hlyA Gene Of Listeria Monocytogenes Research Articles

Simultaneous Detection of Five Foodborne Pathogens Using a Mini Automatic Nucleic Acid Extractor Combined with Recombinase Polymerase Amplification and Lateral Flow Immunoassay.

In recent years, foodborne disease outbreaks have caused huge losses to the economy and have had severe impacts on public health. The accuracy and variety of detection techniques is crucial to controlling the outbreak and spread of foodborne diseases. The need for instruments increases the difficulty of field detection, while manually-handled samples are subject to user error and subjective interpretation. Here, we use a mini automatic nucleic acid extractor combined with recombinant polymerase amplification (RPA) and lateral flow immunoassay (LFIA) for simultaneous quantitative detection of five major foodborne pathogens. The pre-treatment device using the magnetic bead method allows for nucleic acid extraction of the reagent tank without manual operation, which is highly efficient and stable for preventing aerosol contamination. The nuc gene of Staphylococcus aureus, the toxR gene of Vibrio parahaemolyticus, the rfbE gene of Escherichia coli O157:H7, the hlyA gene of Listeria monocytogenes, and the fimY gene of Salmonella enterica were used as target fragments. The labeled antibody concentration is optimized on the LFIA to find the equilibrium point for the binding capacity of the five chemical markers and to efficiently and accurately visualize the bands. The RPA assay shows an optimal performance at 37 °C for 15 min. The optimized RPA-LFIA detection limit can reach 101 CFU/mL. There was no cross-reactivity among forty-eight strains. Furthermore, the average recoveries in spiked food samples were 90.5–104.5%. In summary, the RPA-LFIA established in this study can detect five pathogenic bacteria simultaneously with little dependence on laboratory equipment, and it has promising prospects for screening in low-resource areas.

Molecular characterization and hematological analysis of Listeria monocytogenes infection in dairy cows in Punjab (Pakistan)

Listeriosis is an emerging bacterial disease of animals and humans worldwide, caused by Listeria monocytogenes. The infected dairy cows continuously shed the microbes in their milk, a human being's concern. This study was designed to molecular characterize the Listeria monocytogenes isolated from symptomatic cow's milk of tehsils Samundri, Gujar khan, and Alipur of Punjab. A total of 175 milk samples were collected, pre-enriched and cultured on PALCAM agar. The affirmation of the hlyA gene of Listeria monocytogenes was performed by polymerase chain reaction (PCR) and 3.43% of isolates were found positive. The phylogenetic analysis showed a resemblance of our isolates of Listeria monocytogenes with India (KP965733), the USA (DQ812484), and 3 of our isolates made a clade. The leucocytes and neutrophils count were found significantly increased in listeriosis affected cows. The Chi-square test showed a significant association between poor quality silage feeding and listeriosis. The presence of L.monocytogenes in cow's milk indicates a potential threat to humans. It is further recommended that it should be consistently monitored to ensure food safety.

Multiplex PCR method for simultaneous detection of five pathogenic bacteria closely related to foodborne diseases.

In this study, we describe a multiplex PCR method for the detection of five food-relevant virulence pathogenicity genes of intestinal pathogens. Five pairs of primers were designed based on nuc gene for Staphylococcus aureus, hlyA gene of Listeria monocytogenes, ipaH gene of Shigella flexneri, lysP gene of Yersinia enterocolitica and tpi gene of Clostridium difficile. Conditions were optimized to amplify fragments of those genes simultaneously in one PCR amplification. After developing and optimizing the multiplex PCR reaction system, the specificity and sensitivity of the multiple PCR assays were evaluated. The optimized program is also applied to retail meat for testing. The result indicated that when the annealing temperature was 54°C and the primer concentrations of S. aureus, L. monocytogenes, S. flexneri, Y. enterocolitica and C. difficile are 10, 10, 5, 3 and 2μM, the five strains could expand 484, 345, 204, 156, 88bp of clear fragments, respectively. So was the multiple PCR in artificially contaminated beef produce. All cultures were cultured and separated by traditional methods. The multiplex PCR method offers a rapid, simple, and accurate identification of pathogens and could be used in food safety investigations, clinical diagnosis as well as for the surveillance of the spreading determinants of pathogens in epidemiological studies.

Ultrasensitive cloth-based microfluidic chemiluminescence detection of Listeria monocytogenes hlyA gene by hemin/G-quadruplex DNAzyme and hybridization chain reaction signal amplification.

In this work, a cloth-based chemiluminescence (CL) biosensor has been firstly presented for highlysensitive determination of long PCR amplicons. Under the action of a hybridization chain reaction, a good deal of hemin/G-quadruplex DNAzyme molecules are produced, which can effectively enhance the CL signal. Moreover, effective cloth-based DNA biosensors can be fabricated by sequential wax screen-printing and surface-modification processes. Especially, the integration of a desirable hydrophobic barrier and gravity/capillary flow onto the flow channel of the cloth-based device makes the biosensor easy to be fabricated and to be associated with a flow CL. For the luminol/H2O2-based CL system, the signals are triggered by the hemin/G-quadruplex DNAzyme and are recorded by a low-cost CCD. Under optimized conditions, the determination range of target DNA is 0.002-20,000pM and its limit of detection is calculated to be 1.1fM. The results show that the proposed CL biosensor has a good analytical performance, such as high detectability and specificity, wide linear range, and receivable reproducibility and stability. Finally, the proposed biosensor is proven by the fact that this method can successfully detect the target DNA prepared from the Listeria monocytogenes-spiked milk samples. Therefore, it is believed to have the potential application prospects in food safety and environmental monitoring. Graphical abstract.

Detection of Listeria Spp. and Listeria Monocytogenes in Biological Samples by SYBR Green I and TaqMan Probe-based Real-time PCRs.

IntroductionThe aim of the study was the application and comparison of real-time PCR methods based on the fluorescence of SYBR Green I intercalating dye and TaqMan probes for the detection of the 23S rDNA gene of Listeria spp. and the hlyA gene of Listeria monocytogenes in biological samples of the liver, brain, and blood.Material and MethodsFive strains of L. monocytogenes and single strains of each species L. ivanovii, L. innocua,L. grayi, L. welshimeri, and L. seeligeri were used for the experiments. Additionally, five strains of other species of bacteria were used for evaluation of the specificity of tests. In the first stage of the study SYBR Green I real-time PCRs, one allowing detection of the 23S rDNA gene and two based on the amplification the hlyA gene, were performed. In the next part, three TaqMan probe-based real-time PCRs allowing confirmation of belonging to Listeria spp. and L. monocytogenes were conducted.ResultsThe observation of amplification curves in real-time PCRs enabled the detection of both genes. A high regression coefficient of 0.99 was found for all reactions. Specific amplification products were obtained for the 23S rDNA and hlyA genes which confirm their belonging to Listeria spp. and L. monocytogenes, respectively. Other microbial species did not reveal real-time PCR products.ConclusionBoth real-time PCR methods for the detection of Listeria spp. and L. monocytogenes in biological samples demonstrated a significant sensitivity and high specificity.

Detection of Listeria monocytogenes HlyA gene in Food Samples of Meat by Real-Time PCR In Erbil city

The objectives of this study were to evaluate the detection of  Listeria monocytogenes  in different ready-to-eat foods using real-time PCR (RT-PCR). To monitor L. monocytogenes in foods(meat) Listeria monocytogenes is a gram positive, rod shaped, pathogenic bacterium32 samples (imported frozen breast chicken, 17, local minced beef meat 9 and  imported  minced beef meat 6. were collected from  super  markets in different place in Erbil city . Of all 32samples, 9 samples (28.1%) showed positive signals in RT-PCR. six samples from(imported frozen breast chicken, (35.29%) ,1 samples from  local minced beef meat(11.1%) and two sample from  imported  minced beef meat (33.3%) were determined to be positive for  L. monocytogenes  .  Also the result showed the lowest CT value at (21.57) The results indicate that the RT-PCR detection method with the set of primers and hybridization probe designed in this study should be useful in monitoring for L. monocytogenes hlyA gene in meat

An Improve Protocol for PCR Using LM1 and LM2 Primers for Listeria monocytogenes Detection in Food Matrices.

Several studies have observed that a conventional PCR protocol using primers LM1 and LM2 for the identification of gene hlyA Listeria monocytogenes generates non-specific PCR amplifications and false positives. For this reason in this study, we provide a modified PCR protocol that improves the specificity of the LM1 and LM2 primers.

Application of SYBR Green I and TaqMan probe-based real-time PCRs for the identification of Listeria spp. and Listeria monocytogenes

Abstract The aim of the study was the application and comparison of real-time PCR methods based on the fluorescence of SYBR Green I intercalating dye and TaqMan probes for the detection of the 23S rDNA gene of Listeria spp. and the hlyA gene of Listeria monocytogenes. Five strains of L. monocytogenes and single strains of each of the species: L. ivanovii, L. innocua, L. grayi, L. welshimeri, and L. seeligeri were used for the experiments. Additionally, five strains of other species of bacteria were used for evaluation of the specificity of the tests. QuantiTect SYBR Green PCR and QuantiTect Probe PCR kits were selected for the study. In the first stage of the study, SYBR Green I real-time PCRs were performed under several methods, the first one allowing detection of the 23S rDNA gene and the remainder based on the amplification of the hlyA gene. In the next part, three varied in method TaqMan probe-based real-time PCRs allowing confirmation of strains belonging to Listeria spp. and L. monocytogenes were conducted. The observation of amplification curves in real-time PCR methods enabled the detection of both genes, and these methods demonstrated a significant sensitivity and high specificity. A high regression coefficient of 0.99 was found for all reactions. Specific amplification products were obtained for the 23S rDNA and hlyA genes, which confirmed the tested strains as Listeria spp. and L. monocytogenes respectively. Isolates of other microbial species did not yield real-time PCR products.

A novel paper-based microfluidic enhanced chemiluminescence biosensor for facile, reliable and highly-sensitive gene detection of Listeria monocytogenes

This paper describes the development of a paper-based microfluidic device that detects long DNA amplicons on the basis of hybridization reactions with a covalently immobilized DNA probe and biotin-labeled signal DNA strands, and chemiluminescent (CL) reactions catalyzed by a horseradish peroxidase (HRP)-streptavidin conjugate. The paper-based microfluidic CL biosensor is developed for detection of 198-bp DNA fragments obtained by PCR amplification of the Listeria monocytogenes hlyA gene, where the CL intensity is detected by a simple, inexpensive CCD. To our knowledge, the combined use of the CCD sensing and paper-based microfluidic CL detection is first proposed here. The paper-based device includes wax-screen printed channels, and further explores origami-type folds to create an easy-to-operate valve that makes it possible to successively wash the detection zone in-between hybridization steps. Compared with other paper-based wax-screen-printing methods, the proposed method is faster and needs lower heating temperature. In our DNA biosensor, CL signals generated using a HRP-luminol-H2O2 system are heightened with p-iodine phenol (PIP) and detected with a CCD system. Under optical conditions, a linear range of 1.94×10−1pmol/L to 1.94×104pmol/L is achieved and the limit of detection is found to be 6.3×10−2pmol/L. And, this detection limit is 3–5 orders of magnitude lower than those obtained from other CCD-based microfluidic CL DNA assays. The results show that the proposed integration of paper-based microfluidics, CL method and CCD sensing offers great promises in providing a highly-sensitive, reliable and cost-effective solution for gene biosensing applications.

Using nested PCR to detect the hlyA gene of Listeria monocytogenes in Minas Frescal cow's milk cheese.

To evaluate a nested PCR protocol for Listeria monocytogenes detection, Minas Frescal cow's milk cheeses were produced, artificially inoculated with this pathogen at concentrations ranging from 1 to 1,000 CFU/g, and stored at 4°C for 10 days. The International Organization for Standardization (ISO) standardized method 11290-1/A1 was used to detect L. monocytogenes in the inoculated samples, and DNA was extracted from aliquots (1, 5, and 10 ml) of 1:10 dilution, followed by a nested PCR protocol for the hlyA gene. The ISO standardized reference method and nested PCR both detected L. monocytogenes at all concentrations and during all storage periods; McNemar's test showed no significant difference (P > 0.05). The results indicated that the nested PCR protocol can be used as a screening test to detect L. monocytogenes in Minas Frescal cheese, allowing earlier detection of the pathogen that can later be confirmed by the ISO standardized reference method.

High-level expression of the Listeria monocytogenes listeriolysin O in Escherichia coli and preliminary characterization of the purified protein

Listeriolysin O (LLO) is a cholesterol-binding sulfhydryl-activated hemolysin encoded by Listeria monocytogenes hlyA gene. After analyzing the nucleotide coding sequence of this gene from the ATCC 9525 L. monocytogenes strain, we cloned it in a pET vector for expression in Escherichia coli. Thanks to the optimization of the induction protocol, we achieved a high-level LLO synthesis (about 10% of total cell proteins) in hemolytically active form. The expressed hemolysin was then purified to homogeneity, as revealed by SDS–PAGE and Western blot analysis, by a hydroxyapatite adsorption chromatography, followed by an SP Sepharose ion-exchange chromatography. The recombinant protein showed the same properties determined for LLO purified from L. monocytogenes cultures and the characteristics of the sulfhydryl-activated toxins such as inactivation by oxidation and by reaction with cholesterol. By a combination of the pET expression system and the simple purification method, we obtained a significant amount of toxin (4.5 mg/litre cell culture) in a hemolytically active form (1.25×10 6 HU/mg protein). This procedure can solve the problem of LLO isolation from L. monocytogenes cultures, which is a difficult task, mainly owing to the low levels of toxin released in the culture media. The recombinant hemolysin, purified in sufficient quantities, could be very useful for structural studies and for diagnostic and pharmaceutical applications.

A nucleic acid sequence-based amplification system for detection of Listeria monocytogenes hlyA sequences

A nucleic acid sequence-based amplification system primarily targeting mRNA from the Listeria monocytogenes hlyA gene was developed. This system enabled the detection of low numbers (< 10 CFU/g) of L. monocytogenes cells inoculated into a variety of dairy and egg products after 48 h of enrichment in modified listeria enrichment broth.

A nucleic acid sequence-based amplification (NASBA) system for Listeria monocytogenes and simple method for detection of amplimers

A NASBA system amplifying specific sequences of the Listeria monocytogenes hlyA gene and an immunoenzymatic assay for the detection of amplimers was developed. The immunoenzymatic assay utilized a simple microtiter plate format and an anti-RNA:DNA hybrid antibody for the detection of NASBA product (predominantly RNA) hybridized to an immobilized DNA probe. This highly sensitive isothermal amplification and detection system was reactive with genomic DNA from various L. monocytogenes isolates but not with other Listeria or non-Listeria species.

A simple RNA probe system for analysis of Listeria monocytogenes polymerase chain reaction products

The synthesis of an RNA probe specific for the hlyA gene of Listeria monocytogenes by in vitro transcription from a polymerase chain reaction (PCR)-generated template incorporating bacteriophage T7 promoter sequences is described. This simple method produced a high yield of RNA which hybridized specifically with hlyA PCR products on a membrane, resulting in RNA-DNA hybrids which were detected by an immunoenzymatic assay with an anti-RNA-DNA hybrid antibody. The RNA probe hybridization system was more sensitive in the analysis of the PCR products than was the conventional agarose gel electrophoresis method. When applied to the analysis of PCR samples from cultures of various Listeria and non-Listeria organisms, the RNA probe was reactive in the assay of 62 different L. monocytogenes isolates but not other Listeria species. Among the non-Listeria organisms tested, only Enterococcus faecalis gave a weak positive reaction with more than 10(9) cells per ml. This reactivity disappeared at lower cell densities. This strategy for the synthesis and application of RNA probes should facilitate the analysis of PCR products in the detection of L. monocytogenes and possibly other food pathogens.