Salmonella is the most common cause of bacterial food poisoning in humans worldwide. Thus, rapid and reliable methods for the detection of this pathogen are required. Real-time reverse-transcriptase polymerase chain reaction (rt-RT-PCR), which detects the presence of mRNA (shorter half-life than DNA) has shown great potential for detecting viable pathogens. We recently identified a few new potential specific DNA sequences for Salmonella enterica using a comparative genomics method (Chen et al., 2010). In the present study, we examined the expression of the Salmonella-specific sigDE operon (encoding invasion proteins within the pathogenicity island 5) under typical growth conditions to determine whether sigDE could be a useful viability marker for the bacterium. We then assayed sigDE mRNA from cells heat-treated at 60°C, 100°C, and 121°C (autoclaved), and found that mRNA was degraded in autoclaved bacterial samples. These results showed that the sigDE transcript is a suitable mRNA target for rt-RT-PCR with samples pretreated at 121°C. Thus, an rt-RT-PCR using sigDE primers was developed for the detection of viable Salmonella. An RNA internal amplification control was constructed by overlap extension PCR, synthesized using in vitro transcription with a T7 RNA polymerase promoter, and incorporated into the rt-RT-PCR system to eliminate false-negative results. The rt-RT-PCR system has the capability of specifically detecting all the tested S. enterica serovars, and the detection limit of this assay with cultures of Salmonella Typhimurium ATCC 13311 was 10(1) colony-forming units (CFU)/mL. After 18-h enrichment, sigDE-based rt-RT-PCR could detect as low as 10(0) CFU/mL of Salmonella from egg broth and milk.
Metastasis and invasion, the important characteristics of malignant tumors, are closely associated with a series of changes in the expression of genes and proteins. In this study, we compare mRNA and protein expression in high and low metastasis adenoid cystic carcinoma cell lines by mRNA suppression subtractive hybridization and two-dimensional electrophoresis combined with peptide mass fingerprint analysis. 34 differentially expressed genes were obtained using suppression subtractive hybridization experiments including 6 highly expressed gene sequences in the high metastasis cell line, and 28 in the low metastasis cell line. RNA dot blot hybridization further confirmed the results after excluding false positives. For protein analysis, ten significantly different protein spots were detected using two-dimensional gel electrophoresis technique combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI- TOF-MS). The results then compare with the SWISS PROT database. These results suggest that high tumor metastasis of adenoid cystic carcinoma is associated with multiple genes whose function include angiogenesis, protein synthesis, signal transduction, modulation of cell cycle, molecular chaperones, and immune co-stimulating molecule. Moreover, the results of the phenotypic function-related expression mapping analysis at the mRNA and protein level revealed obvious complementarities, providing important clues for further study of the molecular mechanism of metastasis, metastasis control and possible targets for cancer gene therapy.
Background
Escherichia coli O157: H7, being the cause of hemorrhagic colitis in human, is recognized as one of the most dangerous and widespread foodborne pathogens. A highly specific, sensitive and rapid E. coli O157: H7 detection method need to be developed since the traditional detection methods are complex, costly and time-consuming.
Objective
In this study, a recombinase aided amplification (RAA) assisted CRISPR/Cas12a (RAA-CRISPR/Cas12a) fluorescence platform for specific, sensitive and rapid nucleic acid detection of E. coli O157: H7 was introduced.
Methods
Firstly, the feasibility (components of CRISPR/Cas12a system) of the developed method was evaluated. Then, a total of 34 bacterial strains were used for specificity test, and gradient dilutions of extracted DNA and bacterial solutions of E. coli O157: H7 were prepared for sensitivity test. Thirdly, a real-time PCR assay for detection of the specific wzy gene of E. coli O157: H7 (FDA’s Bacteriological Analytical Manual) was used for sensitivity comparison. Finally, analysis of RAA-CRISPR/Cas12a detection in spiked and 93 real ground beef samples was carried out.
Results
The developed RAA-CRISPR/Cas12a method showed high specificity and the detection could be completed within 30 min (after 4 h enrichment in spiked ground beef samples). The limit of detection (LOD) of bacterial concentrations and genomic DNA was 5.4 × 102 CFU/mL and 7.5 × 10−4 ng/μL, respectively, which exhibited higher sensitivity than RAA-gel electrophoresis and RT-PCR methods. Furthermore, it was shown that E. coli O157: H7 in ground beef samples could be positively detected after 4 h enrichment when the initial bacterial inoculum was 9.0 × 10° CFU/25 g. The detection results of RAA-CRISPR/Cas12a method were 100% consistent with those of the RT-PCR and traditional culture-based methods while screening the E. coli O157: H7 from 93 local collected ground beef samples.
Conclusions
The developed RAA-CRISPR/Cas12a method showed high specificity, high sensitivity, and rapid positive detection of E. coli O157: H7 from ground beef samples.
Highlights
The RAA-CRISPR/Cas12a system proposed in this study provided an alternative molecular tool for quick, specific, sensitive and accurate detection of E. coli O157: H7 in foods.
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