"On-Water" accelerated dearomative cycloaddition via aquaphotocatalysis.

Sulfur(VI) fluoride exchange (SuFEx) has emerged as an innovative click chemistry to harness the pivotal connectivity of sulfonyl fluorides. Synthesizing such alkylated S(VI) molecules through a straightforward process is of paramount importance, and their water-compatibility opens the door to a plethora of applications in biorelevant and materials chemistry. Prior aquatic endeavors have primarily focused on delivering catalysts involving ionic mechanisms, studies regarding visible-light photocatalytic transformation are unprecedented. Herein we report an on-water accelerated dearomative aquaphotocatalysis for heterocyclic alkyl SuFEx hubs. Notably, water exerts a pronounced accelerating effect on the [2 + 2] cycloaddition between (hetero)arylated ethenesulfonyl fluorides and inert heteroaromatics. This phenomenon is likely due to the high-pressure-like reactivity amplification at the water-oil interface. Conventional solvents proved totally ineffective, leading to the isomerization of the starting material.

One-step multiplex real-time RT-PCR for detection and typing of dengue virus.

Previous studies reported that severity of dengue is associated with multiple factors, including secondary infection, age, viral load and infecting serotype and genotype. In addition, other studies have reported that a dengue virus-2 (DENV-2) infection is associated with a prognosis of more severe clinical manifestations than DENV-1 and DENV-4 infections. For these reasons, the ability to identify the DENV serotypes is critical for optimal patient diagnosis and epidemiological studies. In this study, we developed a TaqMan probe-based, one-step real-time reverse transcriptase-polymerase chain reaction (RT-PCR) system for detection and serotyping DENV. Our linear dynamic range (101 to 107 copies/reaction) showed the R2 values of DENV-1, 2, 3 and 4 as 0.998, 0.998, 0.994, and 0.998, respectively. The detection limits of DENV-1, 2, 3, and 4, were 10 copies/reaction, 100 copies/reaction, 10 copies/reaction, and 100 copies/reaction, respectively. Specificity test results indicated that this system is specific for DENV-1, 2, 3, and 4 and does not react with other viruses. Finally, we validated our results with five different real-time PCR instruments. Our results showed that the Ct values of the four serotype templates were similar in five real-time PCR instruments. Thus, this system provides an accurate method for detection and serotyping of DENV, which can be applied in diagnostics, surveillance, and epidemiology. Dengue can be found in many nations with varying socioeconomic and monetary resources. The results of our validation analyses using five different real-time PCR instruments suggest that this method can easily and confidently be used world-wide.

Development of a one-step PCR assay with nine primer pairs for the detection of five diarrheagenic Escherichia coli types.

Certain Escherichia coli (E. coli) strains have the ability to cause diarrheal disease. Five types of diarrheagenic E. coli have been identified, including EHEC, ETEC, EPEC, EAEC, and EIEC. To detect these five diarrheagenic types rapidly, we developed a one-step multiplex PCR (MPPCR) assay using nine primer pairs to amplify nine virulence genes specific to the different virotypes, with each group being represented (i.e., stx1 and stx2 for EHEC, lt, sth, and stp for ETEC, eaeA and bfpA for EPEC, aggR for EAEC, and ipaH for EIEC). The PCR primers were constructed using MultAlin. The sensitivity and specificity of the constructed multiplex PCR primers were measured using DNA isolated from diarrheagenic E. coli strains representing each group. The limits of detection were as follows: 5 × 10(1) CFU/ml for EHEC, 5 × 10(3) CFU/ml for ETEC expressing lt and sth, 5 × 10(4) CFU/ml for ETEC expressing stp, 5 × 102 CFU/ml for EPEC, 5 × 10(4) CFU/ml for EAEC, and 5 × 10(2) CFU/ml for EIEC. To confirm the specificity, C. jejuni, C. perfringens, S. Typhimurium, V. parahaemolyticus, L. monocytogenes, Y. enterocolitica, B. cereus, and S. aureus were used as negative controls, and no amplification was obtained for these. Moreover, this kit was validated using 100 fecal samples from patients with diarrhea and 150 diarrheagenic E. coli strains isolated in Korea. In conclusion, the multiplex PCR assay developed in this study is very useful for the rapid and specific detection of five diarrheagenic E. coli types. This single-step assay will be useful as a rapid and economical method, as it reduces the cost and time required for the identification of diarrheagenic E. coli.

A novel multiplex PCR assay for rapid and simultaneous detection of five pathogenic bacteria: Escherichia coli O157:H7, Salmonella, Staphylococcus aureus, Listeria monocytogenes, and Vibrio parahaemolyticus.

Rapid and sensitive detection techniques for foodborne pathogens are important to the food industry. However, traditional detection methods rely on bacterial culture in combination with biochemical tests, a process that typically takes 4 to 7 days to complete. Thus, this study was conducted to address the issue of time lag inherent in traditional methods by developing a novel PCR assay for each of five foodborne pathogenic bacteria. This new system consists of a simultaneous screening method using multiplex PCR in a single reaction tube for the rapid and sensitive detection of each of the five bacteria. Specific primers for multiplex PCR amplification of the Shiga-like toxin (verotoxin type II), femA (cytoplasmic protein), toxR (transmembrane DNA binding protein), iap (invasive associative protein), and invA (invasion protein A) genes were designed to allow simultaneous detection of Escherichia coli O157:H7, Staphylococcus aureus, Vibrio parahaemolyticus, Listeria monocytogenes, and Salmonella, respectively. To confirm the specificity of each primer pair for the respective target gene, three types of experiments were carried out using boiled cell lysates and their DNAs. In the multiplex PCR with mixed DNA samples, specific bands for corresponding genes were simultaneously detected from a single reaction. The detection of all five foodborne pathogenic bacteria could be completed in less than 24 h with this novel PCR method.