Milk Samples Research Articles

Determination of microbial contamination in raw milk, processed milk and yoghurt consumed in Mbarara city, western part of Uganda

he microbial composition of milk serves as a key indicator of its quality. While its nutritional value is significant, the inherent nature of milk also makes it an ideal environment for the proliferation of microorganisms, some of which lead to human illnesses, while others contribute to the spoilage of milk, making it unsafe for consumption. This research therefore aimed to assess the bacterial contamination content in raw milk, UHT and yoghurt, a total of 36 samples were gathered from various retailers, markets and supermarkets across Mbarara city. Contaminated milk and its products are accountable for human dairy related illnesses. This cross-sectional study was conducted to assess micro bacterial contamination and the presence of selected milk-borne zoonotic pathogens. The study involved 36 samples of milk and milk products mainly raw milk, UHT, or processed milk and yoghurt. Laboratory analysis conducted included Total Plate Count (TPC), Yeasts and molds (YM), staphylococcus, salmonella and coliform, as well as detection of Escherichia coli (E coli), biochemical tests including gram staining were employed to isolate and identify bacteria in these samples. The findings indicated the presence of these types of bacteria in raw milk, Staphylococcus spp (25%), Salmonella spp (33.3%), YM (41.6%), TPC (50%), coliforms (33.3%) and E.coli (25%), for UHT milk the percentage was Staphylococcus spp (0%), Salmonella spp (16.6%), YM (8.3%), TPC(16.6%), coliforms (16.6%) and E.coli (8.3%), finally for yoghurt the results were Staphylococcus spp (8.3%), Salmonella spp (8.3%), YM (16.6%), TPC (25%), coliforms (16.6%) and E.coli (0%). In conclusion, the detection of microbial contamination in raw milk and milk products suggests inadequate sanitary practices and poor storage practices. Products that are contaminated by these pathogenic organisms when consumed may result in food poisoning and a proper awareness among the stakeholders and consumers regarding possible outcomes of consuming contaminated food items is necessary. keywords: Milk, Yoghurt, Food poisoning, Microorganisms, Contamination

An enzyme cascade biosensor based on multiwalled carbon nanotube-RuO2 nanocomposite for selective amperometric determination of lactose in milk samples

An enzyme-based electrochemical biosensor was fabricated for the sensitive determination of lactose. The utilized enzyme cascade system is composed of β-galactosidase (β-Gal) and glucose oxidase (GOx). The ruthenium(IV) oxide (RuO2) presents in MWCNT-RuO2 nanocomposite immobilized on the glassy carbon electrode acts as an electrochemical mediator, resembling a second-generation enzyme biosensor. The functional mechanism of the biosensor was discussed, explaining the chemical oxidation of H2O2, the final product of the enzymatic reaction, by RuO2 and subsequent reoxidation of generated Ru to RuO2 at the electrode surface. This shifts the oxidation of H2O2 to a lower potential magnitude of +0.40 V, enhancing the biosensor’s selectivity. The analytical figures of merit were verified for the developed lactose biosensor through repetitive measurements. The precision of the lactose biosensor was ensured with good reproducibility (RSD % = 2.68) and repeatability (RSD % = 4.12). The selectivity of the biosensor towards various saccharides and ionic species potentially present in milk samples was investigated, and no notable interference effect was detected. Moreover, the accuracy of the lactose biosensor was tested by analyzing spiked samples and a semi-skimmed milk (SS-milk) sample with certified lactose values. The limit of detection (LOD) and limit of quantification (LOQ) were calculated to be (0.036 mM) and (0.121 mM), respectively. Thanks to the short response time of the fabricated lactose biosensor, it was transferred to a screen-printed carbon electrode (SPCE) and successfully employed in flow injection analysis (FIA).

A green, fluorescent probe employing erythrosine-B for tracing the accidental administration of levamisole in milk and plasma samples.

A simple and sensitive fluorescent probe has been developed and optimized to detect the non-intentional administration of levamisole (LVM). LVM is used as an anthelmintic therapy in cows, and hence, its residues appear in the drained milk until 60 hours after administering the drug. Meanwhile, levamisole is known to be an adulterant to cocaine and could be detected in addicts' plasma samples. Owing to its severe side effects, including agranulocytosis, which is lethal in many cases, detection and quantification of LVM in milk and plasma samples are of utmost importance. Therefore, a sensitive and selective analytical method is required for this purpose. This work develops a highly fluorescent probe obtained through the reaction between LVM and erythrosine-B in an acidic medium, where the produced ion pair complex has been measured at 553 nm after excitation at 528 nm. The proposed method provides linearity over the concentration range of 0.5-2.0 μg mL-1 for LVM, with a corresponding detection and quantitation limit of 0.5 and 0.3 μg mL-1. Full validation was performed, permitting the application of the suggested method to perform simple extraction steps. All the applied procedures followed the guidelines offered by green analytical chemistry, where the Green Analytical Procedure Index (GAPI) assessed the greenness of the proposed tool, and the yielded pictograms proved the eco-friendliness of the offered tool.

Visual Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid On-Site Detection of Escherichia coli O157: H7 in Milk Products.

(1) Background: Rapid on-site testing is an effective method for the detection of Escherichia coli O157: H7(E. coli O157: H7) in food ingredients and the environment. (2) Methods: In this study, we developed colorimetric loop-mediated isothermal amplification (LAMP) and immunochromatographic test strips (ICTs) for the rapid and visual detection of E. coli O157: H7. This study designed new specific LAMP primers for E. coli O157: H7 virulence island genes. After the LAMP amplification, the double-stranded DNA target sequence labeled with digoxin and fluorescein isothiocyanate (FITC) at both ends was bound to the anti-digoxin antibody on the gold nanoparticles. Subsequently, it was further bound to the anti-FITC antibody at the T line of the ICTs, forming a positive test result. Hydroxynaphthyl blue dye was directly added to the LAMP amplification product. A blue color indicated positive results, while a purple color indicated negative results. (3) Results: Two visualization methods showed high specificity for the target strains. The visualization tests had sensitivities of 5.7 CFU mL-1, and the detection limit of the Escherichia coli O157: H7 in artificially contaminated milk samples was 5.7 × 102 CFU mL-1, which was consistent with the results of the standard method (LAMP-electrophoresis method) used in commercial inspection. (4) Conclusions: Both methods could be useful in remote and under-resourced areas.

Establishing a surveillance programme for Salmonella Dublin in Austrian dairy herds by comparing herd-level vs. individual animal detection methods

Due to its increasing occurrence in cattle farms in various countries, leading to significant economic losses in affected livestock, Salmonella enterica subspecies enterica serovar Dublin (S. Dublin) has become a highly investigated pathogen in cattle production. In Austria, there have been occasional human cases of S. Dublin as well as an increase in laboratory-confirmed cases in cattle, indicating the need for a screening programme to determine the current status in Austria. The aims of this study were, firstly, to determine the seroprevalence of S. Dublin in dairy herds through bulk milk screenings in two federal states (Salzburg, Tyrol) of Austria. Secondly, the study aimed to identify the infection status of the herds through individual animal and herd level detection, comparing microbiological, molecular and serological detection methods. The results of the study will allow the development of a sampling strategy for a surveillance programme in Austria.A total of 6973 dairy farms were tested through serological bulk milk screening. The seroprevalence for the federal state of Tyrol was 14.8 % and for Salzburg it was 18.2 %, resulting in an average seroprevalence of 16.5 %. At an individual animal level, 205 (11.3 %) animals tested positive for shedding of S. Dublin in the faeces through microbiological detection, and 268 (17.0 %) animals had positive values (ct value ≤ 38) by qPCR. The association between microbiological and molecular detection was statistically significant (p < 0.001), with a calculated kappa value of 0.65 ± 0.27 (p ≤ 0.001), assuming a substantial level of agreement. In 17 herds, where an individual animal tested positive for shedding of S. Dublin, environmental sampling and testing were carried out. At a herd level 16 (94.1 %) out of the 17 participating herds, tested positive for S. Dublin either microbiologically or by molecular assay in boot swab samples. Bulk milk samples from 14 out of the 17 participating herds were analysed for antibodies to S. Dublin and 12 samples (85.7 %) were positive. In total 111 (18.9 %) out of 587 blood samples tested positive for S. Dublin antibodies, demonstrating a statistically significant correlation (p < 0.001) both with microbiological (κ = 0.32 ± 0.49; p ≤ 0.001) and molecular (κ=0.23 ± 0.06; p ≤ 0.001) findings.It was possible to identify S. Dublin by culture from boot swabs in 14 (82.4 %) out of 17 herds and by molecular assay using qPCR in 15 (88.2 %) out of 17 herds, indicating a suitable sample type for screening on a herd level-basis for acute infections, but not for identifying chronic infections or asymptomatic carriers. Other environmental samples, such as sponge-sticks, are only suitable to a limited extent for the detection of S. Dublin. The results of this study demonstrate a moderate S. Dublin prevalence in dairy herds in the selected Austrian regions, signalling further screening and management programmes for the future.

Quantification of pesticide residues in milk by HPLC-UV analysis in Gilan province, Iran

ABSTRACT Eighty milk samples, comprising raw cow’s milk, pasteurised milk, ultra-high temperature milk, and camel milk, were collected randomly from supply centres from December 2022 to March 2023 in Gilan province, Iran. Aldrin, diazinon, and dichlorodiphenyltrichloroethane (DDT) were determined using high-performance liquid chromatography. Aldrin and diazinon were observed in all samples. Furthermore, 87.5% of the milk samples were contaminated with DDT. The raw milk sample showed the greatest concentration of aldrin, measuring 9.31 ± 0.80 µg L−1, and there was no significant difference with the camel milk. The samples of camel milk, raw milk, and ultra-high temperature milk demonstrated the greatest levels of DDT, whereas the lowest concentration was detected in pasteurised milk. In all samples, the maximum residue level (MRL) of Codex Alimentarius and the national Iran standard (20 µg L−1 for DDT) was only exceeded by 25.1% of the milk samples.

Mammary fat globules as a source of mRNA to model alterations in the expression of some milk component genes during lactation in bovines

BackgroundThe milk's nutritional value is determined by its constituents, including fat, protein, carbohydrates, and minerals. The mammary gland's ability to produce milk is controlled by a complex network of genes.Thereby, the fat, protein, and lactose synthesis must be boost in milk to increase milk production efficiency. This can be accomplished by fusing genetic advancements with proper management practices. Therefore, this study aimed to investigate the association between the Lipoprotein lipase (LPL), kappa casein CSN3, and Glucose transporter 1 (GLUT1) genes expression levels and such milk components as fat, protein, and lactose in different dairy breeds during different stages of lactation.MethodsTo achieve such a purpose, 94 milk samples were collected (72 samples from 36 multiparous black-white and red-white Holstein–Friesian (HF) cows and 22 milk samples from 11 Egyptian buffaloes) during the early and peak lactation stages. The milk samples were utilized for milk analysis and genes expressions analyses using non- invasive approach in obtaining milk fat globules (MFGs) as a source of Ribonucleic acid (RNA).ResultsLPL and CSN3 genes expressions levels were found to be significantly higher in Egyptian buffalo than Holstein–Friesian (HF) cows as well as fat and protein percentages. On the other hand, GLUT1 gene expression level was shown to be significantly higher during peak lactation than early lactation. Moreover, lactose % showed a significant difference in peak lactation phase compared to early lactation phase. Also, fat and protein percentages were significantly higher in early lactation period than peak lactation period but lactose% showed the opposite pattern of Egyptian buffalo.ConclusionTotal RNA can be successfully obtained from MFGs. The results suggest that these genes play a role in glucose absorption and lactose synthesis in bovine mammary epithelial cells during lactation. Also, these results provide light on the differential expression of these genes among distinct Holstein–Friesian cow breeds and Egyptian buffalo subspecies throughout various lactation phases.

First Genotyping of Mycobacterium avium Subspecies Paratuberculosis from Large Ruminant Population at Satna District in Madhya Pradesh in India to Bison Type

Background: Paratuberculosis or Johne’s disease in large ruminants is caused due to Mycobacterium avium subspecies paratuberculosis. The bacterial disease in large ruminants is characterized by chronic diarrhea, weakness and emaciation. There are no reports on Paratuberculosis in this area’s large ruminant population and its genotyping. Methods: A total of 76 samples (26 fecal and 50 milk) of large ruminants from Satna district in Madhya Pradesh were investigated (microscopical; for the presence of acid-fast organisms and molecular methods; for gene-specific amplification) for the presence of Mycobacterium avium subspecies paratuberculosis infection. Result: Microscopical examinations of fecal samples revealed 11.54% positivity and milk samples 14% positivity of Mycobacterium avium subspecies paratuberculosis infection. A single fecal and 2 milk samples were found positive in the IS900 polymerase chain reaction test (413 bp product). Specific amplification of 608 bp IS1311 product followed by restriction endonuclease analysis proved the genotype of Mycobacterium avium subspecies paratuberculosis as Bison type.

Resilient Staphylococcus aureus in Mastitis Milk: Insights into Antibiotic Resistance and Biofilm Formation

Background: Staphylococcus aureus is capable of producing a wide range of virulence factors. It is a deadly bacterium that is resistant to the majority of antibiotics that are typically provided. The ability of S. aureus to establish chronic, implant associated infections and our inability to cure them, is directly associated with its biofilm formation, creating an environment where bacteria can grow and persist while protected from the host immune response and antibiotic therapy. Antibiotic resistance of S. aureus is widely spreading with low cure rate. Methods: A total of 62 S. aureus strains were isolated from animal pus, human pus, animal skin, human skin, mastitic milk, normal milk and unprocessed meat samples. The isolates were genotypically confirmed by 23S rRNA ribotyping for S. aureus. Using 14 antibiotics belonging to different generations were tested for their resistance pattern from various sources. Result: All the mastitic milk, animal pus, human pus and unprocessed meat S. aureus isolates from all the places of sampling showed 100% sensitivity towards imipenem while isolates obtained from mastitic milk, human skin, animal skin showed 100% resistance towards ampicillin and ampicillin+sulbactum combination.

Effects of lysine and threonine on milk yield, amino acid metabolism, and fecal microbiota of Yili lactating mares.

The nutritional benefits of mare milk are attracting increasing consumer interest. Limited availability due to low yield poses a challenge for widespread adoption. Although lysine and threonine are often used to enhance protein synthesis and muscle mass in horses, their impact on mare milk yield and nutrient composition remains underexplored. This study investigated the effects of lysine and threonine supplementation on 24 healthy Yili mares, mares at day 30 of lactation, over a 120-day period. The mares were divided into control and three experimental groups (six mares each) under pure grazing conditions. The control group received no amino acid supplementation, while experimental groups received varying daily doses of lysine and threonine: Group I (40 g lysine + 20 g threonine), Group II (60 g lysine + 40 g threonine), and Group III (80 g lysine + 60 g threonine). Supplementation in Group II notably increased milk yield, while Groups I and II showed higher milk fat percentages, and all experimental groups exhibited improved milk protein percentages. Additionally, blood levels of total protein, albumin, triglycerides, and glucose were reduced. Detailed analyses from Group II at peak lactation (day 60) included targeted metabolomics and microbial sequencing of milk, blood, and fecal samples. Amino acid metabolomics assessed amino acid content in mare milk and serum, while 16S rRNA gene sequencing evaluated rectal microbial composition. The results indicated that lysine and threonine supplementation significantly increased levels of threonine and creatine in the blood, and lysine, threonine, glutamine, and alanine in mare milk. Microbial analysis revealed a higher prevalence of certain bacterial families and genera, including Prevotellaceae, p_251_o5, and Rikenellaceae at the family level, and unclassified_p_251_o5, Prevotellaceae_UCG_001, and Rikenellaceae_RC9_gut_group at the genus level. Multi-omics analysis showed positive correlations between specific fecal genera and amino acids in mare milk. For instance, Prevotellaceae_UCG_003, unclassified Bacteroidetes_BS11_gut_group, and Corynebacterium were positively correlated with lysine, while unclassified Prevotellaceae was positively correlated with alanine and threonine, and Unclassified_Bacteroidales_BS11_gut_group was positively correlated with glutamine. In summary, lysine and threonine supplementation in grazing lactating mares enhanced milk production and improved milk protein and fat quality. It is recommended that herders, veterinarians, and technicians consider amino acid content in the diet of lactating mares. The optimal supplementation levels under grazing conditions for Yili horses were determined to be 60 g lysine and 40 g threonine per day. Future research should explore the molecular mechanisms by which these amino acids influence milk protein and lipid synthesis in mare mammary epithelial cells.

Streptococcus raffinosi sp. nov., isolated from human breast milk samples.

Novel Gram-positive, catalase-negative, α-haemolytic cocci were isolated from breast milk samples of healthy mothers living in Hanoi, Vietnam. The 16S rRNA gene sequences of these strains varied by 0-2 nucleotide polymorphisms. The 16S rRNA gene sequence of one strain, designated as BME SL 6.1T, showed the highest similarity to those of Streptococcus salivarius NCTC 8618T (99.4 %), Streptococcus vestibularis ATCC 49124T (99.4 %), and Streptococcus thermophilus ATCC 19258T (99.3 %) in the salivarius group. Whole genome sequencing was performed on three selected strains. Phylogeny based on 631 core genes clustered the three strains into the salivarius group, and the strains were clearly distinct from the other species in this group. The average nucleotide identity (ANI) value of strain BME SL 6.1T exhibited the highest identity with S. salivarius NCTC 8618T (88.4 %), followed by S. vestibularis ATCC 49124T (88.3 %) and S. thermophilus ATCC 19258T (87.4 %). The ANI and digital DNA-DNA hybridization values between strain BME SL 6.1T and other species were below the cut-off value (95 and 70 %, respectively), indicating that it represents a novel species of the genus Streptococcus. The strains were able to produce α-galactosidase and acid from raffinose and melibiose. Therefore, we propose to assign the strains to a new species of the genus Streptococcus as Streptococcus raffinosi sp. nov. The type strain is BME SL 6.1T (=VTCC 12812T=NBRC 116368T).

A ratiometric SERS aptasensor based on catalytic hairpin self-assembly mediated cyclic signal amplification strategy for the reliable determination of E. coli O157:H7.

Aratiometric SERS aptasensor based on catalytic hairpin self-assembly (CHA) mediated cyclic signal amplification strategy was developed for the rapid and reliable determination of Escherichia coli O157:H7. The recognition probe was synthesized by modifying magnetic beads with blocked aptamers, and the SERS probe was constructed by functionalizing gold nanoparticles (Au NPs) with hairpin structured DNA and 4-mercaptobenzonitrile (4-MBN). The recognition probe captured E. coli O157:H7 specifically and released the blocker DNA, which activated the CHA reaction on the SERS probe and turned on the SERS signal of 6-carboxyl-x-rhodamine (ROX). Meanwhile, 4-MBN was used as an internal reference to calibrate the matrix interference. Thus, sensitive and reliable determination and quantification of E. coli O157:H7 was established using the ratio of the SERS signal intensities of ROX to 4-MBN. This aptasensor enableddetection of 2.44 × 102 CFU/mL of E. coli O157:H7 in approximately 3h without pre-culture and DNA extraction. In addition, good reliability and excellent reproducibility were observed for the determination of E. coli O157:H7 in spiked water and milk samples. This study offered a new solution for the design of rapid, sensitive, and reliable SERS aptasensors.

Prevalence and Molecular Characterization of TEM and CTX-M Beta-Lactamase Genes of Escherichia coli Isolated From Cow Milk

Objective: The study objective was to investigate the presence and antibiotic resistance of toxigenic E. coli strains in the raw milk of dairycattle and to determine the prevalence of blaTEM and blaCTX-M genes in the selected isolates. Methods: Two hundred raw milk samples were collected from 20 dairy farms located in Kafr El Sheikh City. Results: Among the samples, 60 were positive for E. coli. The sensitivity of these isolates was detected against different antibiotics. Usingthe disk diffusion test, all the isolates were resistant to at least two beta-lactam antibiotics. The resistance to beta-lactam antibiotics bythe selected E. coli was varied to be highly significant. Five percent of the tested E. coli was highly resistant with a multi-antibiotic resistant (MAR) index, of 1.0, while 8% had the lowest MAR (0.14). Both blaTEM and blaCTX-M resistant genes were detected in isolate No. 52, while, blaCTX-M was detected in isolate No. 34 and blaTEM was detected in isolates No. 9, 21, 28, and 40. The PCR products of blaTEM and blaCTX-M genes were sequenced and deposited in the GenBank of the NCBI database with the accession numbers OR450046 and OR879117, respectively. Conclusion: The macrolides group of antibiotics especially erythromycin should not be a suitable treatment of dairy herds for mastitisby E. coli in Egypt. The majority of E. coli was multiple-antibiotic resistant and co-carried many virulence genes, and it may pose a greatpotential risk to public health.

Prevalence of Neospora caninum and associated risk factors in semi-intensive female water buffaloes reared in Greece, using an ELISA kit on milk samples

Neospora caninum is an apicomplexan (family: Sarcocystidae) protozoan parasite with a global distribution. In the N. caninum life cycle, dogs and other related canids are the definitive hosts, while other animals such as water buffaloes (Bubalus bubalis) constitute the intermediate host for this parasite. In many countries, the water buffalo is of high economic importance, providing valuable high-quality products for human needs. Although knowledge concerning the prevalence of this parasite in intermediate animal host populations is crucial, data from water buffalo are scarce. Keeping this in mind, the aim of this study was to examine the presence and assess the prevalence rates of N. caninum infection in water buffaloes in Northern Greece, where this animal husbandry sector started to raise, as well as to determine associated risk factors, with the application of a commercially available Neospora ISCOM ELISA test kit, developed for the detection of antibodies against N. caninum in milk samples The study was conducted during January–June 2023 in a total of 172 individual raw milk samples, collected from dairy buffaloes, reared under a semi extensive system, in three different farms. Information on the status of N. caninum infection in buffaloes from Greece was so far unknown with a lack of epidemiological or risk factors associated. For the detection of N. caninum, the commercially available Neospora ISCOM ELISA test kit (SANOVIR ®, Svanova Biotech AB, Uppsala, Sweden) was utilized. Results demonstrated the presence of N. caninum in water buffaloes from Greece for the first time. All positive N. caninum animal were asymptomatic, with absence of any disease signs. The overall prevalence of infection was 22.10%, whereas the main risk factors include the presence of dogs as well as the low biosecurity measures.

A novel ratiometric electrochemical sensor based on AuNPs decorated MIL-101(Fe) for simultaneously monitoring typical EDCs in milk

It is challengeable for traditional electrochemical sensors to determine EDCs in food samples due to inadequate reproducibility. Herein, a novel ratiometric electrochemical sensor for monitoring two typical EDCs referred to 17β-estradiol (E2) and bisphenol S (BPS) was developed. Electrodeposition method was utilized for facile decoration of Au nanoparticles (AuNPs) on MIL-101(Fe) to obtain AuNPs@MIL-101(Fe) composite as sensing material. In contrast to common ratiometric approaches, the Fe signal supplied by MIL-101(Fe) was expediently and innovatively adopted as the internal reference tag instead of fixing electroactive molecules. Furthermore, benefited from the synergistic effect of MIL-101(Fe) and AuNPs, AuNPs@MIL-101(Fe) composite exhibited remarkably enhanced electrochemical activity, resulting in strengthened Fe reference signal and facilitated oxidation process of EDCs. With the rise of EDCs’ concentration, the current response of EDCs gradually increased while the Fe signal almost kept constant. Thus, a new ratiometric electrochemical sensor was successfully developed with a wide linear range of 0.039–7.80 µM for EDCs as well as low LOD of 9.8 nM for E2 and 11.2 nM for BPS. Finally, the newly constructed sensor was achieved in evaluating the two targets in milk samples. The developed sensor exhibited high reproducibility, stability, sensitivity and practicability, providing a new idea for constructing ratiometric electrochemical sensors and a new method for EDCs detection.

Prevalence of Neospora caninum and associated risk factors in semi-intensive female water buffaloes reared in Greece, using an ELISA kit on milk samples

Prevalence of Neospora caninum and associated risk factors in semi-intensive female water buffaloes reared in Greece, using an ELISA kit on milk samples

Electrochemical detection of heavy metals in rice, milk and tap water using free-standing carbon felt electrodes

In this work, a carbon felt (CF) was utilized to fabricate electrochemical sensors for the simultaneous detection of heavy metals Cd2+, Pb2+ and Hg2+. The working conditions of CF sensors including thermal activation, electrolytes, and enrichment potentials and times were systematically investigated. Under the optimal detection conditions, the resulting sensors showed good linearity in the concentration ranges of 3–10,000, 2–10,000 and 5–10,000 μg/L for the detection of Cd2+, Pb2+ and Hg2+, corresponding to the detection limits of 1, 0.5, and 1 μg/L, respectively. Meanwhile, the resulting electrochemcial sensor demonstrates excellent reproducibility and anti-interference. In addition, the CF electrodes maintain good stability even after 180 days of storage at room temperature. In real water, rice and milk samples, the CF electrodes have been successfully utilized for the detection of Cd2+, Pb2+ and Hg2+ and the results were in agreement with those obtained from the inductively coupled plasma mass spectrometry.

Allosteric probe-initiated double rolling circle amplification in tandem for sensitive whole-cell detection of E. coli O157:H7

The development of facile, reliable nucleic acid amplification assays for pathogenic bacteria is paramount but still poses a challenge. Here we present a molecular amplification system that integrates the whole-cell recognition of allosteric probes with cascaded rolling circle amplification. Allosteric probe-initiated double rolling circle amplification (AID-RCA) assay involves a three-step process that utilizes allosteric probes, dumbbell templates, and circular templates to recognize, amplify, and detect target bacteria, E. coli O157:H7. The allosteric probes are designed to recognize E. coli O157:H7 without bacterial isolation and nucleic acid extraction. Subsequently, the two-stage in-tandem amplification, that is, dumbbell template-based exponential-rolling circle amplification (DT-RCA) and circular template-based linear-rolling circle amplification (CT-RCA), is initiated and yields powerful fluorescence signals using molecular beacon as a signal probe. The symmetric structure of the dumbbell template endows the AID-RCA assay with good specificity. Cascaded signal amplification boosts its sensitivity. The AID-RCA assay demonstrated high sensitivity in the quantitative detection of E. coli O157:H7(limit of detection 1.6 CFU·mL-1). Compared to real-time PCR, the AID-RCA system exhibited higher sensitivity for low concentrations of E. coli O157:H7. Furthermore, the AID-RCA system can selectively differentiate E. coli O157:H7 from other pathogenic bacteria in complex samples such as drinking water and milk. In a blind validation study using milk samples, the AID-RCA system effectively distinguished between contaminated and pasteurized milk samples. Our method is expected to hold great potential for developing precise, reliable, and efficient analytical platforms to combat pathogenic bacterial pollution and safeguard human health.

Recombinase polymerase amplification combined with lateral flow biosensor for rapid visual detection of Clostridium perfringens in chicken meat and milk.

Clostridium perfringens is one of the major anaerobic pathogen causing food poisoning and animal enteritis. With the rise of antibiotic resistance and the restrictions of the use of antibiotic growth promoting agents (AGPs) in farming, Clostridium enteritis and food contamination have become more common. It is time-consuming and labor-intensive to confirm the detection by standard culture methods, and it is necessary to develop on-site rapid detection tools. In this study, a combination of recombinase polymerase amplification (RPA) and lateral flow biosensor (LFB) was used to visually detect C. perfringens in chicken meat and milk. Two sets of primers were designed for the plc gene of C. perfringens, and the amplification efficiency and specificity of the primers. Selection of primers produces an amplified fragment on which the probe is designed. The probe was combined with the lateral flow biosensor (LFB). The reaction time and temperature of RPA-LFB assay were optimized, and the sensitivity of the assay was assessed. Several common foodborne pathogens were selected to test the specificity of the established method. Chicken and milk samples were artificially inoculated with different concentrations (1 × 102 CFU/mL to 1 × 106 CFU/mL) of C. perfringens, and the detection efficiency of RPA-LFB method and PCR method was compared. RPA-LFB can be completed in 20 min and the results can be read visually by the LFB test strips. The RPA-LFB has acceptable specificity and the lowest detection limit of 100 pg./μL for nucleic acid samples. It was able to stably detect C. perfringens contamination in chicken and milk at the lowest concentration of 1 × 104 CFU/mL and 1 × 103 CFU/mL, respectively. In conclusion, RPA-LFB is specific and sensitive. It is a rapid, simple and easy-to-visualize method for the detection of C. perfringens in food and is suitable for use in field testing work.

Employing a magnetic chitosan/molybdenum disulfide nanocomposite for efficiently removing polycyclic aromatic hydrocarbons from milk samples

This study aimed to develop a highly efficient nanocomposite composed of magnetic chitosan/molybdenum disulfide (CS/MoS2/Fe3O4) for the removal of three polycyclic aromatic hydrocarbons (PAHs)—pyrene, anthracene, and phenanthrene. Novelty was introduced through the innovative synthesis procedure and the utilization of magnetic properties for enhanced adsorption capabilities. Additionally, the greenness of chitosan as a sorbent component was emphasized, highlighting its biodegradability and low environmental impact compared to traditional sorbents. Factors influencing PAH adsorption, such as nanocomposite dosage, initial PAH concentration, pH, and contact time, were systematically investigated and optimized. The results revealed that optimal removal efficiencies were attained at an initial PAH concentration of 150 mg/L, a sorbent dose of 0.045 g, pH 6.0, and a contact time of 150 min. The pseudo-second-order kinetic model exhibited superior fitting to the experimental data, indicating an equilibrium time of approximately 150 min. Moreover, the equilibrium adsorption process followed the Freundlich isotherm model, with kf and n values exceeding 7.91 mg/g and 1.20, respectively. Remarkably, the maximum absorption capacities for phenanthrene, anthracene, and pyrene on the sorbent were determined as 217 mg/g, 204 mg/g, and 222 mg/g, respectively. These findings underscore the significant potential of the CS/MoS2/Fe3O4 nanocomposite for efficiently removing PAHs from milk and other dairy products, thereby contributing to improved food safety and public health.