Alterations of the bile microbiome is associated with progression-free survival in pancreatic ductal adenocarcinoma patients.

BACKGROUND: Patients with pancreatic ductal adenocarcinoma (PDAC) display an altered oral, gastrointestinal, and intra-pancreatic microbiome compared to healthy individuals. However, knowledge regarding the bile microbiome and its potential impact on progression-free survival in PDACs remains limited. METHODS: Patients with PDAC (n = 45), including 20 matched pairs before and after surgery, and benign controls (n = 16) were included prospectively. The characteristics of the microbiomes of the total 81 bile were revealed by 16  S-rRNA gene sequencing. PDAC patients were divided into distinct groups based on tumor marker levels, disease staging, before and after surgery, as well as progression free survival (PFS) for further analysis. Disease diagnostic model was formulated utilizing the random forest algorithm. RESULTS: PDAC patients harbor a unique and diverse bile microbiome (PCoA, weighted Unifrac, p = 0.038), and the increasing microbial diversity is correlated with dysbiosis according to key microbes and microbial functions. Aliihoeflea emerged as the genus displaying the most significant alteration among two groups (p < 0.01). Significant differences were found in beta diversity of the bile microbiome between long-term PFS and short-term PFS groups (PCoA, weighted Unifrac, p = 0.005). Bacillota and Actinomycetota were identified as altered phylum between two groups associated with progression-free survival in all PDAC patients. Additionally, we identified three biomarkers as the most suitable set for the random forest model, which indicated a significantly elevated likelihood of disease occurrence in the PDAC group (p < 0.0001). The area under the receiver operating characteristic (ROC) curve reached 80.8% with a 95% confidence interval ranging from 55.0 to 100%. Due to the scarcity of bile samples, we were unable to conduct further external verification. CONCLUSION: PDAC is characterized by an altered microbiome of bile ducts. Biliary dysbiosis is linked with progression-free survival in all PDACs. This study revealed the alteration of the bile microbiome in PDACs and successfully developed a diagnostic model for PDAC.

Vaginal microbial profile of cervical cancer patients receiving chemoradiotherapy: the potential involvement of Lactobacillus iners in recurrence.

The vaginal microbiome is an immune defense against reproductive diseases and can serve as an important biomarker for cervical cancer. However, the intrinsic relationship between the recurrence and the vaginal microbiome in patients with cervical cancer before and after concurrent chemoradiotherapy is poorly understood. Here, we analyzed 125 vaginal microbial profiles from a patient cohort of stage IB-IVB cervical cancer using 16S metagenomic sequencing and deciphered the microbial composition and functional characteristics of the recurrent and non-recurrent both before and after chemoradiotherapy. We demonstrated that the abundance of beneficial bacteria and stability of the microbial community in the vagina decreased in the recurrence group, implying the unique characteristics of the vaginal microbiome for recurrent cervical cancer. Moreover, using machine learning, we identified Lactobacillus iners as the most important biomarker, combined with age and other biomarkers (such as Ndongobacter massiliensis, Corynebacterium pyruviciproducens ATCC BAA-1742, and Prevotella buccalis), and could predict cancer recurrence phenotype before chemoradiotherapy. This study prospectively employed rigorous bioinformatics analysis and highlights the critical role of vaginal microbiota in post-treatment cervical cancer recurrence, identifying promising biomarkers with prognostic significance in the context of concurrent chemoradiotherapy for cervical cancer. The role of L. iners in determining chemoradiation resistance in cervical cancer warrants further detailed investigation. Our results expand our understanding of cervical cancer recurrence and help develop better strategies for prognosis prediction and personalized therapy.

Global biogeography of microbes driving ocean ecological status under climate change.

Microbial communities play a crucial role in ocean ecology and global biogeochemical processes. However, understanding the intricate interactions among diversity, taxonomical composition, functional traits, and how these factors respond to climate change remains a significant challenge. Here, we propose seven distinct ecological statuses by systematically considering the diversity, structure, and biogeochemical potential of the ocean microbiome to delineate their biogeography. Anthropogenic climate change is expected to alter the ecological status of the surface ocean by influencing environmental conditions, particularly nutrient and oxygen contents. Our predictive model, which utilizes machine learning, indicates that the ecological status of approximately 32.44% of the surface ocean may undergo changes from the present to the end of this century, assuming no policy interventions. These changes mainly include poleward shifts in the main taxa, increases in photosynthetic carbon fixation and decreases in nutrient metabolism. However, this proportion can decrease significantly with effective control of greenhouse gas emissions. Our study underscores the urgent necessity for implementing policies to mitigate climate change, particularly from an ecological perspective.

Effects of different assembly strategies on gene annotation in activated sludge.

Activated sludge comprises diverse bacteria, fungi, and other microorganisms, featuring a rich repertoire of genes involved in antibiotic resistance, pollutant degradation, and elemental cycling. In this regard, hybrid assembly technology can revolutionize metagenomics by detecting greater gene diversity in environmental samples. Nonetheless, the optimal utilization and comparability of genomic information between hybrid assembly and short- or long-read technology remain unclear. To address this gap, we compared the performance of the hybrid assembly, short- and long-read technologies, abundance and diversity of annotated genes, and taxonomic diversity by analysing 46, 161, and 45 activated sludge metagenomic datasets, respectively. The results revealed that hybrid assembly technology exhibited the best performance, generating the most contiguous and longest contigs but with a lower proportion of high-quality metagenome-assembled genomes than short-read technology. Compared with short- or long-read technologies, hybrid assembly technology can detect a greater diversity of microbiota and antibiotic resistance genes, as well as a wider range of potential hosts. However, this approach may yield lower gene abundance and pathogen detection. Our study revealed the specific advantages and disadvantages of hybrid assembly and short- and long-read applications in wastewater treatment plants, and our approach could serve as a blueprint to be extended to terrestrial environments.

Global insights into endophytic bacterial communities of terrestrial plants: Exploring the potential applications of endophytic microbiota in sustainable agriculture.

Endophytic microorganisms are indispensable symbionts during plant growth and development and often serve functions such as growth promotion and stress resistance in plants. Therefore, an increasing number of researchers have applied endophytes for multifaceted phytoremediation (e.g., organic pollutants and heavy metals) in recent years. With the availability of next-generation sequencing technologies, an increasing number of studies have shifted the focus from culturable bacteria to total communities. However, information on the composition, structure, and function of bacterial endophytic communities is still not widely synthesized. To explore the general patterns of variation in bacterial communities between plant niches, we reanalyzed data from 1499 samples in 30 individual studies from different continents and provided comprehensive insights. A group of bacterial genera were commonly found in most plant roots and shoots. Our analysis revealed distinct variations in the diversity, composition, structure, and function of endophytic bacterial communities between plant roots and shoots. These variations underscore the sophisticated mechanisms by which plants engage with their endophytic microbiota, optimizing these interactions to bolster growth, health, and resilience against stress. Highlighting the strategic role of endophytic bacteria in promoting sustainable agricultural practices and environmental stewardship, our study not only offers global insights into the endophytic bacterial communities of terrestrial plants but also underscores the untapped potential of these communities as invaluable resources for future research.

Phages in different habitats and their ability to carry antibiotic resistance genes.

As the most abundant organisms on Earth, phages play a key role in the evolution of bacterial antibiotic resistance. Although previous studies have demonstrated the molecular mechanisms of horizontal gene transfer mediated by mobile genetic elements, our understanding of the intertwined relationships between antibiotic resistance genes (ARGs) and phages is limited. In this study, we analysed 2781 metagenomic samples to reveal the composition and species interactions of phage communities in different habitats as well as their capacity to carry ARGs with health risks. The composition of phage communities varies in different habitats and mainly depends on environmental conditions. Terrestrial habitats display more complex and robust interactions between phages than aquatic and human-associated habitats, resulting in the highest biodiversity of phages. Several types of phages in certain taxa (4.95-7.67%, mainly belonging to Caudoviricetes) have the capacity to carry specific ARGs and display a high potential risk to human health, especially in human-associated habitats. Overall, our results provide insights into the assembly mechanisms of phage communities and their effects on the dissemination of antibiotic resistance.

Stromal score is a promising index in tumor patients' outcome determination.

Background: Immune status is widely acknowledged as a valuable marker for predicting cancer prognosis and therapy response. However, there has been a limited understanding of the stromal landscape in cancer. Methods: By employing ESTIMATE, stromal- and immune-scores were inferred for 6193 tumor samples spanning 12 cancer types sourced from The Cancer Genome Atlas (TCGA). Subsequently, the samples were categorized into seven groups based on their stromal and immune scores. A comparison of prognosis, lymphocyte and stromal cell infiltration, and the response to programmed death ligand 1 (PD-L1) therapy was conducted among these subtypes. Results: It was unveiled by the analysis that, in the majority of cancer types, stromal score exhibited a more potent predictive capability for outcomes compared to the immune score. Furthermore, it was observed that in four cancer types, intermediate immune infiltration coupled with low stromal infiltration correlated with the most favorable overall survival, whereas an unfavorable outcome was predicted in colorectal cancer (CRC) and stomach adenocarcinoma (STAD) when high immune infiltration coexisted with intermediate or high stromal infiltration. Conclusion: In summary, while high immune scores frequently correlate with a positive prognosis, such correlation is not universal. A potential strategy to address the current limitations of the immune score in specific circumstances could involve a focus on stromal scores or a subtle integration of stromal and immune status.

Metagenomic Insight into The Global Dissemination of The Antibiotic Resistome.

The global crisis in antimicrobial resistance continues to grow. Estimating the risks of antibiotic resistance transmission across habitats is hindered by the lack of data on mobility and habitat-specificity. Metagenomic samples of 6092 are analyzed to delineate the unique core resistomes from human feces and seven other habitats. This is found that most resistance genes (≈85%) are transmitted between external habitats and human feces. This suggests that human feces are broadly representative of the global resistome and are potentially a hub for accumulating and disseminating resistance genes. The analysis found that resistance genes with ancient horizontal gene transfer (HGT) events have a higher efficiency of transfer across habitats, suggesting that HGT may be the main driver for forming unique but partly shared resistomes in all habitats. Importantly, the human fecal resistome is historically different and influenced by HGT and age. The most important routes of cross-transmission of resistance are from the atmosphere, buildings, and animals to humans. These habitats should receive more attention for future prevention of antimicrobial resistance. The study will disentangle transmission routes of resistance genes between humans and other habitats in a One Health framework and can identify strategies for controlling the ongoing dissemination and antibiotic resistance.

Effects of dietary supplement with licorice and rutin mixture on production performance, egg quality, antioxidant capacity, and gut microbiota in quails (Turnix tanki).

This study was conducted to evaluate the effect of licorice and rutin on production performance, egg quality, and mucosa antioxidant levels in Chinese yellow quail. A total of 240 Chinese Yellow Quail (400-day-old) were randomly distributed into 5 groups: the Control group, fed with a basic diet; the LR1 group, fed with basal diet supplemented with 300 + 100 mg licorice and rutin mixture/kg diet; the LR2 group, fed with basal diet supplemented with 300 + 200 mg licorice and rutin mixture/kg diet; the LR3 group, fed with basal diet supplemented with 600 + 100 mg licorice and rutin mixture/kg diet and the LR4 group, fed with basal diet supplemented with 600 + 200 mg licorice and rutin mixture/kg diet. Compared with the control, supplementation with the licorice and rutin mixture improved the laying rate and eggshell thickness whereas decreased the feed conversion ratio of quails. Moreover, dietary supplementation with the licorice and rutin mixture improved the antioxidant capacity by increasing the activity of the superoxide dismutase (SOD) level and decreasing the concentration of malondialdehyde (MDA) in the jejunal mucosa. The licorice and rutin mixture altered the composition of intestinal microbiota by influencing the relative abundances of Bacteroidetes and Bacteroides. The relative abundances of the Bacteroidetes were significantly related to the laying rate of quails. In addition, the mixture of licorice and rutin was also effective in reducing the relative abundance of intestinal Proteobacteria and Enterobacter in quails, reducing the accumulation of antibiotic-resistance genes. The results revealed that supplementation of licorice and rutin mixture to the diet improved production performance, egg quality, and antioxidant capacity and modified the composition of intestinal microbiota in quails. This study provides a reference for Chinese herbal additives to promote production performance by modulating quail gut microbes.

A global atlas of marine antibiotic resistance genes and their expression.

Oceans serve as global reservoirs of antibiotic-resistant bacteria and antibiotic resistance genes (ARGs). However, little is known about the traits and expression of ARGs in response to environmental factors. We analyzed 347 metagenomes and 182 metatranscriptomes to determine the distribution, hosts, and expression of ARGs in oceans. Our study found that the diversity and abundance of ARGs varied with latitude and depth. The core marine resistome mainly conferred glycopeptide and multidrug resistance. The hosts of this resistome were mainly limited to the core marine microbiome, with phylogenetic barriers to the horizontal transfer of ARGs, transfers being more frequent within species than between species. Sixty-five percent of the marine ARGs identified were expressed. More than 90% of high-risk ARGs were more likely to be expressed. Anthropogenic activity might affect the expression of ARGs by altering nitrate and phosphate concentrations and ocean temperature. Machine-learning models predict >97% of marine ARGs will change expression by 2100. High-risk ARGs will shift to low latitudes and regions with high anthropogenic activity, such as the Pacific and Atlantic Oceans. Certain ARGs serve a dual role in antibiotic resistance and potentially participate in element cycling, along with other unknown functions. Determining whether changes in ARG expression are beneficial to ecosystems and human health is challenging without comprehensive understanding of their functions. Our study identified a core resistome in the oceans and quantified the expression of ARGs for the development of future control strategies under global change.

Proteomics reveals the underlying mechanism by which the first uneven division affects embryonic development in pig.

One of the most typical abnormal cleavage patterns during early embryonic development is uneven division, but the first uneven division of pig zygote is common. Uneven division results in different daughter cell sizes and an uneven distribution of organelles such as lipid droplet, mitochondria, but the developmental capacity of daughter cells and proteomic changes of daughter cells are still unclear. Therefore, the developmental ability and proteomic quantification were investigated on blastomeres from even division (ED) or uneven division (UD) embryos at 2-cell stage in the present study. Firstly, the developmental ability was affected by the blastomeric size, when compared with medium blastomeres (MBs), the large blastomeres (LBs) with the higher cleavage rate but the small blastomeres (SBs) with the lower rate was observed. Subsequently, proteomic analysis was performed on blastomeres of LBs, MBs and SBs, a total of 109 DEPs were detected, which were involved in protein metabolism and processing, energy metabolism and ribosome. In particular, DEPs in LBs vs. SBs were focused on RNA binding and actin cytoskeletal tissue. Two protein-dense networks associated with RNA binding and cytoskeleton were revealed by further protein-protein interaction (PPI) analysis of DEPs in LBs vs. SBs, that DDX1 related to RNA binding and ACTB related to cytoskeleton were confirmed in UD embryos. Therefore, a briefly information of DEPs in blastomeres of 2-cell stage pig embryos was described in the present study, and it further confirmed that the formation of uneven division of the first cell cycle of pig embryos might be controlled by the cytoskeleton; the developmental capacity of daughter cells might be affected by the energy metabolism, RNA binding and ribosome, and further account for the developmental potential of the whole embryo.

Epigenetic Iinsights into the Senescence of Porcine Fetal Fibroblasts induced by Passaging.

Epigenetic status of fetal fibroblasts (FFs) is one of the crucial factors accounted for the success of somatic cell nuclear transfer and gene editing, which might inevitably be affected by passaging. But few systematic studies have been performed on the epigenetic status of passaged aging cells. Therefore, FFs from large white pig were in vitro passaged to the 5, 10, and 15 (F5, F10, and F15) passages in the present study to investigate the potential alteration of epigenetic status. Results indicated the senescence of FFs occurs with the passaging, as assessed by the weakened growth rate, increased ß-gal expression, and so on. For the epigenetic status of FFs, the higher level both of DNA methylation and H3K4me1, H3K4me2, H3K4me3 was observed at F10, but the lowest level was observed at F15. However, the fluorescence intensity of m6A was significantly higher in F15, but lower (p < 0.05) in F10, and the related mRNA expression in F15 was significantly higher than F5. Further, RNA-Seq indicated a considerable difference in the expression pattern of F5, F10, and F15 FFs. Among differentially expressed genes, not only the genes involved in cell senescence were changed, but also the upregulated expression of Dnmt1, Dnmt3b, Tet1 and dysregulated expression of histone methyltransferases-related genes were detected in F10 FFs. In addition, most genes related to m6A such as METTL3, YTHDF2, and YTHDC1 were significantly different in F5, F10, and F15 FFs. In conclusion, the epigenetic status of FFs was affected by being passaged from F5 to F15.

Antimicrobial Peptides in the Global Microbiome: Biosynthetic Genes and Resistance Determinants.

Antimicrobial peptides are a promising new class of antimicrobials that could address the antibiotic resistance crisis, which poses a major threat to human health. These peptides are present in all kingdoms of life, but especially in microorganisms, having multiple origins in diverse taxa. To date, there has been no global study on the diversity of antimicrobial peptides, the hosts in which these occur, and the potential for resistance to these agents. Here, we investigated the diversity and number of antimicrobial peptides in four main habitats (aquatic, terrestrial, human, and engineered) by analyzing 52,515 metagenome-assembled genomes. The number of antimicrobial peptides was higher in the human gut microbiome than in other habitats, and most hosts of antimicrobial peptides were habitat-specific. The relative abundance of genes that confer resistance to antimicrobial peptides varied between habitats and was generally low, except for the built environment and on human skin. The horizontal transfer of potential resistance genes among these habitats was probably constrained by ecological barriers. We systematically quantified the risk of each resistance determinant to human health and found that nearly half of them pose a threat, especially those that confer resistance to multiple AMPs and polymyxin B. Our results help identify the biosynthetic potential of antimicrobial peptides in the global microbiome, further identifying peptides with a low risk of developing resistance.

Dietary cysteine drives body fat loss via FMRFamide signaling in Drosophila and mouse.

Obesity imposes a global health threat and calls for safe and effective therapeutic options. Here, we found that protein-rich diet significantly reduced body fat storage in fruit flies, which was largely attributed to dietary cysteine intake. Mechanistically, dietary cysteine increased the production of a neuropeptide FMRFamide (FMRFa). Enhanced FMRFa activity simultaneously promoted energy expenditure and suppressed food intake through its cognate receptor (FMRFaR), both contributing to the fat loss effect. In the fat body, FMRFa signaling promoted lipolysis by increasing PKA and lipase activity. In sweet-sensing gustatory neurons, FMRFa signaling suppressed appetitive perception and hence food intake. We also demonstrated that dietary cysteine worked in a similar way in mice via neuropeptide FF (NPFF) signaling, a mammalian RFamide peptide. In addition, dietary cysteine or FMRFa/NPFF administration provided protective effect against metabolic stress in flies and mice without behavioral abnormalities. Therefore, our study reveals a novel target for the development of safe and effective therapies against obesity and related metabolic diseases.

Global distribution of marine microplastics and potential for biodegradation.

Microplastics are a growing marine environmental concern globally due to their high abundance and persistent degradation. We created a global map for predicting marine microplastic pollution using a machine-learning model based on 9445 samples and found that microplastics converged in zones of accumulation in subtropical gyres and near polar seas. The predicted global potential for the biodegradation of microplastics in 1112 metagenome-assembled genomes from 485 marine metagenomes indicated high potential in areas of high microplastic pollution, such as the northern Atlantic Ocean and the Mediterranean Sea. However, the limited number of samples hindered our prediction, a priority issue that needs to be addressed in the future. We further identified hosts with microplastic degradation genes (MDGs) and found that Proteobacteria accounted for a high proportion of MDG hosts, mainly Alphaproteobacteria and Gammaproteobacteria, with host-specific patterns. Our study is essential for raising awareness, identifying areas with microplastic pollution, providing a prediction method of machine learning to prioritize surveillance, and identifying the global potential of marine microbiomes to degrade microplastics, providing a reference for selecting bacteria that have the potential to degrade microplastics for further applied research.

Mutational Bias and Natural Selection Driving the Synonymous Codon Usage of Single-Exon Genes in Rice (Oryza sativa L.).

The relative abundance of single-exon genes (SEGs) in higher plants is perplexing. Uncovering the synonymous codon usage pattern of SEGs will benefit for further understanding their underlying evolutionary mechanism in plants. Using internal correspondence analysis (ICA), we reveal a significant difference in synonymous codon usage between SEGs and multiple-exon genes (MEGs) in rice. But the effect is weak, accounting for only 2.61% of the total codon usage variability. SEGs and MEGs contain remarkably different base compositions, and are under clearly differential selective constraints, with the former having higher GC content, and evolving relatively faster during evolution. In the group of SEGs, the variability in synonymous codon usage among genes is partially due to the variations in GC content, gene function, and gene expression level, which accounts for 22.03%, 5.99%, and 3.32% of the total codon usage variability, respectively. Therefore, mutational bias and natural selection should work on affecting the synonymous codon usage of SEGs in rice. These findings may deepen our knowledge for the mechanisms of origination, differentiation and regulation of SEGs in plants.

CRISPR/Cas12a-Assisted Dual Visualized Detection of SARS-CoV-2 on Frozen Shrimps.

Given the possibility that food contaminated with SARS-CoV-2 might become an infection source, there is an urgent need for us to develop a rapid and accurate nucleic acid detection method for SARS-CoV-2 in food to ensure food safety. Here, we propose a sensitive, specific, and reliable molecular detection method for SARS-CoV-2. It has a mechanism to control amplicon contamination. Swabs from spiked frozen shrimps were used as detection samples, which were processed by heating at 95 °C for 30 s. These preprocessed samples served as the templates for subsequent amplification. A colorimetric LAMP reaction was carried out to amplify both the SARS-CoV-2 target and the MS2 phage simultaneously in one tube. MS2 phage was detected by colorimetric LAMP as the internal control, while SARS-CoV-2 was detected with a CRISPR/Cas12a system. The fluorescence results could be visually detected with an ultraviolet lamp. Meanwhile, uracil was incorporated during the LAMP reaction to provide an amplicon contamination proof mechanism. This test could detect as low as 20 copies of SARS-CoV-2 in one reaction. Additionally, the detection could be finished in 45 min. The test only needs a heating block and an ultraviolet lamp, which shows the potential for field detection.

Integrated slip valve-assisted fluidic chip coupling with CRISPR/Cas12a system for nucleic acid analysis.

Currently, some on-site nucleic acid detection platforms have been developed. However, these platforms still need to be improved in device integration and multiple detection capability. In this work, an integrated dual nucleic acid analysis platform was developed by slip valve-assisted fluidic chip coupled with CRISPR/Cas12a system. All the reagents, including nucleic acid extraction, air-dried loop-mediated isothermal amplification (LAMP) and CRISPR/Cas12a detection reagents, were preloaded on the fluidic chip. Liquids transfer and stirring could be controlled by a slip valve and a syringe. By combining duplex LAMP reaction with two CRISPR detection units, CRISPR/Cas12a-based dual nucleic acid analysis was successfully constructed. Benefiting from high-quality nucleic acid extraction on the chip, as low as 30 copies/reaction of Vibrio parahaemolyticus (V. parahaemolyticus) and 20 copies/reaction of Salmonella typhimurium (S. typhimurium) could be simultaneously detected. Detection results could be observed by the naked eye under a portable ultraviolet lamp. The whole detection procedure was finished within 60 min. This method with integrated nucleic acid analysis, dual detection capability and fluorescence visualized results provides a new solution for on-site nucleic acid analysis.

HPV-related cervical diseases: Alteration of vaginal microbiotas and promising potential for diagnosis.

Vaginal microbiota is closely associated with women's health, however, the correlation between HPV-related cervical disease (HRCD) and vaginal microbiota is still obscure. In this study, patients with HRCD (n = 98) and healthy controls (n = 58) in Hangzhou were recruited, and their vaginal microbiota were collected and analyzed. The composition of the vaginal microbial community was explored, and a disease classification model was developed by random forest algorithm. The results suggested that the diversity of vaginal microbiota was significantly higher in HRCDs than that in healthy controls (p < 0.05). Firmicutes is the dominant phylum in vaginal microbiota, and Lactobacillus was identified as the most altered genus between two groups (p < 0.01). Kyoto Encyclopedia of Genes and Genomes analysis suggested the difference in vaginal microbial community functions between two groups. Furthermore, we identified 10 biomarkers as the optimal marker sets for the random forest model and found a higher probability of disease values in HRCD group in discovery cohort (p < 0.0001), with an area under the receiver operating characteristic curve reaching 89.7% (95% confidence interval: 78.3%-100%). We further validated the model in both validation and independent diagnosis cohorts, confirming its accuracy in the prediction of HRCD. In conclusion, this study revealed the community composition of vaginal microbiota in HRCDs and successfully constructed a diagnostic model for HRCD.

Multiple crRNAs-assisted CRISPR/Cas12a assay targeting cytochrome b gene for amplification-free detection of meat adulteration.

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems have been widely applied in nucleic acid analysis for the high specificity. Coupled with pre-amplification steps, the sensitivity of CRISPR-based detection is greatly improved. However, an extra pre-amplification step not only complicates the detection procedures but may also cause aerosol contaminations in the process of transferring amplified solution into CRISPR system. In this study, we demonstrate that combination of multiple crRNAs in CRISPR/Cas12a system can enhance the detection sensitivity. Based on it, we establish a multiple crRNAs enhanced CRISPR (meCRISPR) method and apply it to meat adulteration identification. Take cytochrome b (Cyt b) gene as a target, meCRISPR method can directly detect as low as 1.13 ng/µL extracted pork DNA and 5% (w/w) pork contamination in pork and beef meat mixtures. There is no cross-reaction with extracted chicken, beef, duck and fish DNA. meCRISPR reaction is incubated at an isothermal temperature, and the detection process can be completed in a designed portable apparatus with a heat block, a light emitting diode and filters. For the simplicity, specificity and sufficient sensitivity of meCRISPR method, it will have great prospects in species identification, food adulteration, and genetically modified food detection.