reference for additional method options and help facilitate the development of rapid and accurate assays. In the future, viable Salmonella detection approaches will become more stable, sensitive, and fast and are expected to play a more significant role in food safety and public health.

Establishment of an efficient transformation system and its application in regulatory mechanism analysis of biological macromolecules in tea plants.

Tea (Camellia sinensis), one of the most important beverage crops originated from China and is now cultivated worldwide, provides numerous secondary metabolites that account for its health benefits and rich flavor. However, the lack of an efficient and reliable genetic transformation system has seriously hindered the gene function investigation and precise breeding of C. sinensis. In this study, we established a highly efficient, labor-saving, and cost-effective Agrobacterium rhizogenes-mediated hairy roots genetic transformation system for C. sinensis, which can be used for gene overexpression and genome editing. The established transformation system was simple to operate, bypassing tissue culture and antibiotic screening, and only took two months to complete. We used this system to conduct function analysis of transcription factor CsMYB73 and found that CsMYB73 negatively regulates L-theanine synthesis in tea plant. Additionally, callus formation was successfully induced using transgenic roots, and the transgenic callus exhibited normal chlorophyll production, enabling the study of the corresponding biological functions. Furthermore, this genetic transformation system was effective for multiple C. sinensis varieties and other woody plant species. By overcoming technical obstacles such as low efficiency, long experimental periods, and high costs, this genetic transformation will be a valuable tool for routine gene investigation and precise breeding in tea plants.

Graphene oxide-assisted optimized narrow-thermal-cycling amplification for accurate detection of Salmonella spp.

Salmonella is a rod-shaped, Gram-negative zoonotic pathogen that poses a serious global socioeconomic and public health threat. Rapid and accurate detection of Salmonella spp. is critical for effective control of its infection. In this study, an accurate, sensitive and specific graphene oxide-assisted accelerated strand exchange amplification (GO-ASEA) method for rapid detection of Salmonella spp. was developed and validated. The detection limit of the GO-ASEA method was 8.6 × 101 fg µL-1 of Salmonella genomic DNA or 1 × 101 CFU g-1 of Salmonella in spiked chicken faeces free of pre-enrichment. And the GO-ASEA method could specifically detect Salmonella spp. without cross-reactivity with other enteric pathogens. In addition, the novel method achieved Salmonella detection within 30 min and was validated using 209 clinical samples, showing its good clinical applicability. Therefore, the GO-ASEA method is a new optional tool for the rapid detection of pathogenic microorganisms, which is ideal for food safety monitoring and high-throughput detection.

Accurate, rapid and highly sensitive detection of African swine fever virus via graphene oxide-based accelerated strand exchange amplification.

African swine fever is an acute, severe and highly contagious infectious disease caused by African swine fever virus (ASFV), posing a huge threat to the global swine industry. Rapid and accurate diagnostic methods are of great significance for the effective prevention and control of ASFV transmission. In this work, we established and evaluated a graphene oxide-based accelerated strand exchange amplification (GO-ASEA) method for rapid, highly sensitive, and quantitative detection of ASFV. The use of GO provided a novel solution reference for improving the specificity of strand exchange amplification and solving the potential false positive problem caused by primer dimers. The detection limit of the GO-ASEA assay was 5.8 × 10-1 copies per µL of ASFV (equal to 2.9 copies per reaction) or 5.8 × 100 copies per µL of ASFV in spiked swine nasal swabs. The selectivity of the GO-ASEA assay was supported by the ASFV DNA reference material and another seven porcine-derived viruses with similar clinical symptoms. The GO-ASEA assay took only about 29 minutes and was validated with 6 inactivated specimens and 52 swine nasal swabs, showing excellent clinical applicability. The novel assay is an accurate and practical method for rapid, highly sensitive detection of ASFV, and can potentially serve as a robust tool in epidemic prevention and point-of-care diagnosis.

Selenium-enriched Bacillus subtilis yb-114246 improved growth and immunity of broiler chickens through modified ileal bacterial composition.

Here, a Selenium-enriched Bacillus subtilis (SEBS) strain was generated and supplemented to broiler chickens' diet, and the impact in ileum bacterial microbiome, immunity and body weight were assessed. In a nutshell, five hundred 1-old old chicken were randomly divided into five groups: control, inorganic Se, Bacillus subtilis (B. subtilis), SEBS, and antibiotic, and colonization with B. subtilis and SEBS in the gastrointestinal tract (GIT) were measured by fluorescence in situ hybridization (FISH) assay and quantitative real-time polymerase chain reaction (qPCR). In summary, Chicks fed SEBS or B. subtilis had higher body weight than the control chicks or those given inorganic Se. SEBS colonized in distal segments of the ileum improved bacterial diversity, reduced the endogenous pathogen burden and increased the number of Lactobacillus sp. in the ileal mucous membrane. Species of unclassified Lachnospiraceae, uncultured Anaerosporobacter, Peptococcus, Lactobacillus salivarius, and Ruminococcaceae_UCG-014, and unclassified Butyricicoccus in the ileal mucous membrane played a key role in promoting immunity. Inorganic Se supplementation also improved bacterial composition of ileal mucous membranes, but to a less extent. In conclusion, SEBS improved performance and immunity of broiler chickens through colonization and modulation of the ileal mucous membrane microbiome.

Selenium enriched Bacillus subtilis yb-1114246 activated the TLR2-NF-κB1 signaling pathway to regulate chicken intestinal ß-defensin 1 expression.

The aim of this study was to investigate the effects and potential signaling pathway of selenium-enriched Bacillus subtilis (SEBS) on beta defensin 1 (BD1) expression in chicken intestine. Chinese Huainan Partridge chickens (500 individuals) were randomly allocated into five groups, including control, inorganic Se, B. subtilis, SEBS, and a mixture of Se and B. subtilis (Se-BS). After 56 d of feeding, chicken ileal mucous membranes were harvested to detect differences in expression of BD1. The results indicated that BD1 was produced in intestinal crypt cells and secreted into the lumen through the villi brush border. BD1 was up-regulated in distal ileum segments colonized by SEBS and B. subtilis. Chicken primary intestinal crypt cells were cultured and grouped into control, inorganic Se, B. subtilis, SEBS, and Se-BS treatments to identify the receptor of B. subtilis. Results indicated that B. subtilis and SEBS were recognized by toll-like receptor 2 (TLR2), stimulating the NF-κB1 signaling pathway to increase expression of BD-1, which was further enhanced when combined with Se. Pro-inflammatory cytokines TNF-α, IL-1ß, and IL-6 were up-regulated with B. subtilis supplementation, and inhibited under the action of Se. In conclusion, B. subtilis and SEBS were recognized by the TLR2 receptor in the ileal mucous membrane, which activated the TLR2-MyD88-NF-κB1 signaling pathway to upregulate BD1 expression. In addition, Se enhanced recognition of B. subtilis and reduced levels of pro-inflammatory factors caused by estrogenic B. subtilis supplementation.

Application of three tailing-based composites in treating comprehensive electroplating wastewater.

Heavy metals and chemical oxygen demand (COD) are major challenging pollutants for most electroplating wastewater treatment plants. A novel composite material, prepared with a mixture of calcium and sodium compounds and tailings, was simply mixed by ratios and used to treat a comprehensive electroplating wastewater with influent COD, total copper (T-Cu), and total nickel (T-Ni) respectively as 690, 4.01, and 20.60 mg/L on average. Operational parameters, i.e. the contact time, pH, mass ratio of calcium and sodium compounds and tailings, were optimized as 30 min, 10.0, and 4:2:1. Removal rates for COD, T-Cu, and T-Ni could reach 71.8, 90.5, and 98.1%, respectively. No significant effect of initial concentrations on removal of T-Cu and T-Ni was observed for the composite material. The adsorption of Cu(II) and Ni(II) on the material fitted Langmuir and Freundlich isotherms respectively. Weight of waste sludge from the calcium/sodium-tailing system after reaction was 10% less than that from the calcium-tailing system. The tailing-based composite is cost-effective in combating comprehensive electroplating pollution, which shows a possibility of applying the tailings in treating electroplating wastewater.

Variation of Microcystis and microcystins coupling nitrogen and phosphorus nutrients in Lake Erhai, a drinking-water source in Southwest Plateau, China.

Lake Erhai is the second largest lake of Southwest China and an important drinking water source. The lake is currently defined as the preliminary stage of eutrophic states, but facing a serious threat with transfer into intensive eutrophication. The present study examined the dynamics of Microcystis blooms and toxic Microcystis in Lake Erhai during 2010, based on quantitative real-time PCR method using 16S rRNA gene specific for Microcystis and microcystin systhesis gene (mcy), and chemical analysis on microcystin (MC) concentrations. Total Microcystis cell abundance at 16 sampling sites were shown as an average of 1.7 × 10(7) cells l(-1) (1.3 × 10(2)-3.8 × 10(9) cells l(-1)). Microcystin LR (MC-LR) and microcystin RR (MC-RR) were the main variants. The strong southwesterly winds, anticlockwise circular flows and geographical characteristics of lake and phytoplankton community succession impacted the distribution patterns of Chl a and MC in the lake. The concentration of Chl a and MC and abundances of total Microsytis and MC-producing Microsystis (MCM) were shown to be positively correlated with pH, DO and TP, negatively correlated with SD, NO3-N, TN/Chl a and TN/TP, and not correlated with NH4-N, TN, dissolved total nitrogen (DTN) and water temperatures. When TN/TP decrease, Microcystis tended to dominate and MC concentrations tended to increase, suggesting that the "TN/TP rule" can be partially applied to explain the correlation between the cyanobacterial blooms and nutrients N and P only within a certain nutrient level. It is speculated that N and P nutrients and the associated genes (e.g., mcy) may jointly drive MC concentration and toxigenicity of Microcystis in Lake Erhai.

Genetic diversity of bloom-forming Microcystis (cyanobacteria) populations in a hyper-eutrophic pond in central China.

Sequencing the environmental rRNA genes of Microcystis populations, such as by internal transcribed spacer (ITS), has been proven to provide a new insight into the genetic diversity of Microcystis in freshwater. In this study, a 19-month monitoring of Microcystis populations in a hyper-eutrophic pond in Wuhan city, China was conducted through molecular method by sequencing ITS fragments from the environmental DNA library. Three hundred twenty ITS genotypes of Microcystis in this pond were identified from a total of 563 sequences, thus exhibiting high genetic diversity of Microcystis in the pond. Dramatic changes and succession in ITS genotypes were also found during the survey period. Despite the absence of significant dominant ITS genotypes in the pond, several main genotypes were found to have been dominated for a short term. However, Microcystis ITS genotype patterns from 2007 to 2008 presented a complicated situation in this pond. The parsimony network (TCS) analysis showed that two groups were formed based on ITS genotypes.