Identification for adulteration of beef with chicken based on single primer-triggered isothermal amplification

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Jiao Chen ◽  
Pansong Zhang ◽  
Haixia Wang ◽  
Yanjing Shi
Keyword(s):  
Limit Of Detection ◽  
Chicken Meat ◽  
Isothermal Amplification ◽  
Work Flow ◽  
Acid Extraction ◽  
Specificity And Sensitivity ◽  
Total Dna ◽  
Meat Species ◽  
Species Specific ◽  
Single Primer

Abstract Adulteration of beef with cheap chicken has become a growing problem worldwide. In this study, a quick, single primer-triggered isothermal amplification (SAMP) combined with a fast nucleic acid extraction method was employed to detect the chicken meat in adulterated beef. Chicken from adulterated beef was identified using the chicken species-specific primer designed according to the Gallus gallus mitochondrial conserved sequences. Our SAMP method displayed good specificity and sensitivity in detecting chicken and beef meat DNA–the limit of detection (LOD) of SAMP is 0.33 pg/μL of chicken and beef total DNA and 2% w/w chicken meat in beef. The whole work flow from DNA extraction to signal detection can be finished within 1 h, fulfilling the requirement of on-site meat species identification.

scholarly journals Multispecies Identification of Oilseed- and Meat-Specific Proteins and Heat-Stable Peptide Markers in Food Products

Molecules ◽  
2021 ◽  
Vol 26 (6) ◽  
pp. 1577
Author(s):  
Klaudia Kotecka-Majchrzak ◽  
Natalia Kasałka-Czarna ◽  
Agata Sumara ◽  
Emilia Fornal ◽  
Magdalena Montowska
Keyword(s):  
Limit Of Detection ◽  
Raw Materials ◽  
Meat Products ◽  
Food Products ◽  
Plant Raw Materials ◽  
Heat Stable ◽  
2S Albumins ◽  
Specific Proteins ◽  
Meat Species ◽  
Species Specific

Consumer demand for both plant products and meat products enriched with plant raw materials is constantly increasing. Therefore, new versatile and reliable methods are needed to find and combat fraudulent practices in processed foods. The objective of this study was to identify oilseed species-specific peptide markers and meat-specific markers that were resistant to processing, for multispecies authentication of different meat and vegan food products using the proteomic LC-MS/MS method. To assess the limit of detection (LOD) for hemp proteins, cooked meatballs consisting of three meat species and hemp cake at a final concentration of up to 7.4% were examined. Hemp addition at a low concentration of below 1% was detected. The LOD for edestin subunits and albumin was 0.9% (w/w), whereas for 7S vicilin-like protein it was 4.2% (w/w). Specific heat-stable peptides unique to hemp seeds, flaxseed, nigella, pumpkin, sesame, and sunflower seeds, as well as guinea fowl, rabbit, pork, and chicken meat, were detected in different meat and vegan foods. Most of the oilseed-specific peptides were identified as processing-resistant markers belonging to 11S globulin subunits, namely conlinin, edestin, helianthinin, pumpkin vicilin-like or late embryogenesis proteins, and sesame legumin-like as well as 2S albumins and oleosin isoforms or selected enzymic proteins.

scholarly journals A Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

2020 ◽  
Vol 21 (8) ◽  
pp. 2826 ◽  
Author(s):  
Renfei Lu ◽  
Xiuming Wu ◽  
Zhenzhou Wan ◽  
Yingxue Li ◽  
Xia Jin ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Limit Of Detection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Qpcr Assay ◽  
N Gene ◽  
Clinical Samples ◽  
Loop Mediated Isothermal Amplification ◽  
Specificity And Sensitivity

COVID-19 has become a major global public health burden, currently causing a rapidly growing number of infections and significant morbidity and mortality around the world. Early detection with fast and sensitive assays and timely intervention are crucial for interrupting the spread of the COVID-19 virus (SARS-CoV-2). Using a mismatch-tolerant amplification technique, we developed a simple, rapid, sensitive and visual reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for SARS-CoV-2 detection based on its N gene. The assay has a high specificity and sensitivity, and robust reproducibility, and its results can be monitored using a real-time PCR machine or visualized via colorimetric change from red to yellow. The limit of detection (LOD) of the assay is 118.6 copies of SARS-CoV-2 RNA per 25 μL reaction. The reaction can be completed within 30 min for real-time fluorescence monitoring, or 40 min for visual detection when the template input is more than 200 copies per 25 μL reaction. To evaluate the viability of the assay, a comparison between the RT-LAMP and a commercial RT-qPCR assay was made using 56 clinical samples. The SARS-CoV-2 RT-LAMP assay showed perfect agreement in detection with the RT-qPCR assay. The newly-developed SARS-CoV-2 RT-LAMP assay is a simple and rapid method for COVID-19 surveillance.

scholarly journals Validation of a Commercial Loop-Mediated Isothermal Amplification (LAMP) Assay for the Rapid Detection of Anisakis spp. DNA in Processed Fish Products

Foods ◽  
2020 ◽  
Vol 9 (1) ◽  
pp. 92 ◽  
Author(s):  
Gaetano Cammilleri ◽  
Vincenzo Ferrantelli ◽  
Andrea Pulvirenti ◽  
Chiara Drago ◽  
Giuseppe Stampone ◽  
...  
Keyword(s):  
Rapid Detection ◽  
Limit Of Detection ◽  
Lamp Assay ◽  
Allergic Diseases ◽  
Isothermal Amplification ◽  
Fish Products ◽  
Loop Mediated Isothermal Amplification ◽  
Specificity And Sensitivity ◽  
Fish Samples ◽  
Anisakis Spp

Parasites belonging to the Anisakis genera are organisms of interest for human health because they are responsible for the Anisakiasis zoonosis, caused by the ingestion of raw or undercooked fish. Furthermore, several authors have reported this parasite to be a relevant inducer of acute or chronic allergic diseases. In this work, a rapid commercial system based on Loop-Mediated Isothermal Amplification (LAMP) was optimised and validated for the sensitive and rapid detection of Anisakis spp. DNA in processed fish products. The specificity and sensitivity of the LAMP assay for processed fish samples experimentally infected with Anisakis spp. larvae and DNA were determined. The LAMP system proposed in this study was able to give positive amplification for all the processed fish samples artificially contaminated with Anisakis spp., giving sensitivity values equal to 100%. Specificity tests provided no amplification for the Contracaecum, Pseudoterranova, or Hysterothylacium genera and uninfected samples. The limit of detection (LOD) of the LAMP assay proposed was 102 times lower than the real-time PCR method compared. To the best of our knowledge, this is the first report regarding the application of the LAMP assay for the detection of Anisakis spp. in processed fish products. The results obtained indicate that the LAMP assay validated in this work could be a reliable, easy-to-use, and convenient tool for the rapid detection of Anisakis DNA in fish product inspection.

scholarly journals Loop-Mediated Isothermal Amplification (LAMP) Method for the Rapid Identification of Dendrobium officinale

2018 ◽  
Author(s):  
Lu Yang ◽  
Hua Zhou ◽  
Huili Lai ◽  
Fei Fu ◽  
Wenru Wu
Keyword(s):  
Color Change ◽  
Dna Barcode ◽  
Its Region ◽  
Rapid Identification ◽  
Isothermal Amplification ◽  
Dendrobium Officinale ◽  
Lamp Method ◽  
Loop Mediated Isothermal Amplification ◽  
Specificity And Sensitivity ◽  
Species Specific

Background: Dendrobium officinale is not only an ornamental plant, but also a valuable medicinal herb that is both effective and widely used in traditional Chinese medicine. However, distinguishing D. officinale from other Dendrobium species is usually a difficult task that need much time and complex technologies due to their very similar external morphologies. The aim of this study is to develop a fast, even on-spot approach to identify D. officinale. Methods: We used DNA barcode-based loop-mediated isothermal amplification (LAMP) method with species-specific LAMP primers targeting the internal transcribed spacer (ITS) region of the rDNA of D. officinale. LAMP reaction time and temperature were optimized and the specificity and sensitivity of LAMP species-specific primers were assessed. Results: This technique showed a high specificity and sensitivity to amplify the genomic DNA of D. officinale and allowed for rapid amplification (within 40 min) of the ITS region under a constant and mild temperature range of 65 °C without using thermocyclers. Besides, by using SYBR® Green I dye as the color developing agent, the color change was easily observed with naked eye. Reaction mixture containing DNA of D. officinale changed from orange to green, while the other Dendrobium species and the negative control retained original orange color. The specificity of this LAMP-based method was confirmed by testing 17 samples of D. officinale and 32 adulterant samples from other Dendrobium species. Conclusions: This LAMP-based rapid identification method does not require expensive equipment or specialized techniques and can be used in field surveys for accurate and fast on site identification.

A novel method for species identification in milk and milk-based products

2008 ◽  
Vol 75 (1) ◽  
pp. 107-112 ◽  
Author(s):  
Silvia Reale ◽  
Angela Campanella ◽  
Amalia Merigioli ◽  
Fabio Pilla
Keyword(s):  
Limit Of Detection ◽  
Bovine Milk ◽  
Buffalo Milk ◽  
Retail Trade ◽  
Detection Rates ◽  
Novel Method ◽  
Species Specific ◽  
Single Primer ◽  
Unambiguous Assignment ◽  
Milk And Dairy Products

This study describes a method for species-specific detection of animal DNA from different species (cattle, sheep, goat, water buffalo) in milk and dairy products. A primer set was designed in conserved region on the basis of the alignment of the sequence codifying the genomic κ-casein gene in order to amplify all four species with a single primer pair. Polymorphisms were detected via minisequencing with extension primers designed in conserved sequences for haplotype determination that allow unambiguous assignment to each species. The method was successfully applied to the detection of raw and pasteurized milk from the four different species considered as well as to cheese products from the retail trade. Estimation of the limit of detection was carried out using a progression of dilutions of genomic DNA as well as DNA isolated from milk of a known number of somatic cells from different species in order to be able to achieve detection rates as low as 0·1% bovine milk mixed with buffalo milk.

Ferromagnetic Resonance Biosensor for Homogeneous and Volumetric Detection of DNA

2017 ◽  
Author(s):  
Bo Tian ◽  
Peter Svedlindh ◽  
Mattias Strömberg ◽  
Erik Wetterskog
Keyword(s):  
Magnetic Nanoparticles ◽  
Ferromagnetic Resonance ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Rolling Circle Amplification ◽  
Resonance Field ◽  
Isothermal Amplification ◽  
Detection Sensitivity ◽  
Serum Samples ◽  
Rolling Circle

In this work, we demonstrate for the first time, a ferromagnetic resonance (FMR) based homogeneous and volumetric biosensor for magnetic label detection. Two different isothermal amplification methods, <i>i.e.</i>, rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP) are adopted and combined with a standard electron paramagnetic resonance (EPR) spectrometer for FMR biosensing. For RCA-based FMR biosensor, binding of RCA products of a synthetic Vibrio cholerae target DNA sequence gives rise to the formation of aggregates of magnetic nanoparticles. Immobilization of nanoparticles within the aggregates leads to a decrease of the net anisotropy of the system and a concomitant increase of the resonance field. A limit of detection of 1 pM is obtained with an average coefficient of variation of 0.16%, which is superior to the performance of other reported RCA-based magnetic biosensors. For LAMP-based sensing, a synthetic Zika virus target oligonucleotide is amplified and detected in 20% serum samples. Immobilization of magnetic nanoparticles is induced by their co-precipitation with Mg<sub>2</sub>P<sub>2</sub>O<sub>7</sub> (a by-product of LAMP) and provides a detection sensitivity of 100 aM. The fast measurement, high sensitivity and miniaturization potential of the proposed FMR biosensing technology makes it a promising candidate for designing future point-of-care devices.<br>

Detectability of Plasma Proteins in SRM Measurements

2018 ◽  
Vol 16 (1) ◽  
pp. 74-81 ◽  
Author(s):  
Olga I. Kiseleva ◽  
Elena A. Ponomarenko ◽  
Yulia A. Romashova ◽  
Ekaterina V. Poverennaya ◽  
Andrey V. Lisitsa
Keyword(s):  
Blood Plasma ◽  
Human Plasma ◽  
Limit Of Detection ◽  
Human Blood Plasma ◽  
Analytical Sensitivity ◽  
Plasma Proteome ◽  
Protein Targets ◽  
Blood Plasma Sample ◽  
Specificity And Sensitivity ◽  
Human Plasma Proteome

Background: Liquid chromatography coupled with targeted mass spectrometry underwent rapid technical evolution during last years and has become widely used technology in clinical laboratories. It offers confident specificity and sensitivity superior to those of traditional immunoassays. However, due to controversial reports on reproducibility of SRM measurements, the prospects of clinical appliance of the method are worth discussing. </P><P> Objective: The study was aimed at assessment of capabilities of SRM to achieve a thorough assembly of the human plasma proteome. </P><P> Method: We examined set of 19 human blood plasma samples to measure 100 proteins, including FDA-approved biomarkers, via SRM-assay. </P><P> Results: Out of 100 target proteins 43 proteins were confidently detected in at least two blood plasma sample runs, 36 and 21 proteins were either not detected in any run or inconsistently detected, respectively. Empiric dependences on protein detectability were derived to predict the number of biological samples required to detect with certainty a diagnostically relevant quantum of the human plasma proteome. </P><P> Conclusion: The number of samples exponentially increases with an increase in the number of protein targets, while proportionally decreasing to the logarithm of the limit of detection. Analytical sensitivity and enormous proteome heterogeneity are major bottlenecks of the human proteome exploration.

scholarly journals Multiplex End-Point PCR for the Detection of Three Species of Ophiosphaerella Causing Spring Dead Spot of Bermudagrass

Plant Disease ◽  
2019 ◽  
Vol 103 (8) ◽  
pp. 2010-2014 ◽  
Author(s):  
J. Francisco Iturralde Martinez ◽  
Francisco J. Flores ◽  
Alma R. Koch ◽  
Carla D. Garzón ◽  
Nathan R. Walker
Keyword(s):  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Temperature Cycling ◽  
Molecular Technique ◽  
Correct Identification ◽  
Internal Transcribed Spacer Region ◽  
Spring Dead Spot ◽  
End Point ◽  
Specificity And Sensitivity ◽  
Species Specific

A multiplex end-point polymerase chain reaction (PCR) assay was developed for identifying the three-fungal species in the genus Ophiosphaerella that cause spring dead spot (SDS), a devastating disease of bermudagrass. These fungi are difficult to identify by morphology because they seldom produce pseudothecia. To achieve species-specific diagnosis, three pairs of primers were designed to identify fungal isolates and detect the pathogen in infected roots. The internal transcribed spacer region, the translation elongation factor 1-α, and the RNA polymerase II second-largest subunit were selected as targets and served as templates for the design of each primer pair. To achieve uniform melting temperatures, three to five random nucleotide extensions (flaps) were added to the 5′ terminus of some of the designed specific primers. Temperature cycling conditions and PCR components were standardized to optimize specificity and sensitivity of the multiplex reaction. Primers were tested in multiplex on DNA extracted from axenic fungal cultures and from field-collected infected and uninfected roots. A distinct amplicon was produced for each Ophiosphaerella sp. tested. The DNA from Ophiosphaerella close relatives and other common bermudagrass pathogens did not amplify during the multiplex assay. Metagenomic DNA from infected bermudagrass produced species-specific amplicons while DNA extracted from noninfected roots did not. This multiplex end-point PCR approach is a sensitive and specific molecular technique that allows for correct identification of SDS-associated Ophiosphaerella spp. from field-collected roots.

scholarly journals Highly Sensitive Detection of Campylobacter spp. in Chicken Meat using a Silica Nanoparticle Enhanced Dot Blot DNA Biosensor

2020 ◽  
Author(s):  
Priya Vizzini ◽  
Marisa Manzano ◽  
Carole Farre ◽  
Thierry Meylheuc ◽  
Carole Chaix ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Point Of Care ◽  
Silica Nanoparticle ◽  
Chicken Meat ◽  
Dot Blot ◽  
Dna Biosensors ◽  
Point Of Care Diagnostics ◽  
Detection Probe ◽  
Highly Sensitive Detection ◽  
User Friendly

AbstractPaper-based DNA biosensors are powerful tools in point-of-care diagnostics since they are affordable, portable, user-friendly, rapid and robust. However, their sensitivity is not always as high as required to enable DNA quantification. To improve the response of standard dot blots, we have applied a new enhancement strategy that increases the sensitivity of assays based on the use of biotinylated silica-nanoparticles (biotin-Si-NPs). After immobilization of a genomic Campylobacter DNA onto a paper membrane, and addition of a biotinylated-DNA detection probe, hybridization was evidenced using streptavidin-conjugated to horseradish peroxidase (HRP) in the presence of luminol and H2O2. Replacement of the single biotin by the biotin-Si-NPs boosted on average a 30 fold chemiluminescent read-out of the biosensor. Characterization of biotin-Si-NPs onto a paper with immobilized DNA was done using a scanning electron microscope. A limit of detection of 3 pg/μL of DNA, similar to the available qPCR kits, is achieved, but it is cheaper, easier and avoids inhibition of DNA polymerase by molecules from the food matrices. We demonstrated that the new dot blot coupled to biotin-Si-NPs successfully detected Campylobacter from naturally contaminated chicken meat, without needing a PCR step. Hence, such an enhanced dot blot paves the path to the development of a portable and multiplex paper based platform for point-of-care screening of chicken carcasses for Campylobacter.Graphical abstract

Sign in / Sign up

Export Citation Format

Share Document