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Objective: In this study, a colorimetric sensing system based on different aptamers and AuNPs was constructed for the rapid quantitative detection of aflatoxin M1 (AFM1) in milk. The detection performance of AFM1 aptamers with 21 (A21) and 72 base (A72) lengths commonly used in literature reports was evaluated in actual samples. Methods: AuNPs solution was prepared by sodium citrate reduction. After adding AFM1 aptamer and AFM1 standard solution, the aptamer specifically combined with AFM1 to form a special three-dimensional structure. With the addition of NaCl solution, the stability of AuNPs solution was disrupted and aggregation occurred, resulting in color change of the solution. AFM1 was quantitatively detected by measuring the absorbance value and absorption spectrum of AuNPs solution. Results: Under the optimum condition, the limits of detection of AuNPs-aptasensor based on A21 and A72 aptamers were 25.26 µg/L and 5.77 µg/L, respectively, with the linear range of 10~800 µg/L. Moreover, the AuNPs-aptasensor had good selectivity and specificity. The recoveries of AuNPs-aptasensor based on A21 and A72 aptamers were 95.7%~103.6% and 94.3%~98.2%, respectively. Conclusion: The A72 performed better than A21 in detecting AFM1 in milk, which would be related to the length, configuration and affinity of the aptamer. The colorimetric sensing method established based on aptamers A72 was simple, fast, sensitive, highly specific, and visualized, providing data reference for the application of AFM1 aptamers in dairy products.

Health concerns and financial losses caused by mushroom poisoning have been reported worldwide. Amanita citrinoannulata, a poisonous mushroom commonly found in China, results in a toxic reaction in humans after mistaken ingestion. To reduce the mistaken ingestion of poisonous mushrooms and to improve clinical diagnosis of mushroom poisoning, a rapid mushroom species identification method is required. Such identification methods could be advantageous in the identification of other poisonous mushroom species. This study developed two rapid and sensitive methods for the detection of A. citrinoannulata utilizing colorimetric and real-time loop-mediated isothermal amplification (LAMP) technology and specifically designed primers for the internal transcribed spacer (ITS) genes of A. citrinoannulata. The methods demonstrated high sensitivity as 0.2 ng of A. citrinoannulata DNA could be detected, with no cross-reaction with 41 non-target mushroom species. The entire detection process could be completed within 40 min without requiring complex instruments and can be observed by the naked eye. Therefore, these novel methods can be used for the identification of fresh and cooked mushroom samples and vomit samples, which contain only 1% A. citrinoannulata. Furthermore, these methods facilitate the detection of mushroom poisoning, and thus, have potential to reduce the number of mushroom poisoning-related deaths worldwide.

Caused by Salmonella pullorum, pullorosis is a bacterial disease threatening the poultry industry and has been listed as the bacterial disease to be eliminated by the government. However, antibiotic treatment of pullorosis has become increasingly difficult, resulting in severe influences on the sustainable development of poultry. Abuse of antibiotics may cause global drug-resistant problems. Hence, early diagnosis of young chickens and accurate treatment of sick chickens are urgently needed. Traditional serotyping for Salmonella detection is costly and labor-intensive, whereas other commonly used plate agglutination test methods often cause physical injury of chickens. Therefore, a rapid and nondamaging detection method is of great significance for early diagnosis, which is the key step in accurate medication and elimination of pullorosis. In this study, we propose a novel lateral flow nucleic acid assay (LFNAA) system combining recombinase polymerase amplification (RPA) for the detection of S. pullorum. In this method, the DNA of S. pullorum strains was quickly amplified by RPA under 37°C, and then, the RPA products were added onto the LFNAA sample pad until the final results could be observed by naked eyes within 3 min. The proposed assay is fast and delivers visible results to naked eyes in field test. The limit of detection for genomic DNA was 5 × 10−3 ng/μL, indicating high sensitivity. In addition, the proposed LFNAA system is cost-effective, efficient, and nondamaging to chicks in the field test. This system provides technical support for early diagnosis of S. pullorum in the poultry and paves a way for future precision medicine to avoid the global drug-resistance issues.

With widespread use of antibiotics in the aquaculture industry, bacterial resistance has recently attracted increasing attention. Continuous emergence of multi-resistant bacteria has greatly threatened human and animal health, as well as the quality and safety of livestock products. To control bacterial resistance, the effect of bacterial resistance needs to be well understood. The purpose of this study was to explore the factors influencing Escherichia coli (E. coli) drug resistance in large-scale pig farms. In this study, 296 strains of E. coli isolated and identified from large-scale pig farms in Beijing were used as the research objects. In vitro drug sensitivity tests were used to determine the sensitivity to 10 antibiotics of pig-derived E. coli. SPSS logistic regression was employed to analyze the effects of the season, pig type, sampling point (medication type) and sampling location on resistance and multi-drug resistance of E. coli from pigs. The degrees of drug resistance to 10 antibiotics of the 296 strains of pig-derived E. coli were varied, their resistance rates were between 4.05 and 97.64%, and their multi-drug resistance was appalling, with the highest resistance to six antibiotics being 26.35%. The isolated strains were proven more resistant to tetracyclines, penicillin and chloramphenicol, which are commonly used for disease prevention in pig farms, and less resistant to quinolones and aminoglycosides, which are not used in pig farms. The resistance of the isolated strains in spring and summer was generally higher than that in winter. E. coli resistance in piglets, fattening pigs and sows was more serious than that in nursery and sick pigs. The results showed that the season, type of medication and type of pig had an influence on the pig-derived E. coli resistance, among which the type of medication was the most influencing factor.

Chlorophyllum molybdites is a kind of common poisonous mushroom in China that is widely distributed in different areas. Food poisoning caused by accidentally eating C. molybdites has become more frequent in recent years. In 2019, there were 55 food poisoning incidents caused by eating this mushroom in China. Mushroom poisoning continues to be a common health issue of global concern. When mushroom poisoning occurs, an effective, simple, and rapid detection method is required for accurate clinical treatment or forensic analysis. For the first time, we established a loop-mediated isothermal amplification (LAMP) assay for the visual detection of C. molybdites. A set of specific LAMP primers was designed, and the specificity was confirmed against 43 different mushroom species. The LAMP method could detect as low as 1 pg of genomic DNA. Boiled mushrooms and artificial gastric-digested mushroom samples were prepared to test the applicability of the method, and the results showed that as low as 1% C. molybdites in boiled and digested samples could be successfully detected. The LAMP method can also be completed within 45 min, and the reaction results could be directly observed based on a color change under daylight by the naked eye. Therefore, the LAMP assay established in this study provides an accurate, sensitive, rapid, and low-cost method for the detection of C. molybdites.

Traceability of milk origin in China is conducive to the implementation of the protection of regional products. In order to distinguish milk from different geographical distances in China, we traced the milk of eight farms in four neighboring provinces of China (Inner Mongolia autonomous region, Hebei, Ningxia Hui autonomous and Shaanxi), and multivariate data analysis was applied to the data including elemental analysis, stable isotope analysis and fatty acid analysis. In addition, orthogonal partial least squares discriminant analysis (OPLS-DA) is used to determine the optimal classification model, and it is explored whether the combination of different technologies is better than a single technical analysis. It was confirmed that in the inter-provincial samples, the combination of the two techniques was better than the analysis using a single technique (fatty acids: R2 = 0.716, Q2 = 0.614; fatty acid-binding isotopes: R2 = 0.760, Q2 = 0.635). At the same time, milk produced by farms with different distances of less than 11 km in each province was discriminated, and the discriminant distance was successfully reduced to 0.7 km (Ningxia Hui Autonomous Region: the distance between the two farms was 0.7 km, R2 = 0.771, Q2 = 0.631). For short-distance samples, the combination multiple technologies are not completely superior to a single technique, and sometimes, it is easy to cause model over-fitting.

Type-B aflatoxins (AFB1 and AFB2) frequently contaminate food, especially nuts and fried figs, and seriously threaten human health; hence, it is necessary for the newly rapid and sensitive detection methods to prevent the consumption of potentially contaminated food. Here, a lateral flow aptasensor for the detection of type-B aflatoxins was developed. It is based on the use of fluorescent dye Cy5 as a label for the aptamer, and on the competition between type-B aflatoxins and the complementary DNA of the aptamer. This is the first time that the complementary strand of the aptamer has been used as the test line (T-line) to detect type-B aflatoxins. In addition, the truncated aptamer was used to improve the affinity with type-B aflatoxins in our study. Therefore, the lengths of aptamer and cDNA probe were optimized as key parameters for higher sensitivity. In addition, binding buffer and organic solvent were investigated. The results showed that the best pair for achieving improved sensitivity and accuracy in detecting AFB1 was formed by a shorter aptamer (32 bases) coupled with the probe complementary to the AFB1 binding region of the aptamer. Under the optimal experimental conditions, the test strip showed an excellent linear relationship in the range from 0.2 to 20 ng/mL with a limit of detection of 0.16 ng/mL. This aptamer-based strip was successfully applied to the determination of type-B aflatoxins in spiked and commercial peanuts, almonds, and dried figs, and the recoveries of the spiked samples were from 93.3%−112.0%. The aptamer-complementary strand-based lateral flow test strip is a potential alternative tool for the rapid and sensitive detection of type-B aflatoxins in nuts and dried figs. It is of help for monitoring aflatoxins to avoid the consumption of unsafe food.

Ochratoxin A (OTA) is a mycotoxin identified as a contaminant in grains and wine throughout the world, and convenient, rapid and sensitive detection methods for OTA have been a long-felt need for food safety monitoring. Herein, we presented a new competitive format based lateral flow strip fluorescent aptasensor for one-step determination of OTA in corn samples. Briefly, biotin-cDNA was immobilized on the surface of a nitrocellulose filter on the test line. Without OTA, Cy5-labeled aptamer combined with complementary strands formed a stable double helix. In the presence of OTA, however, the Cy5-aptamer/OTA complexes were generated, and therefore less free aptamer was captured in the test zone, leading to an obvious decrease in fluorescent signals on the test line. The test strip showed an excellent linear relationship in the range from 1 ng·mL−1 to 1000 ng·mL−1 with the LOD of 0.40 ng·mL−1, IC15 value of 3.46 ng·mL−1 and recoveries from 96.4% to 104.67% in spiked corn samples. Thus, the strip sensor developed in this study is an acceptable alternative for rapid detection of the OTA level in grain samples.

Colorimetric aptasensors using unmodified gold nanoparticles (AuNPs) have attracted much attention because of their low cost, simplicity, and practicality, and they have been developed for various targets in the past several years. However, previous research has focused on developing single-target assays. Here, we report the development of a homogeneous multiplex aptasensor by using more than one class of aptamers to stabilize AuNPs. Using sulfadimethoxine (SDM), kanamycin (KAN) and adenosine (ADE) as example targets, a KAN aptamer (750 nM), an SDM aptamer (250 nM) and an ADE aptamer (500 nM) were mixed at a 1∶1∶1 volume ratio and adsorbed directly onto the surface of unmodified AuNPs by electrostatic interaction. Upon the addition of any of the three targets, the conformation of the corresponding aptamer changed from a random coil structure to a rigid folded structure, which could not adsorb and stabilize AuNPs. The AuNPs aggregated in a specific reaction buffer (20 mM Tris-HCl containing 20 mM NaCl and 5 mM KCl), which led to a color change from red to purple/blue. These results demonstrate that the multiplex colorimetric aptasensor detected three targets simultaneously while maintaining the same sensitivity as a single-target aptasensor for each individual target. The multiplex aptasensor could be extended to other aptamers for various molecular detection events. Due to its simple design, easy operation, fast response, cost effectiveness and lack of need for sophisticated instrumentation, the proposed strategy provides a powerful tool to examine large numbers of samples to screen for a small number of potentially positive samples containing more than one analyte, which can be further validated using sophisticated instruments.

The ability to detect sequence-specific single-strand DNA (ssDNA) in complex, contaminant-ridden samples, using a fluorescent method directly without a DNA extraction and PCR step could simplify the detection of pathogens in the field and in the clinic. Here, we have demonstrated a simple label-free sensing strategy to detect ssDNA by employing its complementary ssDNA, S1 nuclease and nucleic acid fluorescent dyes. Upon clearing away redundant complementary ssDNA and possibly mismatched double strand DNA by using S1 nuclease, the fluorescent signal-to-noise ratio could be increased dramatically. It enabled the method to be adaptable to three different types of DNA fluorescent dyes and the ability to detect target ssDNA in complex, multicomponent samples, like tissue homogenate. The method can distinguish a two-base mismatch from avian influenza A (H1N1) virus. Also, it can detect the appearance of 50 pM target ssDNA in 0.5 µg · mL(-1) Lambda DNA, and 50 nM target ssDNA in 5 µg · mL(-1) Lambda DNA or in tissue homogenate. It is facile and cost-effective, and could be easily extended to detect other ssDNA with many common nucleic acid fluorescent dyes.

Ochratoxin A(OTA) is found to be one of the predominant contaminating mycotoxins in a wide variety of food commodities. To avoid the risk of OTA consumption, the detection and quantitation of OTA level are of great significance. Based on the fact that ssDNA aptamer has the ability to form a double-strand structure with its complementary sequence, a simple and rapid aptamer-based label-free approach for highly sensitive and selective fluorescence detection of OTA was developed by using ultra-sensitive double-strand DNA specific dyes PicoGreen. The results showed that as low as 1 ng/mL of OTA could be detected with a dynamic range of more than 5 orders of magnitude which satisfies the requirements for OTA maximum residue limit in various food regulated by European Commission. With the specificity of aptamer, the assay exhibited high selectivity for OTA against two other analogues (N-acetyl-l-phenylalanine and zearalenone). We also tested the aptasensor practicability using real sample of 1% beer spiked with a series of concentration of OTA and the results show good tolerance to matrix effect. All detections could be achieved in less than 30 min, which provides a simple, quick and sensitive detection method for OTA screening in food safety and could be easily extend to other small molecular chemical compounds detection which aptamer has been selected.

A simple method to extract DNA from hair shafts was developed by using enzymatic laundry powder at the first step of the process. The whole extraction can be finished in less than 2 hours. The simple extraction reagent proposed here contains only two cheap components: ordinary enzymatic laundry powder and PCR buffer. After extraction, an ultra sensitive fluorescent nucleic acid stain, PicoGreen, was used for quantifying trace amount of double-stranded DNA in the solution extracted. For further validation of DNA extraction, four primers were employed to amplify DNA microsatellite loci. Both fluorescence spectroscopy and PCR results suggested that this method can extract DNA from hair shafts with good efficiency and repeatability. The study will greatly facilitate the use of hair shafts in future for DNA analyses on genome-wide scale.

Steroid metabolism is important in various species. The accumulation of androgen metabolite, androstenone, in pig adipose tissue is negatively associated with pork flavor, odour and makes the meat unfit for human consumption. The 17β-hydroxysteroid dehydrogenase type 7 (17βHSD7) expressed abundantly in porcine liver, and it was previously suggested to be associated with androstenone levels. Understanding the enzymes and metabolic pathways responsible for androstenone as well as other steroids metabolism is important for improving the meat quality. At the same time, metabolism of steroids is known to be species- and tissue-specific. Therefore it is important to investigate between-species variations in the hepatic steroid metabolism and to elucidate the role of 17βHSD7 in this process. Here we used an effective methodological approach, liquid chromatography coupled with mass spectrometry, to investigate species-specific metabolism of androstenone, testosterone and beta-estradiol in HepG2 cell line, and pig cultured hepatocytes. Species- and concentration-depended effect of steroids on 17βHSD7 gene expression was also investigated. It was demonstrated that the investigated steroids can regulate the 17βHSD7 gene expression in HepG2 and primary cultured porcine hepatocytes in a concentration-dependent and species-dependent pattern. Investigation of steroid metabolites demonstrated that androstenone formed a 3'-hydroxy compound 3β-hydroxy-5α-androst-16-ene. Testosterone was metabolized to 4-androstene-3,17-dione. Estrone was found as the metabolite for β-estradiol. Inhibition study with 17βHSD inhibitor apigenin showed that apigenin didn't affect androstenone metabolism. Apigenin at high concentration (50 µM) tends to inhibit testosterone metabolism but this inhibition effect was negligible. Beta-estradiol metabolism was notably inhibited with apigenin at high concentration. The study also established that the level of testosterone and β-estradiol metabolites was markedly increased after co-incubation with high concentration of apigenin. This study established that 17βHSD7 is not the key enzyme responsible for androstenone and testosterone metabolism in porcine liver cells.