MnO2 Nanozyme-Mediated CRISPR-Cas12a System for the Detection of SARS-CoV-2.

The CRISPR-Cas system was developed into a molecular diagnostic tool with high sensitivity, low cost, and high specificity in recent years. Colorimetric assays based on nanozymes offer an attractive point-of-care testing method for their low cost of use and user-friendly operation. Here, a MnO2 nanozyme-mediated CRISPR-Cas12a system was instituted to detect SARS-CoV-2. MnO2 nanorods linked to magnetic beads via a single-stranded DNA (ssDNA) linker used as an oxidase-like nanozyme inducing the color change of 3,3',5,5'-tetramethylbenzidine, which can be distinguished by the naked eye. The detection buffer color will change when the Cas12a is activated by SARS-CoV-2 and indiscriminately cleave the linker ssDNA. The detection limit was 10 copies per microliter and showed no cross-reaction with other coronaviruses. The nanozyme-mediated CRISPR-Cas12a system shows high selectivity and facile operation, with great potential for molecular diagnosis in point-of-care testing applications.

Ultrasensitive SARS-CoV-2 diagnosis by CRISPR-based screen-printed carbon electrode.

Early and accurate diagnosis of SARS-CoV-2 was crucial for COVID-19 control and urgently required ultra-sensitive and rapid detection methods. CRISPR-based detection systems have great potential for rapid SARS-CoV-2 detection, but detecting ultra-low viral loads remains technically challenging. Here, we report an ultrasensitive CRISPR/Cas12a-based electrochemical detection system with an electrochemical biosensor, dubbed CRISPR-SPCE, in which the CRISPR ssDNA reporter was immobilized onto a screen-printed carbon electrode. Electrochemical signals are detected due to CRISPR cleavage, giving enhanced detection sensitivity. CRISPR-SPCE enables ultrasensitive SARS-CoV-2 detection, reaching as few as 0.27 copies µL-1. Moreover, CRISPR-SPCE is also highly specific and inexpensive, providing a fast and simple SARS-CoV-2 assay.

Development and evaluation of a meat mitochondrial metagenomic (3MG) method for composition determination of meat from fifteen mammalian and avian species.

BACKGROUND: Bioassessment and biomonitoring of meat products are aimed at identifying and quantifying adulterants and contaminants, such as meat from unexpected sources and microbes. Several methods for determining the biological composition of mixed samples have been used, including metabarcoding, metagenomics and mitochondrial metagenomics. In this study, we aimed to develop a method based on next-generation DNA sequencing to estimate samples that might contain meat from 15 mammalian and avian species that are commonly related to meat bioassessment and biomonitoring. RESULTS: In this project, we found the meat composition from 15 species could not be identified with the metabarcoding approach because of the lack of universal primers or insufficient discrimination power. Consequently, we developed and evaluated a meat mitochondrial metagenomics (3MG) method. The 3MG method has four steps: (1) extraction of sequencing reads from mitochondrial genomes (mitogenomes); (2) assembly of mitogenomes; (3) mapping of mitochondrial reads to the assembled mitogenomes; and (4) biomass estimation based on the number of uniquely mapped reads. The method was implemented in a python script called 3MG. The analysis of simulated datasets showed that the method can determine contaminant composition at a proportion of 2% and the relative error was < 5%. To evaluate the performance of 3MG, we constructed and analysed mixed samples derived from 15 animal species in equal mass. Then, we constructed and analysed mixed samples derived from two animal species (pork and chicken) in different ratios. DNAs were extracted and used in constructing 21 libraries for next-generation sequencing. The analysis of the 15 species mix with the method showed the successful identification of 12 of the 15 (80%) animal species tested. The analysis of the mixed samples of the two species revealed correlation coefficients of 0.98 for pork and 0.98 for chicken between the number of uniquely mapped reads and the mass proportion. CONCLUSION: To the best of our knowledge, this study is the first to demonstrate the potential of the non-targeted 3MG method as a tool for accurately estimating biomass in meat mix samples. The method has potential broad applications in meat product safety.

Evaluation of the differences between biofilm and planktonic Brucella abortus via metabolomics and proteomics.

This study analyzed the difference between biofilm and planktonic Brucella abortus using metabolomics and proteomics. Brucella abortus was cultured in different media to induce Brucella abortus biofilm formation and planktonic cells, followed by metabolomics and proteomics analyses for these two samples. Significant differential metabolites were identified, followed by KEGG pathway analysis. Differentially expressed proteins were identified, followed by subcellular localization, GO annotation, and KEGG pathway enrichment. Additionally, a correlation analysis of metabolomics and proteomics was performed. Metabolomics analysis showed 7682 positive and 4433 negative metabolites, including 188 positive and 117 negative significant differential metabolites. These differential metabolites were enriched in fatty acid/unsaturated fatty acid biosynthesis and linoleic acid metabolism. Proteomics analysis revealed 1759 proteins, including 486 differentially expressed proteins, which were enriched in various metabolic and degradation-related pathways. Subcellular localization showed that 74.3% of the differential proteins were cytoplasmic proteins. Correlation analysis showed that 1-palmitoyl-2-oleoyl-phosphatidylglycerol had the most significant correlations with proteins, followed by cytosine. Both metabolites correlated with the protein Q57EI7 (RbsB-1, ribose ABC transporter). One common pathway, fatty acid biosynthesis, was identified by both proteomics and metabolomics analyses that involved the metabolites, oleic acid, and protein Q57DK3 (biotin carboxylase). There were metabolomic and proteomic differences between Brucella abortus biofilm and planktonic cells, and these results provide novel insights into the biofilm-forming process of Brucella abortus.

Comparative proteomic and genomic analyses of Brucella abortus biofilm and planktonic cells.

The present study aimed to explore the differences in protein and gene expression of Brucella abortus cultured under biofilm and planktonic conditions. The proteins unique to biofilms and planktonic B. abortus were separated by two­dimensional (2­D) electrophoresis and then identified by matrix­assisted laser desorption/ionization­tandem time of flight­mass spectrometry (MALDI­TOF/TOF­MS). High­throughput sequencing and bioinformatic analyses were performed to identify differentially expressed genes between B. abortus cultured under biofilm and planktonic conditions. The proteins and genes identified by proteomic and genomic analyses were further evaluated via western blot and reverse transcription­quantitative polymerase chain reaction (RT­qPCR) analyses. 2­D electrophoresis identified 20 differentially expressed protein spots between biofilms and planktonic cells, which corresponded to 18 individual proteins (12 downregulated and 6 upregulated) after MALDI­TOF/TOF­MS analysis, including elongation factor Tu and enolase. RT­qPCR analysis revealed that all of the 18 genes were downregulated in biofilms compared with planktonic cells. Western blot analysis identified 9 downregulated and 3 upregulated proteins. High­throughput sequencing and bioinformatic analyses identified 14 function and pathway­associated genes (e.g., BAbS19_I14970). RT­qPCR analysis of the 14 genes showed that they were upregulated in biofilm compared with in planktonic state. In conclusion, these differentially expressed genes may play important roles in bacterial defense, colonization, invasion, and virulence.

Development of a multiplex TaqMan probe-based real-time PCR for discrimination of variant and classical porcine epidemic diarrhea virus.

Since October 2010, porcine diarrhea outbreaks have occurred widely, resulting in major losses in suckling piglets in China. A variant porcine epidemic diarrhea virus (PEDV), characterized by base deletion and insertion in the S gene, compared to classical PEDV CV777, was shown to be responsible for this outbreak. In this study, a multiplex TaqMan probe-based real-time PCR was developed for detecting PEDV and differentiating the variant from classical PEDV, by using two sets of primers and probes based on the S gene of PEDV. The limits of detection of both variant and classical PEDV were 5×10(2) DNA copies. Specificity was determined using eight other viral pathogens of swine. Reproducibility was evaluated using standard dilutions, with coefficients of variation <1.4%. Standard dilutions included in each test allowed quantification of the amount of PEDV. Among 42 intestinal samples from pigs with severe watery diarrhea, 36 variant PEDV and three classical PEDV samples were detected, with viral loads of 10(2)-10(8) copies/µl and 10(3)-10(5) copies/µl, respectively, which suggested that the variant PEDV was prevalent in China. The multiplex TaqMan probe-based real-time PCR should be a useful tool for quantifying viral load, detecting PEDV, and differentiating variant from classical PEDV.

[Rapid detection of salmonella in food by using fluorescently labeled phage O-I].

OBJECTIVE: To develop a rapid detection method for Salmonella in food by using specific Salmonella-phage O-I. METHODS: One hundred bacteria strains and 120 food sample isolates were infected using fluorescently labeled O-I phage genome with SYBR gold stain (a nucleic acid dye, 1 x working solution), then were observed under epi-fluorescence microscopy. The sensitivity of the method was tested. RESULTS: Among the 100 strains infected with O-I/SYBR gold stain, 40 Salmonella strains exhibited rod fluorescence. Other bacteria including 10 Proteus, 20 Shigella, 20 E. coli and 10 Staphylococcus did not exhibit this feature The sensitivity of detecting Salmonella was 10 CFU/100 microL. The detection for 120 food samples by using the O-I/SYBR gold stain had similar results to those by using the biochemical method. CONCLUSION: Fluorescent-labeled O-I phage could rapidly, sensitively and specifically detect Salmonella species in food samples.