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1.
Anal Chem ; 96(16): 6282-6291, 2024 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-38595038

RESUMO

Respiratory tract infections (RTIs) pose a grave threat to human health, with bacterial pathogens being the primary culprits behind severe illness and mortality. In response to the pressing issue, we developed a centrifugal microfluidic chip integrated with a recombinase-aided amplification (RAA)-clustered regularly interspaced short palindromic repeats (CRISPR) system to achieve rapid detection of respiratory pathogens. The limitations of conventional two-step CRISPR-mediated systems were effectively addressed by employing the all-in-one RAA-CRISPR detection method, thereby enhancing the accuracy and sensitivity of bacterial detection. Moreover, the integration of a centrifugal microfluidic chip led to reduced sample consumption and significantly improved the detection throughput, enabling the simultaneous detection of multiple respiratory pathogens. Furthermore, the incorporation of Chelex-100 in the sample pretreatment enabled a sample-to-answer capability. This pivotal addition facilitated the deployment of the system in real clinical sample testing, enabling the accurate detection of 12 common respiratory bacteria within a set of 60 clinical samples. The system offers rapid and reliable results that are crucial for clinical diagnosis, enabling healthcare professionals to administer timely and accurate treatment interventions to patients.


Assuntos
Infecções Respiratórias , Infecções Respiratórias/diagnóstico , Infecções Respiratórias/microbiologia , Humanos , Técnicas Analíticas Microfluídicas/instrumentação , Dispositivos Lab-On-A-Chip , Técnicas de Amplificação de Ácido Nucleico , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Bactérias/isolamento & purificação , Bactérias/genética , Recombinases/metabolismo , Automação , Infecções Bacterianas/diagnóstico
2.
Int J Nanomedicine ; 19: 2429-2440, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38476285

RESUMO

Purpose: COVID-19 is rampant throughout the world, which has caused great damage to human lives and seriously hindered the development of the global economy. Aiming at the treatment of SARS-CoV-2, in this study, we proposed a novel fenobody strategy based on ferritin (Fe) self-assembly technology. Methods: The neutralizing nanobody H11-D4 of SARS-CoV-2 fused to the C-terminus of end-modified human ferritin was expressed in E. coli and silkworm baculovirus expression systems. A large number of nanoparticles were successfully self-assembled in silkworms, while relatively few nanoparticles can be observed in the treated products from E. coli by electron microscopy. Subsequently, the fenobody's expression level and neutralizing activity were then evaluated. Results: The results showed that the IC50 of H11-D4 and fenobody Fe-H11-D4 expressed in E. coli were 171.1 nmol L-1 and 20.87 nmol L-1, respectively. However, the IC50 of Fe-HD11-D4 expressed in silkworms was 1.46 nmol L-1 showing better neutralization activity. Conclusion: Therefore, fenobodies can be well self-assembled in silkworm baculovirus expression system, and ferritin self-assembly technology can effectively improve nanobody neutralization activity.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Ferritinas , Escherichia coli , Anticorpos Neutralizantes , Anticorpos Antivirais
3.
Food Chem ; 443: 138507, 2024 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-38277932

RESUMO

Rapid, accurate, and sensitive analytical methods for the detection of food fraud are now an urgent requirement in the global food industry to ensure food quality. In response to this demand, a centrifugal integrated purification-CRISPR array for meat adulteration (CIPAM) was established. In detail, CIPAM system combines microneedles for DNA extraction and RAA-CRISPR/Cas12a integrated into a centrifugal microfluidic chip for the detection of meat adulteration. The RAA-CRISPR/Cas12a reaction reagents were pre-embedded into the different reaction chambers on the microfluidic chip to achieve the streamline of operations, markedly simplifying the detection process. The whole reaction was completed within 30 min with a detection limit of 0.1 % (w/w) in pig, chicken, duck, and lamb products. Referring to the results of the standard method, CIPAM system achieved 100 % accuracy. The automatic multiplex detection process implemented in the developed CIPAM system met the needs of food regulatory authorities.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Carne , Animais , Ovinos , Suínos/genética , Carne/análise , Qualidade dos Alimentos , Técnicas de Amplificação de Ácido Nucleico/métodos
4.
J Agric Food Chem ; 71(36): 13518-13526, 2023 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-37658470

RESUMO

A figure-actuated microfluidic biosensor was developed for the rapid and sensitive detection of Salmonella typhimurium using immunomagnetic separation to separate target bacteria and rolling circle amplification (RCA) combined with CRISPR/Cas12a to amplify the detection signal. The magnetic nanoparticles (MNPs) modified with the capture antibodies (MNPs@Ab1) and RCA primer linked with recognized antibodies (primer@Ab2) were first used to react with S. typhimurium, resulting in the formation of MNPs@Ab1-S. typhimurium-primer@Ab2 complexes. Then, the RCA and CRISPR/Cas12a reagents were successively pumped into the chamber and incubated at the appropriate conditions. With the help of a 3D-printed signal detector, the fluorescence signal was collected and analyzed using the smartphone APP for the determination of bacterial concentration. This biosensor exhibited a wide linear range for the detection of S. typhimurium with a low limit of detection of 1.93 × 102 CFU/mL and a mean recovery of about 106% in the spiked milk sample.


Assuntos
Separação Imunomagnética , Salmonella typhimurium , Salmonella typhimurium/genética , Sistemas CRISPR-Cas , Microfluídica , Anticorpos
5.
Anal Chem ; 95(35): 13391-13399, 2023 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-37610722

RESUMO

Early detection of foodborne bacteria is urgently needed to ensure food quality and to avoid the outbreak of foodborne bacterial diseases. Here, a kind of metal-organic framework (Zr-MOF) modified with Pt nanoparticles (Pt-PCN-224) was designed as a peroxidase-like signal amplifier for microfluidic biosensing of foodborne bacteria. Taking Escherichia coli (E. coli) O157:H7 as a model, a linear range from 2.93 × 102 to 2.93 × 108 CFU/mL and a limit of detection of 2 CFU/mL were obtained. The whole detection procedure was integrated into a single microfluidic chip. Water, milk, and cabbage samples were successfully detected, showing consistency with the results of the standard culture method. Recoveries were in the range from 90 to 110% in spiked testing. The proposed microfluidic biosensor realized the specific and sensitive detection of E. coli O157:H7 within 1 h, implying broad prospects of MOF with biomimetic enzyme activities for biosensing.


Assuntos
Escherichia coli O157 , Doenças Transmitidas por Alimentos , Humanos , Microfluídica , Bactérias , Amplificadores Eletrônicos , Biomimética
6.
Anal Chem ; 95(35): 13368-13375, 2023 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-37610723

RESUMO

A multifunctional platform that meets the demands of both bacterial detection and elimination is urgently needed because of their harm to human health. Herein, a "sense-and-treat" biosensor was developed by using immunomagnetic beads (IMBs) and AgPt nanoparticle-decorated PCN-223-Fe (AgPt/PCN-223-Fe, PCN stands for porous coordination network) metal-organic frameworks (MOFs). The synthesized AgPt/PCN-223-Fe not only exhibited excellent peroxidase-like activity but also could efficiently kill bacteria under near infrared (NIR) irradiation. This biosensor enabled the colorimetric detection of E. coli O157:H7 in the range of 103-108 CFU/mL with a limit of detection of 276 CFU/mL, accompanied with high selectivity, good reproducibility, and wide applicability in diverse real samples. Furthermore, the biosensor possessed a highly effective antibacterial rate of 99.94% against E. coli O157:H7 under 808 nm light irradiation for 20 min. This strategy can provide a reference for the design of novel versatile biosensors for bacterial discrimination and antibacterial applications.


Assuntos
Escherichia coli O157 , Estruturas Metalorgânicas , Humanos , Reprodutibilidade dos Testes , Bactérias , Antibacterianos
7.
Anal Chem ; 95(20): 7993-8001, 2023 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-37156096

RESUMO

To achieve high-throughput ultrasensitive detection of mycotoxins in food, a functional DNA-guided transition-state CRISPR/Cas12a microfluidic biosensor (named FTMB) was successfully constructed. The signal transduction CRISPR/Cas12a strategy in FTMB has utilized DNA sequences with a specific recognition function and activators to form trigger switches. Meanwhile, the transition-state CRISPR/Cas12a system was constructed by adjusting the composition ratio of crRNA and activator to achieve a high response for low concentrations of target mycotoxins. On the other hand, the signal enhancement of FTMB has efficiently integrated the signal output of quantum dots (QDs) with the fluorescence enhancement effect of photonic crystals (PCs). The construction of universal QDs for the CRISPR/Cas12a system and PC films matching the photonic bandgap produced a significant signal enhancement by a factor of 45.6. Overall, FTMB exhibited a wide analytic range (10-5-101 ng·mL-1), low detection of limit (fg·mL-1), short detection period (∼40 min), high specificity, good precision (coefficients of variation <5%), and satisfactory practical sample analysis capacity (the consistency with HPLC at 88.76%-109.99%). It would provide a new and reliable solution for the rapid detection of multiple small molecules in the fields of clinical diagnosis and food safety.


Assuntos
Técnicas Biossensoriais , Micotoxinas , Microfluídica , Bioensaio , Cromatografia Líquida de Alta Pressão , Transdução de Sinais , Sistemas CRISPR-Cas
8.
Process Biochem ; 129: 200-208, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37007452

RESUMO

Spike (S) protein, a homotrimeric glycoprotein, is the most important antigen target for SARS-CoV-2 vaccines. A complete simulation of the advanced structure of this homotrimer during subunit vaccine development is the most likely method to improve its immunoprotective effects. In this study, preparation strategies for the S protein receptor-binding domain, S1 region, and ectodomain trimer nanoparticles were designed using ferritin nanoparticle self-assembly technology. The Bombyx mori baculovirus expression system was used to prepare three nanoparticle vaccines with high expression levels recorded in silkworms. The results in mice showed that the nanoparticle vaccine prepared using this strategy could induce immune responses when administered via both the subcutaneous administration and oral routes. Given the stability of these ferritin-based nanoparticle vaccines, an easy-to-use and low-cost oral immunization strategy can be employed in vaccine blind areas attributed to shortages of ultralow-temperature equipment and medical resources in underdeveloped areas. Oral vaccines are also promising candidates for limiting the spread of SARS-CoV-2 in domestic and farmed animals, especially in stray and wild animals.

9.
Foods ; 12(7)2023 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-37048374

RESUMO

The preparation of novel antioxidant peptides from food raw materials is one of the research focuses, but there are fewer studies on the preparation of antioxidant peptides from walnut meal, a by-product of processing walnuts. This study analyzed the antioxidant properties and protective effects of walnut protein hydrolyzed by alkaline protease and trypsin on the oxidative stress of HT22 cells. The peptides were identified by UPLC-MS/MS, and the anti-oxidative peptides were screened based on virtual computer tools. The potential anti-oxidative stress mechanism of the walnut polypeptide on HT22 cells was explored by molecular docking. The results revealed that walnut protein hydrolysates (WPH) with molecular weights of less than 1 kDa had good antioxidant properties and inhibited oxidative damage of HT22 cells by regulating the levels of reactive oxygen species (ROS) and antioxidant enzyme catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px). Six of the ninety identified new peptides showed good solubility, non-toxicity, and bioactivity. The molecular docking results showed that the six peptides could dock with Keap1 successfully, and EYWNR and FQLPR (single-letter forms of peptide writing) could interact with the binding site of Nrf2 in the Keap1-Kelch structural domain through hydrogen bonds with strong binding forces. The results of this study provided important information on the antioxidant molecular mechanism of the walnut polypeptide and provided a basis for further development of walnut antioxidant polypeptide products.

10.
J Colloid Interface Sci ; 635: 503-513, 2023 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-36599247

RESUMO

HYPOTHESIS: Selenium (Se)-containing compound is sensitive to redox stimulation, showing hydrophobic-hydrophilic reversible transition. Introduction of such compound into honeycomb film could confer on it redox-switchable surface wettability, which is expected to control cell adhesion/detachment behavior. EXPERIMENTS: Didodecyl selenide was designed and mixed with polystyrene to prepare honeycomb films using "breath figure" method. The film microstructures were characterized by scanning electron microscope and atomic force microscopy, and the arrangement of Se atoms in honeycomb film was determined by X-ray photoelectron spectroscopy and energy dispersive spectrometry. The variation of film wettability upon the alternating stimulation of H2O2 and Vc was examined. Then the cell adhesion, proliferation, and controlled detachment on honeycomb films were conducted. FINDINGS: The introduction of didodecyl selenide helps to form ordered honeycomb film, and Se atoms were found to located on the bottom, pore walls, and top surface of the film. The presence of didodecyl selenide not only greatly improves film biocompatibility by enhancing cell thioredoxin reductase activity, but also imparts the film with H2O2-/vitamin C-regulated tunable wettability that controls cell adhesion and detachment. H2O2 treatment produces a hydrophilic surface for cell adhesion and proliferation, whereas the addition of vitamin C generates hydrophobic surfaces and allows cells to detach while remaining alive with high activity.


Assuntos
Selênio , Adesão Celular , Selênio/farmacologia , Peróxido de Hidrogênio , Oxirredução , Ácido Ascórbico , Propriedades de Superfície
11.
Talanta ; 253: 123980, 2023 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-36201954

RESUMO

As a major threat to food safety due to their pathogenicity, foodborne bacteria have received much attention. In this paper, we present a one-step and wash-free microfluidic biosensor platform by smartphone for simultaneous multiple foodborne bacteria target single-stranded DNA (ssDNA) detection. This technology is based on the fluorescence resonance energy transfer (FRET) between the graphene oxide (GO) and fluorescence molecules modified capture ssDNA of the target bacteria ssDNA (ctDNA) which were coated on the microfluidic chips. The fluorescence recovery was recorded by a smartphone fluorescent detector. With an optimal analytical performance, the platform realized the detection of four kinds of bacteria ssDNA simultaneously within 5 min, with the limits of detection (LODs) of 0.17, 0.18, 0.27, and 0.17 nM, respectively. And the throughput analysis of trace amounts of foodborne bacteria ssDNA in milk and water samples were successfully detected. This one-step and wash-free microfluidic biosensor can be used as a tool for food safety analysis.


Assuntos
DNA de Cadeia Simples , Microfluídica , Bactérias
12.
Biosens Bioelectron ; 220: 114885, 2023 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-36410158

RESUMO

Foodborne pathogens have raised significant concerns in human public health. Rapid, high-sensitive, low-cost, and easy-to-use testing methods for food safety are needed. In this study, we developed a finger-actuated microfluidic biosensor (FA-MB) for multiplexed detection of Bacillus cereus and other six common foodborne pathogens based on one-pot CRISPR/Cas12a combined with recombinase aided amplification (RAA). Wells for RAA and CRISPR/Cas12a were isolated to avoid interference, while finger-actuated one-way control valves were incorporated to fulfill the unidirectional flow of RAA products to the CRISPR/Cas12a reaction wells, realizing one-pot RAA-CRISPR/Cas12a assay. The final fluorescent signal was acquired and processed by a smartphone. Under selected experimental conditions, seven pathogenic bacteria could be tested in about 1 h with the limits of detection (LODs) below 500 CFU/mL. Recoveries ranged from 90% to 116% of the spiked samples were readily achieved. The proposed FA-MB is highly integrated and easy-to-use, and could be used for rapid, high-sensitive point of care (POC) testing without the external driving device, suitable for resource-constrained settings.


Assuntos
Técnicas Biossensoriais , Contaminação de Alimentos , Microfluídica , Sistemas CRISPR-Cas/genética , Hidrolases , Recombinases/genética , Contaminação de Alimentos/análise , Microbiologia de Alimentos , Bacillus cereus/isolamento & purificação
13.
Food Res Int ; 162(Pt A): 112032, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36461252

RESUMO

With the rapid development of logistics, a growing number of pathogenic microorganisms has the means to spread worldwide using food as a carrier; thus, there is an urgent need to develop effective detection strategies to ensure food safety. By combining novel markers identified by pan-genome analysis and a digital recombinase-aided amplification (RAA) detection method based on a microfluidic chip, a strategy of high-fidelity target-based microfluidic identification (HFTMI) has been developed. Herein, a proof-of-concept study of HFTMI for rapid pathogen detection of V. parahaemolyticus was investigated. Specific primers designed for the gene group_41170 identified in the pan-genome analysis showed high sensitivity and a broad spectrum for the detection of V. parahaemolyticus. Different power systems were investigated to increase the partition rate on specifically designed chamber-based digital chips. The performance of HFTMI was greatly improved compared with qPCR. Collectively, this novel HFTMI system provides more reliable guidance for food safety testing.


Assuntos
Vibrio parahaemolyticus , Primers do DNA , Alimentos , Microfluídica , Vibrio parahaemolyticus/genética
14.
Polymers (Basel) ; 14(22)2022 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-36432932

RESUMO

The development of semi-aromatic polyamides with excellent mechanical properties has always been a popular research avenue. In this work, the semi-aromatic polyamide 12T (PA12T) with the maximum tensile strength of 465.5 MPa was prepared after stretching at 210 °C 4.6 times. Wide-angle X-ray diffraction (WAXD) and small-angle X-ray scattering (SAXS) were used to characterize the structural evolution of semi-aromatic polyamide 12T (PA12T) after stretching at different stretching temperatures and stretching ratios. The formation mechanism of this change in mechanical properties was investigated from different aspects of the aggregated structure such as crystal morphology, crystal orientation and crystallinity. The relevant characterization results show that the crystal structure, crystal orientation and crystallinity of PA12T were the highest when the sample was pre-stretched at 210 °C, which is crucial for improving the mechanical properties of PA12T. These findings will provide important guidance for the preparation of polymer materials with excellent mechanical properties.

15.
Anal Chem ; 94(48): 16787-16795, 2022 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-36398979

RESUMO

A portable microfluidic biosensor was developed for the detection of E. coli O157:H7 using finger actuation. The chip was assembled with three functional zones, immunomagnetic separation, nucleic acid extraction and purification, and signal detection. First, antibody-modified magnetic nanoparticles (MNPs) were used to separate the target bacteria from the sample. The captured bacteria were then lysed and silica-coated MNPs were used to absorb DNA, followed by washing and eluting to obtain purified DNA. The obtained DNA was subjected to amplification and fluorescence detection based on the recombinase polymerase amplification-clustered regularly interspaced short palindromic repeat-associated protein/Cas12a reaction. The fluorescence images were collected and analyzed using a smartphone app under a 3D-printed detection device. It could quantitatively detect E. coli O157:H7 from 102 to 108 CFU/mL in 2.5 h with a limit of detection (LOD) of 10 CFU/mL. The recovery rate ranged from 104 to 120%. Overall, the biosensor realizes "sample-in and answer-out" assay for E. coli O157:H7 and eliminates the need for external pumps and skilled personnel.


Assuntos
Técnicas Biossensoriais , Escherichia coli O157 , Escherichia coli O157/genética , Microfluídica , Técnicas Biossensoriais/métodos , Separação Imunomagnética/métodos , Limite de Detecção
16.
Infect Genet Evol ; 103: 105332, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35811034

RESUMO

Interferon (IFN), a critical antiviral cytokine produced by pathogens-induced cells, plays an important role in host innate immune system. In this study, to investigate the inhibition effect of IFN on avian influenza virus (AIV), Chicken Embryo Fibroblasts (CEFs) was infected by H9N2 AIV. The pre-immune state and transcriptome analysis have been observed and performed. The result showed chicken interferon gamma (chIFN-γ) have the most inhibitory effect on H9N2 virus among three types of chicken interferons (chIFNs). Inhibition of chIFN-γ on H9N2 virus was verified by indirect immunofluorescence, RT-qPCR and western blot. The possible signaling pathways induced by chIFN-γ with or without virus were analyzed by transcriptome. The transcriptome data were compared among H9N2-infected, chIFN-γ-treated, chIFN-γ + H9N2-treated, and Control groups. In summary, RNA-sequencing (RNA-seq) data suggested that H9N2 virus infection resulted in corresponding response of certain defensive, inflammatory and metabolism pathways to the virus replication in CEFs. Furthermore, while CEFs were treated with chIFN-γ, many immune-related signaling pathways in cells are affected and altered. Antiviral genes involved in these immune pathways such as interferon regulatory factors, chemokines, interferon-stimulated genes (ISGs) and transcription factors were significantly up-regulated, and showed significant antiviral responses. Compared with virus infected CEFs alone, pretreatment with IFN induced the expression of antiviral genes and activated related antiviral pathways, inhibited the viral replication as result. Our study provided functional annotations for antiviral genes and the basis for studying the mechanism of chIFN-γ mediated response against H9N2 AIV.


Assuntos
Vírus da Influenza A Subtipo H9N2 , Vírus da Influenza A , Influenza Aviária , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Embrião de Galinha , Galinhas , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Vírus da Influenza A Subtipo H9N2/genética , Vírus da Influenza A/genética , Interferon gama , Interferons/genética
17.
Front Microbiol ; 13: 820431, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35602063

RESUMO

Mining novel specific molecular targets and establishing efficient identification methods are significant for detecting Pseudomonas aeruginosa, which can enable P. aeruginosa tracing in food and water. Pangenome analysis was used to analyze the whole genomic sequences of 2017 strains (including 1,000 P. aeruginosa strains and 1,017 other common foodborne pathogen strains) downloaded from gene databases to obtain novel species-specific genes, yielding a total of 11 such genes. Four novel target genes, UCBPP-PA14_00095, UCBPP-PA14_03237, UCBPP-PA14_04976, and UCBPP-PA14_03627, were selected for use, which had 100% coverage in the target strain and were not present in nontarget bacteria. PCR primers (PA1, PA2, PA3, and PA4) and qPCR primers (PA12, PA13, PA14, and PA15) were designed based on these target genes to establish detection methods. For the PCR primer set, the minimum detection limit for DNA was 65.4 fg/µl, which was observed for primer set PA2 of the UCBPP-PA14_03237 gene. The detection limit in pure culture without pre-enrichment was 105 colony-forming units (CFU)/ml for primer set PA1, 103 CFU/ml for primer set PA2, and 104 CFU/ml for primer set PA3 and primer set PA4. Then, qPCR standard curves were established based on the novel species-specific targets. The standard curves showed perfect linear correlations, with R 2 values of 0.9901 for primer set PA12, 0.9915 for primer set PA13, 0.9924 for primer set PA14, and 0.9935 for primer set PA15. The minimum detection limit of the real-time PCR (qPCR) assay was 102 CFU/ml for pure cultures of P. aeruginosa. Compared with the endpoint PCR and traditional culture methods, the qPCR assay was more sensitive by one or two orders of magnitude. The feasibility of these methods was satisfactory in terms of sensitivity, specificity, and efficiency after evaluating 29 ready-to-eat vegetable samples and was almost consistent with that of the national standard detection method. The developed assays can be applied for rapid screening and detection of pathogenic P. aeruginosa, providing accurate results to inform effective monitoring measures in order to improve microbiological safety.

18.
Anal Methods ; 14(14): 1445-1453, 2022 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-35332354

RESUMO

Foodborne illnesses caused by Salmonella represent a significant public health problem worldwide. The aim of this study was to establish multiplex PCR (mPCR) for the rapid identification of Salmonella serogroups B, C1, C2, D, and E as well as for the serovars enteritidis and typhimurium. Employing pan-genome analysis and PCR verification, B-rfbJ, C1-9679, C2-pimB, D-rfbJ, E-rfbC, and four genes (SE18636, SE16574, SE2599, and SE13329) were identified as specific target genes for Salmonella serogroups B, C1, C2, D, E, and S. enteritidis, respectively. Thereafter, three novel mPCR assays (one of 3-mPCR and two of 2-mPCR) were successfully developed to identify these bacteria based on the target genes and another S. typhimurium-specific STM4495 gene. The primers targeting C1-9679, C2-pimB, and E-rfbC genes specific to the serogroups C1, C2, and E, respectively, constituted a 3-mPCR, while the other two 2-mPCRs, respectively, consisting primers specific to serogroup D and S. enteritidis (D-rfbJ and SE16574), and serogroup B and S. typhimurium-specific primers (B-rfbJ and STM4495), were also designed. The specificity of each mPCR was further evaluated by using non-target strains. The detection limits of mPCRs were approximately 103-104 CFU mL-1 in pure culture and 104-105 CFU g-1 in spiked chicken meat. In addition, mPCR assays could correctly detect target Salmonella in food samples. These results suggest that specific targets could be mined efficiently through a pan-genome analysis tool, and the novel mPCR assays developed in this study offer a promising technique for rapid and accurate detection of five serogroups of Salmonella (B, C1, C2, D, and E) and two serovars (S. enteritidis and S. typhimurium).


Assuntos
Reação em Cadeia da Polimerase Multiplex , Salmonella enteritidis , Salmonella enteritidis/genética , Sorogrupo
19.
Anal Chim Acta ; 1201: 339657, 2022 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-35300793

RESUMO

Strict monitoring of Salmonella serotypes is the most effective way to limit the risk of its transmission to humans. In this study, a microfluidic genoserotyping strategy was developed for the rapid and cost-effective detection of 11 common Salmonella serotypes from retail food samples, i.e. Typhimurium, Derby, Indiana, Agona, Rissen, Braenderup, Hadar, Enteritidis, Weltevreden, Meleagridis, and Pomona. The programmable LAMP in the strategy can complete the whole detection process within 40 min and avoid false positive results. The limit of detection was 102 or 103 CFU/mL. Referring to the results of standard culture method, the LAMP-chip was verified with 100% accuracy on testing 688 Salmonella and 22 non-Salmonella strains. This simple, portable and accurate microfluidic genoserotyping strategy is fully adapted to the needs of food regulatory authorities and has wide application prospect in food safety field.


Assuntos
Microbiologia de Alimentos , Microfluídica , Inocuidade dos Alimentos , Humanos , Salmonella/genética , Sorogrupo
20.
Front Microbiol ; 12: 664810, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34234757

RESUMO

Salmonella is a widely distributed foodborne pathogen. The use of Salmonella phages as biocontrol agents has recently gained significant interest. Because the Salmonella genus has high diversity, efforts are necessary to identify lytic Salmonella phages focusing on different serovars. Here, five Salmonella phages were isolated from soil samples, and vB_SalP_TR2 was selected as a novel phage with high lytic potential against the host Salmonella serovar Albany, as well as other tested serovars, including Corvallis, Newport, Kottbus, and Istanbul. Morphological analyses demonstrated that phage vB_SalP_TR2 belongs to the Podoviridae family, with an icosahedral head (62 ± 0.5 nm in diameter and 60 ± 1 nm in length) and a short tail (35 ± 1 nm in length). The latent period and burst size of phage vB_SalP_TR2 was 15 min and 211 PFU/cell, respectively. It contained a linear dsDNA of 71,453 bp, and G + C content was 40.64%. Among 96 putative open reading frames detected, only 35 gene products were found in database searches, with no virulence or antibiotic resistance genes being identified. As a biological control agent, phage vB_SalP_TR2 exhibited a high temperature and pH tolerance. In vitro, it lysed most S. Albany after 24 h at 37°C with multiplicities of infection of 0.0001, 0.001, 0.01, 0.1, 1, 10, and 100. In food matrices (milk and chicken meat), treatment with phage vB_SalP_TR2 also reduced the number of S. Albany compared with that in controls. These findings highlighted phage vB_SalP_TR2 as a potential antibacterial agent for the control of Salmonella in food samples.

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