Rapid and visual detection of Staphylococcus aureus in milk using a recombinase polymerase amplification-lateral flow assay combined with immunomagnetic separation.

AIMS: The aim of this study was to develop a novel approach using lateral flow recombinase polymerase amplification (RPA-LF) combined with immunomagnetic separation (IMS) for the rapid detection of Staphylococcus aureus in milk. METHODS AND RESULTS: Under optimum conditions, the average capture efficiency values for S. aureus strains (104 colony-forming units [CFU] per ml) was above 95.0% in PBST and ~80% in milk within 45 min with 0.7 mg immunomagnetic beads. The RPA-LF assay, which comprised DNA amplification via RPA at 39°C for 10 min and visualization of the amplicons through LF strips for 5 min, detected S. aureus within 15 min. The method only detected S. aureus and did not show cross-reaction with other bacteria, exhibiting a high level of specificity. Sensitivity experiments confirmed a detection limit of RPA-LF assay as low as 600 fg per reaction for the S. aureus genome (corresponding to approximately 36 CFU of S. aureus), which was about 16.7-fold more sensitive than that of the conventional polymerase chain reaction method. When RPA-LF was used in combination with IMS to detect S. aureus inoculated into artificially contaminated milk, it exhibited a detection limit of approximately 40 CFU per reaction. CONCLUSIONS: The newly developed IMS-RPA-LF method enabled detection of S. aureus at levels as low as 40 CFU per reaction in milk samples without culture enrichment for an overall testing time of only 70 min. SIGNIFICANCE AND IMPACT OF THE STUDY: The newly developed IMS-lateral flow RPA-LF assay effectively combines sample preparation, amplification and detection into a single platform. Because of its high sensitivity, specificity and speed, the IMS-RPA-LF assay will have important implications for the rapid detection of S. aureus in contaminated food.

Biological and transcriptional studies reveal VmeL is involved in motility, biofilm formation and virulence in Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a marine pathogen thought to be the leading cause of seafood-borne gastroenteritis globally, urgently requiring efficient management methods. V. parahaemolyticus encodes 12 resistance/nodulation/division (RND) efflux systems. However, research on these systems is still in its infancy. In this study, we discovered that the inactivation of VmeL, a membrane fusion protein within the RND efflux systems, led to reduction of the ability of biofilm formation. Further results displayed that the decreased capacity of Congo red binding and the colony of ΔvmeL is more translucent compared with wild type strains, suggested reduced biofilm formation due to decreased production of biofilm exopolysaccharide upon vmeL deletion. In addition, the deletion of vmeL abolished surface swarming and swimming motility of V. parahaemolyticus. Additionally, deletion of vmeL weakened the cytotoxicity of V. parahaemolyticus towards HeLa cells, and impaired its virulence in a murine intraperitoneal infection assay. Finally, through RNA-sequencing, we ascertained that there were 716 upregulated genes and 247 downregulated genes in ΔvmeL strain. KEGG enrichment analysis revealed that quorum sensing, bacterial secretion systems, ATP-binding cassette transporters, and various amino acid metabolism pathways were altered due to the inactivation of vmeL. qRT-PCR further confirmed that genes accountable to the type III secretion system (T3SS1) and lateral flagella were negatively affected by vmeL deletion. Taken together, our results suggest that VmeL plays an important role in pathogenicity, making it a good target for managing infection with V. parahaemolyticus.

Transcriptional silencing is associated with extensive methylation of the CMV promoter following adenoviral gene delivery to muscle.

BACKGROUND: Although the transient nature of transgene expression using first-generation adenovirus (Ad) vectors is well known, the exact mechanisms responsible for this phenomenon are uncertain. METHODS: Rats were given intramuscular (i.m.) injections of a first-generation Ad containing the human fibroblast growth factor 4 (hFGF-4) gene driven by the cytomegalovirus (CMV) promoter and enhancer (CMV-PE). The copy number of hFGF-4 mRNA and viral DNA was measured in the same muscles by quantitative RT-PCR and quantitative PCR at times between 1 h and 84 days after virus injection. Quantitative Southern blot analysis for the intact hFGF-4 transcription unit DNA was also performed, and the methylation status of the CMV-PE DNA in the muscle was determined using bisulfite sequencing. RESULTS: The copy number of hFGF-4 mRNA peaked at 6 h then decreased 56-fold by 24 h, and a further 240-fold between days 3 and 28. Although the viral DNA copy number also decreased 23-fold between 6 h and 28 days, the ratio of copies of hFGF-4 mRNA per copy of viral DNA decreased 385-fold over this period. Methylation of the CMV-PE DNA in the muscle at both CpG and non-CpG sites was observed 24 h after virus administration and had increased at day 7. CONCLUSIONS: Decreased transcription associated with extensive methylation of the CMV-PE was the major mechanism responsible for the decrease in transgene mRNA levels. Strategies for preventing transcriptional silencing will be valuable for improving the duration of transgene expression from adenoviral vectors.