Simultaneous detection of breast cancer biomarkers circROBO1 and BRCA1 based on a CRISPR-Cas13a/Cas12a system.

Breast cancer is reported to be one of the most lethal cancers in women, and its multi-target detection can help improve the accuracy of diagnosis. In this work, a cluster regularly interspaced short palindromic repeats (CRISPR)-Cas13a/Cas12a-based system was established for the simultaneous fluorescence detection of breast cancer biomarkers circROBO1 and BRCA1. CRISPR-Cas13a and CRISPR-Cas12a were directly activated by their respective targets, resulting in the cleavage of short RNA and DNA reporters, respectively, thus the signals of 6-carboxyfluorescein (FAM) and 6-carboxy-xrhodamine (ROX) were restored. As the fluorescence intensities of FAM and ROX were dependent on the concentrations of circROBO1 and BRCA1, respectively, synchronous fluorescence scanning could achieve one-step detection of circROBO1 and BRCA1 with detection limits of 0.013 pM and 0.26 pM, respectively. The system was highly sensitive and specific, holding high diagnostic potential for the detection of clinical samples. Furthermore, the competing endogenous RNA mechanism between circROBO1 and BRCA1 was also explored, providing a reliable basis for the intrinsic regulatory mechanism of breast cancer.

IM-IAD: Industrial Image Anomaly Detection Benchmark in Manufacturing.

Image anomaly detection (IAD) is an emerging and vital computer vision task in industrial manufacturing (IM). Recently, many advanced algorithms have been reported, but their performance deviates considerably with various IM settings. We realize that the lack of a uniform IM benchmark is hindering the development and usage of IAD methods in real-world applications. In addition, it is difficult for researchers to analyze IAD algorithms without a uniform benchmark. To solve this problem, we propose a uniform IM benchmark, for the first time, to assess how well these algorithms perform, which includes various levels of supervision (unsupervised versus fully supervised), learning paradigms (few-shot, continual and noisy label), and efficiency (memory usage and inference speed). Then, we construct a comprehensive IAD benchmark (IM-IAD), which includes 19 algorithms on seven major datasets with a uniform setting. Extensive experiments (17 017 total) on IM-IAD provide in-depth insights into IAD algorithm redesign or selection. Moreover, the proposed IM-IAD benchmark challenges existing algorithms and suggests future research directions. For reproducibility and accessibility, the source code is uploaded to the website: https://github.com/M-3LAB/open-iad.

Surface plasmon resonance detection of UV irradiation-induced DNA damage and photoenzymatic repair processes through specific interaction between consensus double-stranded DNA and p53 protein.

DNA damage, such as DNA lesions and strand breaks, impairs normal cell functions and failure in the DNA repair process could lead to gene mutation, cell apoptosis and disease occurrence. p53 is a tumor suppressor and DNA-binding protein, and DNA damage might affect their interaction and the subsequent p53 function. Herein, real-time monitoring of DNA damage and repair processes through DNA-p53 protein interaction was performed by surface plasmon resonance (SPR). The target DNA with consecutive pyrimidine nucleobases was first damaged upon UVC (254 nm) irradiation and then photoenzymatically repaired under UVA (365 nm) irradiation. The as-formed double-stranded (ds) DNA between probe DNA and normal, damaged or repaired target DNA was immobilized on the sensor chips, followed by the injection of p53 protein. By measuring the SPR signals under different cases, the DNA damage and repair processes could be conveniently monitored. The SPR signals were inversely proportional to the UVC doses ranging from 0.021 to 1.26 kJ m-2, providing a viable means for the quantification of the DNA damage level. The binding affinity between p53 and the dsDNA formed upon the hybridization of probe DNA and normal, damaged, or photoenzymatically repaired target DNA was estimated. This is the first report on measuring the equilibrium dissociation constant (KD) between the p53 protein and the dsDNA with photodamaged or repaired target sequences. The sensing strategy by SPR thus opens a new avenue for real-time measurement of the DNA damage and the repair processes.

A Combination of Novel Nucleic Acid Cross-Linking Dye and Recombinase-Aided Amplification for the Rapid Detection of Viable Salmonella in Milk.

Salmonella, as an important foodborne pathogen, can cause various diseases, such as severe enteritis. In recent years, various types of nucleicacid-intercalating dyes have been utilized to detect viable Salmonella. However, in principle, the performance of existing nucleic acid dyes is limited because they depend on the integrity of cell membrane. Herein, based on the metabolic activity of bacteria, a novel DNA dye called thiazole orange monoazide (TOMA) was introduced to block the DNA from dead bacteria. Recombinase-aided amplification (RAA) was then performed to detect viable Salmonella in samples. In this study, the permeability of TOMA to the cell membrane of Salmonella was evaluated via confocal laser scanning microscopy and fluorescence emission spectrometry. The limit of detection (LOD) of the TOMA-RAA method was 2.0 × 104 CFU/mL in pure culture. The feasibility of the TOMA-RAA method in detecting Salmonella was assessed in spiked milk. The LOD for Salmonella was 3.5 × 102 CFU/mL after 3 h of enrichment and 3.5 × 100 CFU/mL after 5 h of enrichment. The proposed TOMA-RAA assay has great potential to be applied to accurately detect and monitor foodborne pathogens in milk and its byproducts.

A novel photoreactive DNA-binding dye for detecting viable Klebsiella pneumoniae in powdered infant formula.

In addition to Cronobacter spp., Klebsiella pneumoniae is another opportunistic bacterial pathogen present in powdered infant formula (PIF) that can cause pneumonia, septicemia, and other diseases. In this study, a rapid and specific method based on a fluorescence probe was developed for detecting viable K. pneumoniae in PIF samples via the combination of recombinase-aided amplification (RAA) with thiazole orange monoazide (TOMA) dye (the TOMA-RAA assay hereafter). As a novel photosensitive DNA-intercalating dye, TOMA was used to penetrate bacterial cells, including both dead and viable cells, as verified by confocal laser scanning microscopy and fluorescent emission spectrometry. Importantly, the RAA assay exhibited good performance in detecting K. pneumoniae within 40 min at 39°C. Under optimal conditions, the TOMA-RAA assay can detect as low as 2.6 × 103 cfu/mL of K. pneumoniae in pure culture and 2.3 × 104 cfu/g of K. pneumoniae in spiked PIF sample. After 3 h of pre-enrichment, 3 × 100 cfu/g of K. pneumoniae can be detected. Furthermore, the TOMA-RAA assay displayed an excellent anti-interference ability to nontarget bacteria. In short, the proposed method has great potential application for the rapid and accurate detection of viable K. pneumoniae in PIF.

Hybrid RCA-DLS assay combined with aPCR for sensitive Salmonella enteritidis detection.

Salmonella infection could come from eating contaminated meat or raw eggs, and drinking milk or water contaminated by Salmonella enteritidis. Therefore, it is necessary to explore a fast and easy method for the detection of S. enteritidis in these diverse samples. For this purpose, a novel particle size sensing tool was designed for ultrasensitive and accurate S. enteritidis detection. This assay consisted of rolling circle amplification (RCA) with dynamic light scattering (DLS) using gold nanoparticles (AuNPs) modified with DNA probe as DNA-AuNPs as the capture surface into a hybrid RCA-DLS assay combined with asymmetric polymerase chain reaction (aPCR) and subsequent detection. Under optimal experimental conditions, the novel hybrid RCA-DLS assay combined with aPCR for S. enteritidis reached a limit of detection (LOD) as low as 3 × 100 CFU/mL in pure culture. In spiked milk samples, the LOD was 2.0 × 100 CFU/mL without pre-enriched bacteria. The total time of RCA-DLS assay was about 6 h which including genomic DNA extraction, aPCR, RCA and DLS determination. The hybrid RCA-DLS assay combined with aPCR holds promise in the specific and sensitive S. enteritidis detection.

Real-time recombinase-aided amplification with improved propidium monoazide for the rapid detection of viable Escherichia coli O157:H7 in milk.

Escherichia coli O157:H7, the causative agent of thrombotic thrombocytopenic purpura and hemolytic uremic syndrome in humans, generates a effective harm to community health because of its high pathogenicity. A real-time recombinase-aided amplification (rRAA) is an emerging method for nucleic acid detection. However, genomic DNA of bacteria could exist in food and the environment for a long time after death and could be amplified by rRAA assay, resulting in false-positive signal; thus, developing a fast and sensitive method is necessary to detect viable foodborne pathogens in food products. In our research, rRAA assay coupled with an enhanced nucleic acid binding dye named improved propidium monoazide (PMAxx) was established and applied in viable E. coli O157:H7 identification in skim milk. The PMAxx could eliminate interference from dead bacteria by permeating impaired membranes and covalently linking to DNA to prevent DNA amplification. The PMAxx-rRAA assay was performed with high sensitivity and good specificity. The PMAxx-rRAA assay could detect as low as 5.4 × 100 cfu/mL of viable E. coli O157:H7 in pure culture, and 7.9 × 100 cfu/mL of viable E. coli O157:H7 in skim milk. In addition, the PMAxx-rRAA assay was performed in the presence of a high concentration of dead bacteria or nontarget bacteria in skim milk to verify the capacity to resist interference from dead bacteria and nontarget bacteria. Therefore, the established PMAxx-rRAA assay is a valuable tool for the identification of viable E. coli O157:H7 in complex food matrix.

The fluorescent probe-based recombinase-aided amplification for rapid detection of Escherichia coli O157:H7.

Escherichia coli O157:H7 (E. coli O157:H7) is a common foodborne morbigenous microorganism, which can spread through fecal-oral transmission. Humans can be infected by ingesting foods and water contaminated with E. coli O157:H7, which can cause various symptoms. In present study, we have successfully developed a quick and hypersensitive fluorescent probe-based Recombinase-aided amplification (RAA) method and applied in E. coli O157:H7 detection at 39 °C in 20 min. The sensitivity of the assay in pure E. coli O157:H7 suspension was 5.6 × 100 CFU/mL. The fluorescent probe-based RAA assay was further applied in three samples, and the limit of detection (LOD) in skimmed milk, lettuces and lake water was 5.4 × 101 CFU/mL, 7.9 × 101 CFU/mL and 5.2 × 101 CFU/mL, separately. This method showed a high sensitivity and short detection time, which has the feasible application in on-site test in real samples.

A simple and sensitive aptasensor with rolling circle amplification for viable Cronobacter sakazakii detection in powdered infant formula.

Cronobacter sakazakii is a foodborne, emerging opportunistic pathogen that causes severe bacteremia, necrotizing enterocolitis, and sepsis with a mortality rate of up to 80%. In this study, we developed a simple and sensitive fluorescent turn-off aptasensor with rolling circle amplification assay for viable C. sakazakii detection in powdered infant formula. The results showed that the proposed aptasensor has good performance and specificity for detecting viable C. sakazakii in pure culture and powdered infant formula samples within 3 h. Under the optimal reaction conditions, there is a linear relationship between fluorescent intensity at 490 nm and logarithmic concentration of C. sakazakii in the range of 2.7 × 105 to 2.7 × 102 cfu/mL, with a limit of detection of 2.7 × 102 cfu/mL in pure culture. The proposed aptasensor achieved a recovery of 104 to 111% in pure culture, and 96 to 107% in spiked powdered infant formula samples. The proposed aptasensor does not require complicated DNA extraction steps or antibodies, and can be performed at 37°C, making it a convenient and sensitive strategy for C. sakazakii detection.

Vancomycin-modified poly-l-lysine magnetic separation combined with multiplex polymerase chain reaction assay for efficient detection of Bacillus cereus in milk.

In this study, a new vancomycin (Van)-modified poly-l-lysine (PLL) magnetic bead (MB) technique was developed for isolation of gram-positive bacteria. The method combines magnetic separation with a multiplex PCR (mPCR) assay and gel electrophoresis for easy and rapid detection of Bacillus cereus. Vancomycin was used as a molecular ligand between the MB and the d-alanyl-d-alanine moieties on the cell wall surface of B. cereus. The PLL served as a flexible molecular tether between the MB and Van that reduced steric hindrance maintaining the biological activity of Van. The MB-PLL-Van capture nanoprobes exhibited excellent capture and isolation efficiency for B. cereus in spiked milk matrix samples without interference from the complex food matrix. The subsequent mPCR assay showed high specificity for the 4 target genes in B. cereus, the entFM, cesB, cer, and 16S rRNA genes, that were used to achieve efficient genotyping and detection. Under optimum conditions, the limit of detection reached 103 cfu/mL, with a dynamic range of detection at 103 to 107 cfu/mL in pure culture. Application of the MB-PLL-Van mediated mPCR assay for B. cereus in milk matrix samples achieved results similar to those of the pure culture. In addition, with a 6-h pre-enrichment of B. cereus that was spiked in milk matrix samples, the limit of detection reached 101 cfu/mL. The MB-PLL-Van mediated mPCR assay developed in this study could be used as a universal technology platform for the efficient enrichment and genotyping of gram-positive bacteria.

Simultaneous detection of Salmonella spp., Pseudomonas aeruginosa, Bacillus cereus, and Escherichia coli O157:H7 in environmental water using PMA combined with mPCR.

A multiplex polymerase chain reaction (mPCR) with propidium monoazide (PMA) and internal amplification control (IAC) for the simultaneous detection of waterborne pathogens Salmonella spp., Pseudomonas aeruginosa, Bacillus cereus, and Escherichia coli O157:H7, was developed. This PMA-IAC-mPCR assay used four new specific primers based on the genes for invA, ecfX, cesB, and fliC, respectively. A 16S rRNA primer was chosen for IAC to eliminate false negative results. The photosensitive dye, propidium monoazide (PMA) was used to exclude signals from dead bacteria that could lead to false positive results. In pure culture, the limits of detection (LOD) were 101 CFU/ml for P. aeruginosa, 102 CFU/ml for both Salmonella spp. and E. coli O157:H7, and 103 CFU/ml for B. cereus, respectively. In addition, with a 6-8 h enrichment of all four bacteria that were combined in a mixture that was spiked in water sample matrix, the LOD was 3 CFU/ml for Salmonella spp., 7 CFU/ml for E. coli O157:H7, 10 CFU/ml for B. cereus and 2 CFU/ml for P. aeruginosa. This PMA-IAC-mPCR assay holds potential for application in the multiplex assay of waterborne pathogens.

Dual-signal amplification strategy: Universal asymmetric tailing-PCR triggered rolling circle amplification assay for fluorescent detection of Cronobacter spp. in milk.

Cronobacter spp. are important opportunistic foodborne pathogens in powdered infant formula that cause many serious diseases in neonates and infants. In this study, a novel assay based on dual signal amplification strategy was developed by coupling asymmetric tailing PCR (AT-PCR) with rolling circle amplification (RCA) for the detection of Cronobacter spp. in milk. The tailing single-stranded DNA was generated through AT-PCR and used to initiate RCA, generating tandem repetitive G-quadruplex sequences. In the presence of the fluorescence dye thioflavin T that could intercalate into the G-quadruplex structures, the fluorescence signal was detected with a microplate reader. The AT-PCR coupled with RCA assay was specific for Cronobacter spp. detection because of the highly specific primers chosen for the AT-PCR. The limits of detection were 4.3 × 101 cfu/mL in pure culture and 4.5 × 102 cfu/mL in spiked milk, respectively. The fixed sequences designed in the hairpin DNA allowed this AT-PCR coupled with RCA assay to serve as a universal platform for the detection of other pathogens by modifying the specificity of the PCR primers.

Rapid and quantitative detection of viable emetic Bacillus cereus by PMA-qPCR assay in milk.

Emetic Bacillus cereus is one of the causative agents of foodborne diseases which can cause vomiting-type food poisoning after ingestion of contaminated food. To minimize B. cereus food poisoning, propidium monoazide (PMA) combined with quantitative polymerase chain reaction (qPCR) called PMA-qPCR was applied for detecting viable emetic B. cereus in milk. The cereulide synthetase gene of emetic B. cereus (cesB) was chosen for the primer, and PMA treatment was optimized at 3 µg/mL to inhibit the PCR amplification of DNA from dead cells. Under optimized assay parameters, the limit of detection (LOD) using this method were 102 CFU/mL in both pure culture and in spiked milk matrix. The cycle threshold (Ct) values obtained for this assay was not significantly affected by the presence of non-target bacteria such as E. coli O157:H7 which indicated the high selectivity of the assay for emetic B. cereus. The PMA-qPCR assay used in this study has the potential for sensitive detection of viable emetic B. cereus in milk.