scholarly journals Bushmeat Species Identification: Recombinase Polymerase Amplification (RPA) Combined with Lateral Flow (LF) Strip for Identification of Formosan Reeves’ Muntjac (Muntiacus reevesi micrurus)

Animals ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 426
Author(s):  
Yun-Hsiu Hsu ◽  
Wei-Cheng Yang ◽  
Kun-Wei Chan

The identification of animal species of meat in meat products is of great concern for various reasons, such as public health, religious beliefs, food allergies, legal perspectives, and bushmeat control. In this study, we developed a new technique to identify Formosan Reeves’ muntjac in meat using recombinase polymerase amplification (RPA) in combination with a lateral flow (LF) strip. The DNA extracted from a piece of Formosan Reeves’ muntjac meat was amplified by a pair of specific primers based on its mitochondrial cytochrome b gene for 10 min at a constant temperature ranging from 30 to 45 °C using RPA. Using the specific probe added to the RPA reaction system, the amplified products were visualized on the LF strip within 5 min. The total operating time from quick DNA extraction to visualizing the result was approximately 30 min. The RPA-LF system we designed was efficient when using boiled, pan-fried, roasted, stir-fried, or stewed samples. The advantages of simple operation, speediness, and cost-effectiveness make our RPA-LF method a promising molecular detection tool for meat species identification of either raw or variously cooked Formosan Reeves’ muntjac meat. It is also possible to apply this method to identify the meat of other wildlife sources.

2018 ◽  
Vol 25 (1) ◽  
pp. 38-46
Author(s):  
Aysun Türkanoǧlu Özçelik ◽  
Semiramis Yılmaz ◽  
Sevda Gökbora ◽  
Mehmet İnan

Meat is one of the most important basic foodstuffs in human nutrition. Nowadays, adulteration and authenticity are common problems for meat products. Identification of meat species is important in terms of consumer protection and prevention of adulteration. There are different methods to determine adulteration of meat and meat products. These methods are histological controls, serological tests, and quantitative polymerase chain reaction. In this study, species identification and quantification analysis of meat and meat products were done by using horse-, donkey-, and bovine-specific primers with quantitative polymerase chain reaction method. Triple meat mixtures containing horse and donkey meat ranging from 0.1 to 50% levels were prepared within a bovine mixture for using species identification and quantification analysis. The method specificity was confirmed by melting curve analysis. In conclusion, quantitative polymerase chain reaction is an easy, rapid, and reliable method for meat species identification, and with this study an applicable method was developed for the detection and quantification of equine-originated meat in bovine meat products.


PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e8083 ◽  
Author(s):  
Tingting Dai ◽  
Tao Hu ◽  
Xiao Yang ◽  
Danyu Shen ◽  
Binbin Jiao ◽  
...  

Phytophthora hibernalis, the causal agent of brown rot of citrus fruit, is an important worldwide pathogen and a quarantine pest in China. Current diagnosis of the disease relies on disease symptoms, pathogen isolation and identification by DNA sequencing. However, symptoms caused by P. hibernalis can be confused with those by other Phytophthora and fungal species. Moreover, pathogen isolation, PCR amplification and sequencing are time-consuming. In this study, a rapid assay including 20-min recombinase polymerase amplification targeting the Ypt1 gene and 5-min visualization using lateral flow dipsticks was developed for detecting P. hibernalis. This assay was able to detect 0.2 ng of P. hibernalis genomic DNA in a 50-µL reaction system. It was specific to P. hibernalis without detection of other tested species including P. citrophthora, P. nicotianae, P. palmivora and P. syringae, four other important citrus pathogens. Using this assay, P. hibernalis was also detected from artificially inoculated orange fruits. Results in this study indicated that this assay has the potential application to detect P. hibernalis at diagnostic laboratories and plant quarantine departments of customs, especially under time- and resource-limited conditions.


Thrita ◽  
2021 ◽  
Vol In Press (In Press) ◽  
Author(s):  
Peyman Gholamnezhad ◽  
Hamed Ahari ◽  
Gholamreza Nikbakht Brujeni ◽  
Seyed Amir Ali Anvar ◽  
Abbas Ali Motalebi

Background: Real-time polymerase chain reaction (PCR) and high-resolution melting (HRM) analysis are currently considered as reliable techniques for the species identification of meat-based products and widely used to detect meat adulteration. Objectives: To examine the validity of real-time PCR and HRM analysis to identify meat species in meat-based products. Methods: Meat samples from five species (i.e., cattle, sheep, chicken, turkey, and wild pig) were purchased. Minced meat from the animal species of interest was prepared at the purities of 10%, and 20% and also were prepared as single and mixtures of two species. For molecular assessments, DNA samples were extracted from all the meat samples and subjected to real-time PCR by amplifying a mitochondrial cytochrome b specific for each species. Results: All the meat species studied in this research were successfully detected in the mixed meat samples when separately examined by real-time PCR. High-resolution melting analysis showed that all the meat species of interest were efficiently distinguished when examined simultaneously. Conclusions: The data presented here shows that the real-time PCR and HRM analysis are reliable methods for the identification of meat species used in meat products.


Animals ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 1765
Author(s):  
Ming Fu ◽  
Quanwang Zhang ◽  
Xiang Zhou ◽  
Bang Liu

Meat adulteration has become a global social problem. In order to protect consumers from meat adulteration, several methods have been developed to identify meat species. However, the conventional methods are labor-intensive, time-consuming and require instruments. In the present study, a rapid and visual method based on recombinase polymerase amplification (RPA) and multiplex lateral flow dipstick (MLFD) was developed to detect duck ingredient in adulterated beef. Using recombinase and strand displacement polymerase enable RPA to amplify different double-labeled DNA amplicons at room temperature, which can be further detected by MLFD. The whole reaction process can be finished within 35 min, and the results can be determined by naked eyes. As low as 5% of duck ingredient in adulterated beef can be easily measured. Moreover, we confirmed that our new method held good potential in the detection of commercially processed meat samples. In conclusion, this study reported a useful animal derived meat adulteration detection method, which have potential application in future.


Author(s):  
Evrim Güneş Altuntaş ◽  
Ebru Deniz ◽  
Beycan Ayhan ◽  
Kezban Candogan ◽  
Duygu Ozel Demiralp

Meat is one of the main nutrition source in the human diet with its excellent protein, vitamin and mineral contents. Despite its advantages, being high-priced makes meat products open to adulteration. Meat products are mixed food types which can contain different species of meat. However, mixing two or more types of meats is not always allowed by laws. On the other hand, replacement high quality meats with cheaper meat types are a cost lowering way for the producers. The commonly consumed meat types differ from country to country, but generally economical, ethnic and religion concerns are in the foreground. In this case, species identification techniques are gaining importance. Although some techniques depending on DNA or spectroscopy have been developed for many years, choosing the best method for species identification is still among the controversial issues today. Thus, the currently used methods and promising techniques in this area were discussed in this review.


Separations ◽  
2021 ◽  
Vol 8 (8) ◽  
pp. 116
Author(s):  
Peyman Gholamnezhad ◽  
Hamed Ahari ◽  
Gholamreza Nikbakht Brujeni ◽  
Seyed Amir Ali Anvar ◽  
Abbasali Motallebi

The current study aimed to examine a real-time PCR assay with high-resolution melting (HRM) analysis for the species identification of minced meat samples. Meat samples from several animal species were purchased and minced separately or as a mixture of two species. DNA was extracted from all meat samples and subjected to real-time PCR assay by amplifying species-specific mitochondrial cytochrome b regions. Regarding the meat mixtures, two separate melting curves with specific melt peak temperatures (Tm) were detected. Additionally, DNA from each species was quantified, based on the calibration curves. The results showed that a real-time PCR assay with HRM analysis is suitable for the species identification of meat products, and could be used for the detection of meat frauds.


Molecules ◽  
2021 ◽  
Vol 26 (6) ◽  
pp. 1577
Author(s):  
Klaudia Kotecka-Majchrzak ◽  
Natalia Kasałka-Czarna ◽  
Agata Sumara ◽  
Emilia Fornal ◽  
Magdalena Montowska

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.


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