A Disposable and Multi-Chamber Film-Based PCR Chip for Detection of Foodborne Pathogen

Since the increment of the threat to public health caused by foodborne pathogens, researches have been widely studied on developing the miniaturized detection system for the on-site pathogen detection. In the study, we focused on the development of portable, robust, and disposable film-based polymerase chain reaction (PCR) chip containing a multiplex chamber for simultaneous gene amplification. In order to simply fabricate and operate a film-based PCR chip, different kinds of PCR chambers were designed and fabricated using polyethylene terephthalate (PET) and polyvinyl chloride (PVC) adhesive film, in comparison with commercial PCR, which employs a stereotyped system at a bench-top scale. No reagent leakage was confirmed during the PCR thermal cycling using the film PCR chip, which indicates that the film PCR chip is structurally stable for rapid heat cycling for DNA amplification. Owing to use of the thin film to fabricate the PCR chip, we are able to realize fast thermal transfer from the heat block that leads to short PCR amplification time. Moreover, using the film PCR chip, we could even amplify the target pathogen with 10 CFU mL−1. The artificially infected milk with various concentration of Bacillus cereus was successfully amplified on a single film PCR chip. On the basis of the reliable results, the developed film PCR chip could be a useful tool as a POCT device to detect foodborne pathogens via genetic analysis.

Download Full-text

Utilizing the Public GenomeTrakr Database for Foodborne Pathogen Traceback v1

10.17504/protocols.io.bxw3ppgn ◽

2021 ◽

Author(s):

Ruth E Timme ◽

Maria Sanchez ◽

Marc Allard

Keyword(s):

Real Time ◽

Foodborne Pathogens ◽

Pathogen Detection ◽

Foodborne Pathogen ◽

Reference Database ◽

Surveillance Program ◽

Environmental Isolates ◽

The Public ◽

The World ◽

The Usa

This protocol outlines the all the steps necessary to become a GenomeTrakr data contributor. GenomeTrakr is an international genomic reference database of mostly food and environmental isolates from foodborne pathogens. The data and analyses are housed at the National Center for Biotechnology Information (NCBI), which is a database freely available to anyone in the world. The Pathogen Detection browser at NCBI computes daily cluster results adding the newly submitted data to the existing phylogenetic clusters of closely related genomes. Contributors to this database can see how their new isolates are related to the real-time foodborne pathogen surveillance program established in the USA and a few other countries, and at the same time adding valuable new data to the reference database. ------ Although originally published as a Chapter in Methods and Protocols, Foodborne Bacterial Pathogens, the protocol has since been adapted and split into four separate protocols all of which are contained in this collection.

Download Full-text

Applications of Nanotechnology in Sensor-Based Detection of Foodborne Pathogens

Sensors ◽

10.3390/s20071966 ◽

2020 ◽

Vol 20 (7) ◽

pp. 1966 ◽

Cited By ~ 3

Author(s):

Harsh Kumar ◽

Kamil Kuča ◽

Shashi Kant Bhatia ◽

Kritika Saini ◽

Ankur Kaushal ◽

...

Keyword(s):

Foodborne Pathogens ◽

Pathogen Detection ◽

Foodborne Pathogen ◽

Health Issues ◽

Detection And Identification ◽

Specificity And Sensitivity ◽

Food Borne ◽

Contaminated Food ◽

Working Principle ◽

Effective Use

The intake of microbial-contaminated food poses severe health issues due to the outbreaks of stern food-borne diseases. Therefore, there is a need for precise detection and identification of pathogenic microbes and toxins in food to prevent these concerns. Thus, understanding the concept of biosensing has enabled researchers to develop nanobiosensors with different nanomaterials and composites to improve the sensitivity as well as the specificity of pathogen detection. The application of nanomaterials has enabled researchers to use advanced technologies in biosensors for the transfer of signals to enhance their efficiency and sensitivity. Nanomaterials like carbon nanotubes, magnetic and gold, dendrimers, graphene nanomaterials and quantum dots are predominantly used for developing biosensors with improved specificity and sensitivity of detection due to their exclusive chemical, magnetic, mechanical, optical and physical properties. All nanoparticles and new composites used in biosensors need to be classified and categorized for their enhanced performance, quick detection, and unobtrusive and effective use in foodborne analysis. Hence, this review intends to summarize the different sensing methods used in foodborne pathogen detection, their design, working principle and advances in sensing systems.

Download Full-text

A Portable Automatic Endpoint Detection System for Amplicons of Loop Mediated Isothermal Amplification on Microfluidic Compact Disk Platform

Sensors ◽

10.3390/s150305376 ◽

2015 ◽

Vol 15 (3) ◽

pp. 5376-5389 ◽

Cited By ~ 20

Author(s):

Shah Uddin ◽

Fatimah Ibrahim ◽

Abkar Sayad ◽

Aung Thiha ◽

Koh Pei ◽

...

Keyword(s):

Pathogen Detection ◽

Detection System ◽

Liquid Crystal Display ◽

Foodborne Pathogen ◽

Isothermal Amplification ◽

Detection Methods ◽

Endpoint Detection ◽

Compact Disk ◽

Loop Mediated Isothermal Amplification ◽

The Impact

In recent years, many improvements have been made in foodborne pathogen detection methods to reduce the impact of food contamination. Several rapid methods have been developed with biosensor devices to improve the way of performing pathogen detection. This paper presents an automated endpoint detection system for amplicons generated by loop mediated isothermal amplification (LAMP) on a microfluidic compact disk platform. The developed detection system utilizes a monochromatic ultraviolet (UV) emitter for excitation of fluorescent labeled LAMP amplicons and a color sensor to detect the emitted florescence from target. Then it processes the sensor output and displays the detection results on liquid crystal display (LCD). The sensitivity test has been performed with detection limit up to 2.5 × 10−3 ng/µL with different DNA concentrations of Salmonella bacteria. This system allows a rapid and automatic endpoint detection which could lead to the development of a point-of-care diagnosis device for foodborne pathogens detection in a resource-limited environment.

Download Full-text

Group-specific differentiation of Rhizobium from clover species by PCR amplification of 23S rDNA sequences

Canadian Journal of Microbiology ◽

10.1139/w98-114 ◽

1998 ◽

Vol 44 (11) ◽

pp. 1102-1105

Author(s):

Mesfin Tesfaye ◽

F Brian Holl

Keyword(s):

Root Nodule ◽

Bacterial Species ◽

Pcr Amplification ◽

Dna Amplification ◽

Specific Detection ◽

Rdna Sequences ◽

Rhizobium Spp ◽

Polymerase Chain ◽

Inoculation Group ◽

Identification Tool

Two 20-bp primers that provide group-specific detection of Rhizobium spp. by polymerase chain reaction (PCR) have been used to differentiate strains that belong to different effectiveness groups within the Rhizobium-Trifolium cross-inoculation group. The target for DNA amplification was a 370-bp fragment of the 23S rDNA region. Analysis of additional root-nodule forming, as well as root-associated bacterial species by PCR-primer assay revealed that variability within this 20-bp segment of the 23S rDNA region may be widespread and provide an effective identification tool. Our data suggest that strains of Rhizobium isolated from the perennial clover Trifolium semipilosum may be phylogenetically more closely related to Rhizobium etli.Key words: Rhizobium, Trifolium, detection, PCR, rDNA.

Download Full-text

Double Fluorescent-amplification Refractory Mutation Detection (dF-ARMS) of the Factor V Leiden and Prothrombin Mutations

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614422 ◽

1999 ◽

Vol 81 (01) ◽

pp. 76-80 ◽

Cited By ~ 10

Author(s):

Caroline Maher ◽

Dolores Crowley ◽

Carmel Cullen ◽

Carmel Wall ◽

David Royston ◽

...

Keyword(s):

Mutation Detection ◽

Detection System ◽

Factor V Leiden ◽

Pcr Amplification ◽

Factor V ◽

Direct Dna Sequencing ◽

Genescan Analysis ◽

Assay Design ◽

Polymerase Chain ◽

Compound Heterozygotes

SummarySimultaneous fluorescent [F] detection of the factor V Leiden (G1691A) and the prothrombin 3’-untranslated region (G20210A) mutations were performed in a single tube polymerase chain reaction (PCR). Amplification refractory mutation detection system (ARMS) formed the basis of this assay design. Fluorescent-labelled primers incorporated into amplicons during the reaction facilitated detection directly by GeneScan analysis without further manipulation. To test the efficacy of this double [F]-ARMS (dF-ARMS) method, 48 patients with unexplained thrombotic tendencies were investigated for their factor V Leiden and prothrombin genotypes. These results corresponded exactly with data achieved using the more conventional methods of restriction fragment length polymorphism (RFLP)-PCR and direct DNA sequencing. Three out of the 48 patients in this group were found to be compound heterozygotes.

Download Full-text

Detection ofTrypanosoma congolenseandTrypanosoma bruceisubspecies by DNA amplification using the polymerase chain reaction

Parasitology ◽

10.1017/s0031182000061023 ◽

1989 ◽

Vol 99 (1) ◽

pp. 57-66 ◽

Cited By ~ 168

Author(s):

D. R. Moser ◽

G. A. Cook ◽

Diane E. Ochs ◽

Cheryl P. Bailey ◽

Melissa R. McKane ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Dna Sequences ◽

Large Scale ◽

Nuclear Dna ◽

Pcr Amplification ◽

Dna Amplification ◽

Detection Methods ◽

Chain Reaction ◽

African Trypanosomes ◽

Polymerase Chain

SUMMARYThe nuclear DNA ofTrypanosoma congolensecontains a family of highly conserved 369 base pair (bp) repeats. The sequences of three cloned copies of these repeats were determined. An unrelated family of 177 bp repeats has previously been shown to occur in the nuclear DNA ofTrypanosoma brucei brucei(Sloofet al.1983a). Oligonucleotides were synthesized which prime the specific amplification of each of these repetitive DNAs by the polymerase chain reaction (PCR). Amplification of 10% of the DNA in a single parasite ofT. congolenseorT. bruceispp. produced sufficient amplified product to be visible as a band in an agarose gel stained with ethidium bromide. This level of detection, which does not depend on the use of radioactivity, is about 100 times more sensitive than previous detection methods based on radioactive DNA probes. The oligonucleotides did not prime the amplification of DNA sequences in other trypanosome species nor inLeishmania, mouse or human DNAs. Amplification of DNA from the blood of animals infected withT. congolenseand/orT. bruceispp. permitted the identification of parasite levels far below that detectable by microscopic inspection. Since PCR amplification can be conducted on a large number of samples simultaneously, it is ideally suited for large-scale studies on the prevalence of African trypanosomes in both mammalian blood and insect vectors.

Download Full-text

A newly developed paper embedded microchip based on LAMP for rapid multiple detections of foodborne pathogens

BMC Microbiology ◽

10.1186/s12866-021-02223-0 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Mimi Zhang ◽

Jinfeng Liu ◽

Zhiqiang Shen ◽

Yongxin Liu ◽

Yang Song ◽

...

Keyword(s):

Centrifugal Force ◽

Foodborne Pathogens ◽

Pathogen Detection ◽

Point Of Care ◽

Foodborne Pathogen ◽

Cost Effective ◽

Reaction Chamber ◽

Salmonella Spp ◽

Culture Methods ◽

Detection Technology

Abstract Background Microfluidic chip detection technology is considered a potent tool for many bioanalytic applications. Rapid detection of foodborne pathogens in the early stages is imperative to prevent the outbreak of foodborne diseases, known as a severe threat to human health. Conventional bacterial culture methods for detecting foodborne pathogens are time-consuming, laborious, and lacking in pathogen diagnosis. To overcome this problem, we have created an embedded paper-based microchip based on isothermal loop amplification (LAMP), which can rapidly and sensitively detect foodborne pathogens. Results We embed paper impregnated with LAMP reagent and specific primers in multiple reaction chambers of the microchip. The solution containing the target pathogen was injected into the center chamber and uniformly distributed into the reaction chamber by centrifugal force. The purified DNA of Escherichia coli O157:H7, Salmonella spp., Staphylococcus aureus, and Vibrio parahaemolyticus has been successfully amplified and directly detected on the microchip. The E. coli O157:H7 DNA was identified as low as 0.0134 ng μL− 1. Besides, the potential of this microchip in point-of-care testing was further tested by combining the on-chip sample purification module and using milk spiked with Salmonella spp.. The pyrolyzed milk sample was filtered through a polydopamine-coated paper embedded in the inside of the sample chamber. It was transported to the reaction chamber by centrifugal force for LAMP amplification. Then direct chip detection was performed in the reaction chamber embedded with calcein-soaked paper. The detection limit of Salmonella spp. in the sample measured by the microchip was approximately 12 CFU mL− 1. Conclusion The paper embedded LAMP microchip offers inexpensive, user-friendly, and highly selective pathogen detection capabilities. It is expected to have great potential as a quick, efficient, and cost-effective solution for future foodborne pathogen detection.

Download Full-text

DETECTION AND APPLICATION OF MICROFLUIDIC ISOTHERMAL AMPLIFICATION ON CHIP

Journal of Innovative Optical Health Sciences ◽

10.1142/s1793545808000248 ◽

2008 ◽

Vol 01 (02) ◽

pp. 257-265 ◽

Cited By ~ 3

Author(s):

GUOLIANG HUANG ◽

XIAOYONG YANG ◽

JIANG ZHU ◽

SHUKUAN XU ◽

CHENG DENG ◽

...

Keyword(s):

Detection System ◽

Dna Amplification ◽

Isothermal Amplification ◽

Nucleic Acid Amplification ◽

Amplification Method ◽

Amplification Process ◽

Different Temperatures ◽

System Temperature ◽

Polymerase Chain ◽

On Chip

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method. Compared with the widely utilized polymerase chain reaction (PCR), LAMP has higher speed and efficiency as well as lower requirement for system temperature control because the whole amplification process is isothermal and no efforts are needed to switch between different temperatures. In this paper, we designed and fabricated different kinds of polycarbonate (PC) microfluid chips, explored appropriate reaction condition for LAMP in microenvironment (1 nL → 10 μL), and developed a microfluidic isothermal amplification detection system. The DNA optimal amplification temperature is obtained; the starting time of exponential amplification of DNA is put forward farther. The optimal condition of DNA amplification in microenvironment, with a little reaction materials and early starting exponential amplification time of DNA are very important for clinic DNA detection and the application of Lab-on-a-Chip.

Download Full-text

Molecular Bird Sexing on Fischeri Lovebird (Agapornis fischeri) by Using Polymerase Chain Reaction

BIO Web of Conferences ◽

10.1051/bioconf/20202004003 ◽

2020 ◽

Vol 20 ◽

pp. 04003

Author(s):

Azalea Dyah Argarini ◽

Herjuno Ari Nugroho ◽

Medania Purwaningrum ◽

Aris Haryanto

Keyword(s):

Sex Chromosome ◽

Pcr Amplification ◽

Dna Amplification ◽

Total Sample ◽

Z Chromosome ◽

Agarose Gel Electrophoresis ◽

Chain Reaction ◽

Male And Female ◽

Pcr Products ◽

Polymerase Chain

Fischeri Lovebird (Agapornis fischeri) found originally in Africa which has spread to many countries. In Indonesia, Fischeri Lovebird is popular as a pet animal. This lovebird is a monomorphic bird, so it is difficult to differentiate morphologically between male and female birds. In general, a male lovebird has ZZ homozygotes, whereas females' lovebird has ZW heterozygous of their sex chromosome. These sex chromosomes set used as study targets for molecular bird sexing of many species of birds because this method is effective and simple to perform. This method targeted to amplify the Chromodomain Helicase DNA-binding (CHD) gene, which found into the sex chromosome of male and female birds. The objective of this study was to rapid molecular bird sexing of Fischeri Lovebird by using PCR methods. Research samples were collected from feather calamus of A. fischeri. The total sample was 11 feathers from A. fischeri. which were collected three to six feathers for each lovebird. Then the research was followed by DNA extraction from calamus feathers, DNA amplification by PCR and agarose gel electrophoresis of PCR products and visualization of PCR predicts by UV-Transilluminator in darkroom. It concluded that PCR amplification using NP, MP and P2 primers produced double DNA bands in size of 400 bp on Z chromosome and bp on W chromosome for female Fischeri Lovebird, whereas for male Fischeri Lovebird only produced a single DNA band in size of 400 bp on Z chromosome. From eleven samples of Fischeri Lovebird showed a total of five females and six male Fischeri Lovebirds.

Download Full-text