Establishment of Multiple-PCR for Diagnosing Three Food-Borne Pathogenic Bacteria in Aquatic Products

A rapid multiplex-PCR method was established in order to detect three foodborne pathogenic bacteria including Salmonella typli，Vibrio parahaemolyticus and Listeria monocytogenes in aquatic products. Three pairs of oligonucleotide primers were designed for multiplex-PCR amplification according to gene coding invasion protein A of Salmonella typli，gene coding immune response of Vibrio parahaemolyticus and regulation histone gene of Listeria monocytogenes. The amplified fragment sizes of these three bacteria were 213 bp, 369 bp and 517bp, respectively．The specificity of the multiplex- PCR was high and the minimum detection limit reached 103 CFU/mL. The multiplex-PCR method was used to analyze three aquatic product samples compared with national standard methods, the coincidence rate of two methods reached 100%. The method developed in this study had high sensitivity and specificity, which could be applied for the rapid detection and molecular epidemiology survey of food-borne pathogenic bacteria in aquatic products．

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A Rapid and Sensitive Europium Nanoparticle-Based Lateral Flow Immunoassay Combined with Recombinase Polymerase Amplification for Simultaneous Detection of Three Food-Borne Pathogens

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph18094574 ◽

2021 ◽

Vol 18 (9) ◽

pp. 4574

Author(s):

Kai Chen ◽

Biao Ma ◽

Jiali Li ◽

Erjing Chen ◽

Ying Xu ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Vibrio Parahaemolyticus ◽

Pathogenic Bacteria ◽

Simultaneous Detection ◽

Lateral Flow ◽

Recombinase Polymerase Amplification ◽

Quantitative Detection ◽

Bacterial Strains ◽

Food Borne Pathogens ◽

Food Borne

Food-borne pathogens have become an important public threat to human health. There are many kinds of pathogenic bacteria in food consumed daily. A rapid and sensitive testing method for multiple food-borne pathogens is essential. Europium nanoparticles (EuNPs) are used as fluorescent probes in lateral flow immunoassays (LFIAs) to improve sensitivity. Here, recombinase polymerase amplification (RPA) combined with fluorescent LFIA was established for the simultaneous and quantitative detection of Listeria monocytogenes, Vibrio parahaemolyticus, and Escherichia coliO157:H7. In this work, the entire experimental process could be completed in 20 min at 37 °C. The limits of detection (LODs) of EuNP-based LFIA–RPA were 9.0 colony-forming units (CFU)/mL for Listeria monocytogenes, 7.0 CFU/mL for Vibrio parahaemolyticus, and 4.0 CFU/mL for Escherichia coliO157:H7. No cross-reaction could be observed in 22 bacterial strains. The fluorescent LFIA–RPA assay exhibits high sensitivity and good specificity. Moreover, the average recovery of the three food-borne pathogens spiked in food samples was 90.9–114.2%. The experiments indicate the accuracy and reliability of the multiple fluorescent test strips. Our developed EuNP-based LFIA–RPA assay is a promising analytical tool for the rapid and simultaneous detection of multiple low concentrations of food-borne pathogens.

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The Study of Multiplex PCR Conditions for Detection of Three Food-Borne Bacterial Pathogens

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.236-238.2803 ◽

2011 ◽

Vol 236-238 ◽

pp. 2803-2809

Author(s):

Ai Li ◽

Guo Xing Yang ◽

Wei Zhang

Keyword(s):

Staphylococcus Aureus ◽

Listeria Monocytogenes ◽

Multiplex Pcr ◽

Bacterial Pathogens ◽

Simultaneous Detection ◽

Pcr Amplification ◽

Orthogonal Experimental Design ◽

Food Borne ◽

Amplification System ◽

Four Levels

To establish a rapid, sensitive and specific multiplex PCR method for the simultaneous detection ofStaphylococcus aureus,Salmonellaspp, andlisteria monocytogenes. Three pairs of primers have been designed according to theStaphylococcus aureusnucgene,Salmonellaspp IpaBgene,listeria monocytogenes inlAgene. Orthogonal experimental design was used to determine Multiplex PCR amplification system for Food-borne Bacterial Pathogens of four factors (Taq DNA polymerase, Mg2+, dNTP and primers) from four levels; three DNA fragments of 210bp,280bp and 476bp were amplified. The specificity and the sensitivity of this method was valued. Template was prepared using FTA filter; the three food-borne Bacterial Pathogens were simultaneously detected by the multiplex PCR technology which have been designed; The sensitivity of this method was 3.0×102cfu/mL forStaphylococcus aureus, 2.0×102cfu/mL forSalmonellaspp, and 3.5×102cfu/mL forlisteria monocytogenes. This method lies on its accuracy, rapidity and efficiency in the diagnosis, so it could be a useful method for the simultaneous detection of the three species of bacteria in food.

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Biofilm Formation and Associated Genes in Listeria Monocytogenes

THE INDIAN JOURNAL OF VETERINARY SCIENCES AND BIOTECHNOLOGY ◽

10.21887/ijvsbt.v12i3.7079 ◽

2017 ◽

Vol 12 (3) ◽

Author(s):

S. R. Warke ◽

V. C. Ingle ◽

N. V. Kurkure ◽

P. A. Tembhurne ◽

Minakshi Prasad ◽

...

Keyword(s):

Public Health ◽

Listeria Monocytogenes ◽

Multiplex Pcr ◽

Food Processing ◽

Biofilm Formation ◽

Milk Samples ◽

Biofilm Production ◽

Food Borne ◽

Serious Infections ◽

Microtiter Assay

Listeria monocytogenes, an opportunistic food borne pathogen can cause serious infections in immunocompromised individuals. L. monocytogenes is capable of producing biofilm on the surface of food processing lines and instruments.The biofilm transfers contamination to food products and impose risk to public health. In the present study biofilm producing ability of L. monocytogenes isolates were investigated phenotypically and genotypically by microtiter assay and multiplex PCR, respectively. Out of 38 L. monocytogenes isolates 14 were recovered from animal clinical cases, 12 bovine environment and 12 from milk samples. A total of 3 (21.42%) clinical, 2 (16.66%) environment and 3 (25%) milk samples respectively, revealed biofilm production in microtiter assay. Cumulative results showed that 23 (60.52%) out of 38 strains of L. monocytogenes were positive for luxS and flaA gene and 1 (2.63%) was positive only for the flaA gene.

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Development of multiplex PCR assay for rapid detection Listeria monocytogenes in clinical samples

Tạp chí Y học Dự phòng ◽

10.51403/0868-2836/2020/246 ◽

2021 ◽

Vol 30 (4) ◽

pp. 20-26

Author(s):

Le Thanh Huong ◽

Ha Thi Phuong Mai ◽

Hoang Thi Thu Ha ◽

Nguyen Dong Tu ◽

Bui Tien Sy ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Multiplex Pcr ◽

Rapid Detection ◽

Pathogenic Bacteria ◽

Vulnerable Groups ◽

Clinical Samples ◽

Specific Gene ◽

Pcr Assay ◽

Multiplex Pcr Assay ◽

Specificity And Sensitivity

Listeria monocytogenes is widely present in the natural environment. This bacteria can cause infections in both humans and animals. In humans, the most vulnerable groups to be infected with L. monocytogenes are the elderly, people with an impaired immune system and chronically illness, pregnant women, and newborn babies. The aim of this study was to develop a multiplex PCR assay for the rapid detection of L. monocytogenes in mock clinical samples. A pair of primers were designed for detection of L. monocytogenes based on prs, a Listeria genus specific gene, and hly, a hemolysin gene. The specificity of the primers were tested by using different L. monocytogenes strains and other common pathogenic bacteria. The results showed that L. monocytogenes strains were positive in the detection and other tested strains were negative in mock (spiked) clinical samples. The sensitivity of multiplex PCR assay was 102 CFU/ml per reaction. The specificity and sensitivity of multiplex PCR technology for detecting L. monocytogenes in mock (spiked) clinical samples were high, and the assay could be completed within 1.5 hours. Therefore, this established multiplex PCR provides a rapid and reliable method and will be useful for the detection of L. monocytogenes in mock clinical samples.

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369 AMPLIFICATION OF THE SRY GENE FOR SEXING OF BUFFALO (BUBALUS BUBALIS) EMBRYOS USING NESTED MULTIPLEX PCR

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab369 ◽

2007 ◽

Vol 19 (1) ◽

pp. 300

Author(s):

M. Zhang ◽

Q. Fu ◽

W. S. Qin ◽

H. Y. Zheng ◽

Y. Q. Lu ◽

...

Keyword(s):

Multiplex Pcr ◽

Internal Control ◽

Pcr Amplification ◽

Single Copy ◽

Bubalus Bubalis ◽

Sex Identification ◽

Sry Gene ◽

Embryo Sex ◽

Pcr Method ◽

Male Specific

In mammals, the Y chromosome-linked SRY gene is responsible for male sex determination. Therefore, a logical approach for embryo sex identification is to amplify the male-specific single-copy SRY gene. The objective of this study was to design specific primers for amplification of buffalo SRY gene and develop a reliable PCR method for sex identification of buffalo embryos. Genomic DNA was extracted from swamp buffalo (Bubalus bubalis) peripheral blood. A pair of primers based on the sequence of Holstein bovine SRY gene (forward, 52-GTTTGCCTTATGGATTTATT-32; reverse, 52-TCTACTTTAGCCTATTTG-32) was used to amplify whole buffalo SRY gene. This amplified fragment was isolated and constructed into plasmids for sequencing. Two pairs of primers, S1/S2 (forward, 52-CCATGAACGCCTTCATTTTGTG-32; reverse, 52-ACGAGGTCGATATTTATAGC CC-32) and S3/S4 (forward, 52-AAGCAGCTGGGCTATGAGTGGAA-32; reverse, 52-ACGAGGTCGATATTTATAGCCC-32), were designed based on the SRY sequence above. Simultaneously, the G3PDH gene was amplified to serve as an internal control for both male and female embryos. A multiplex-nested-PCR system was optimized by varying the following parameters individually: concentration of Mg2+, dNTPs, primers, and different cycles number. Twenty-seven IVF morulae were identified with the optimal PCR procedure after biopsy. Accuracy of PCR amplification was verified by dot blotting. The sex of 24 embryos fertilized with Y-sperm separated by flow cytometry was also examined. Results indicated that the optimal procedure of Nested-Multiplex-PCR consisted of 1.5 mM Mg2+, 100 �M dNTPs, 0.5 �M SA3/SA4 primers, and 0.25 �M GA3/GA4 primers, and 35 cycles. Accuracy of identification was 100% for 27 IVF morulae; 14 were judged as males and 13 were females. The result of blotting confirmed that the accuracy of amplification was 100%. The proportion of males was 83.3% (20/24) in embryos fertilized with Y-sperm. This confirms that the PCR system targeting the SRY gene can be used for accurate sex identification of buffalo embryos. This study was supported by grants from the Foundation of Guangxi Key Laboratory for Subtropical Bio-Resource Conservation and Utilization (SB0403) and the Guangxi Department of Science and Technology (0626001-3-1).

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A Multiplex-PCR Method for Strain Identification and Detailed Phylogenetic Analysis of AY-Group Phytoplasmas

Plant Disease ◽

10.1094/pdis-03-13-0216-re ◽

2014 ◽

Vol 98 (3) ◽

pp. 299-305 ◽

Cited By ~ 7

Author(s):

Shigeyuki Kakizawa ◽

Yoichi Kamagata

Keyword(s):

Phylogenetic Analysis ◽

Multiplex Pcr ◽

16S Rdna ◽

Pathogenic Bacteria ◽

Target Genes ◽

Direct Sequencing ◽

Pcr Amplification ◽

Single Copy ◽

Strain Identification ◽

Specific Detection

Phytoplasmas are plant pathogenic bacteria that cause devastating losses in the yield of diverse crops worldwide. Specific detection and strain identification of phytoplasmas is important to prevent the spread of phytoplasma-induced diseases. Hence, methods to rapidly detect these organisms are important for pest control. Polymerase chain reaction (PCR) methods using phytoplasma-specific primers are widely used to detect phytoplasmas from infected plants and insects because they are highly sensitive, easily handled, and have a variety of analytical secondary applications. The phytoplasma 16S rDNA was widely used as a target of the PCR detection method; however, further target genes and more rapid methods have been required for more specific detection of phytoplasmas. Here, we developed a multiplex-PCR system to amplify several phytoplasma genes. We designed 36 primers, based on the genome sequence of ‘Candidatus Phytoplasma asteris’, to amplify 18 single-copy genes covering wide regions of the phytoplasma genome. Nine genes could be simultaneously amplified in a single PCR. This multiplex-PCR was applied to DNAs from 10 phytoplasma strains belonging to the AY-group, and different amplification patterns were obtained between strains, suggesting that this method would allow us to differentiate phytoplasmas at the strain level. Direct sequencing was also possible after the multiplex-PCR amplification by a modified sequencing method. Detailed phylogenetic analysis was performed using concatenated sequences, and evolutionary relationships among four Japanese isolates were revealed, where these strains could not be distinguished by their 16S rDNA. Thus, this multiplex-PCR system is useful for rapid strain identification and detailed phylogenetic analysis of phytoplasmas.

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Selection of aptamers targeted to food‐borne pathogenic bacteria Vibrio parahaemolyticus

Food Science & Nutrition ◽

10.1002/fsn3.1677 ◽

2020 ◽

Vol 8 (7) ◽

pp. 3835-3842

Author(s):

Lan Wang ◽

Shuxia Lyu ◽

Ganyu Gu ◽

Samantha Bolten

Keyword(s):

Vibrio Parahaemolyticus ◽

Pathogenic Bacteria ◽

Food Borne ◽

Selection Of

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Comparison on the growth heterogeneity of Vibrio parahaemolyticus coupled with strain sources and genotypes analyses in different oligotrophic conditions

Journal of Food Protection ◽

10.4315/jfp-21-089 ◽

2021 ◽

Author(s):

Jun Shi ◽

Wei Zhao ◽

Jing Xie ◽

Yongheng Zhu ◽

Yingjie Pan ◽

...

Keyword(s):

Vibrio Parahaemolyticus ◽

Specific Growth ◽

Growth Curves ◽

Gompertz Model ◽

Growth Variability ◽

Aquatic Products ◽

Theoretical Support ◽

Food Borne ◽

Modified Gompertz Model

Vibrio parahaemolyticus is an important food-borne pathogen in aquatic products, which can survive long-term in an oligotrophic environment and maintain pathogenicity. In this study, the growth curves of 38 strains of V.parahaemolyticus (pathogenic and environmental strains) under different oligotrophic conditions (tryptone soy broth (TSB), TSB diluted 2, 4, and 6 times medium) were simulated and their growth heterogeneity was compared. The growth kinetic parameters (maximum specific growth rate ( µ max ) and lag time (LT)) were calculated by the modified Gompertz model. The results showed that oligotrophic conditions affected the growth variability of strains, and the coefficient of variation (CV) of all strains reached the maximum in the 4-fold dilution of TSB. Under different oligotrophic conditions, the LT of the pathogenic strains was shorter than that of the environmental strains, while the µ max of the environmental strains was greater. This indicated that pathogenic strains were more adaptable to the nutrient-deficient environment. The analysis of different genotypes revealed that the strains with genotype tlh + /tdh + /trh − showed a greater growth variability in oligotrophic environments. These results provided theoretical support for the accuracy of the risk assessment of aquatic products.

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Detection of Emerging Food Pathogens in Chicken Meat Using Multiplex Polymerase Chain Reaction

Journal of Agricultural Science ◽

10.5539/jas.v8n9p217 ◽

2016 ◽

Vol 8 (9) ◽

pp. 217 ◽

Cited By ~ 1

Author(s):

Satheesh Raja ◽

Appa Rao ◽

Narendra Babu ◽

Thamizhannal Thamizhannal

Keyword(s):

Listeria Monocytogenes ◽

Multiplex Pcr ◽

Dna Extraction ◽

Campylobacter Jejuni ◽

Simultaneous Detection ◽

Pcr Amplification ◽

Extraction Process ◽

Chicken Meat ◽

Rapid Screening ◽

Culture Methods

A study was undertaken to develop a multiplex PCR (m-PCR) protocol for simultaneous detection of Campylobacter jejuni and Listeria monocytogenes in chicken meat. The extraction of DNA was carried out using commercial DNA extraction kit, Phenol Chloroform and boiling method. Samples with OD ratio (260:280) between 1.7 and 1.9 were considered good in terms of concentration and purity and were used for PCR amplification. DNA extraction kit and Phenol Chloroform method revealed good OD value were used for sample extraction process. PCR and m-PCR amplification was carried out using genus specific primers were designed by targeting its Hyp (500 bp) and prfA (290 bp) gene for Campylobacter jejuni and Listeria monocytogenes respectively. Electrophoresis of amplified PCR products and gel documentation revealed 500 bp and 290 bp in 2% Agarose. The multiplex PCR technique was standardized using the reference strains with the similar amplification procedure. The minimum detection level (sensitivity) by mPCR for Campylobacter jejuni and Listeria monocytogenes was found to be 0.2 ng/ul and 1.0 ng/ul of DNA in a reaction mixture (25 ul). The developed multiplex PCR technique could detect Campylobacter jejuni and Listeria monocytogenes upto 3 × 105 and 3 × 104 CFU/ml of artificially inoculated meat homogenate. Around 60 chicken meat samples were collected from different regions of chennai and were screened for the presence of Campylobacter jejuni and Listeria monocytogenes. All the samples screened were not positive either for Campylobacter jejuni and Listeria monocytogenes. The negative samples were further checked by culture methods and good correlation between these two methods was observed. Hence, the m-PCR technique developed in this study can be used as a rapid screening test for detection of Campylobacter jejuni and Listeria monocytogenes from chicken meat within 24 hours.

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