scholarly journals Evaluation of High Resolution Melting (HRM) Analysis for Meat Species Identification of Raw and Cooked Meat

Separations ◽  
2021 ◽  
Vol 8 (8) ◽  
pp. 116
Author(s):  
Peyman Gholamnezhad ◽  
Hamed Ahari ◽  
Gholamreza Nikbakht Brujeni ◽  
Seyed Amir Ali Anvar ◽  
Abbasali Motallebi
Keyword(s):  
High Resolution ◽  
Real Time ◽  
Species Identification ◽  
Real Time Pcr ◽  
High Resolution Melting ◽  
Meat Products ◽  
Pcr Assay ◽  
Hrm Analysis ◽  
Mitochondrial Cytochrome B ◽  
Meat Samples

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.

scholarly journals Real-time PCR High-resolution Melting Analysis for the Species Identification of Meat Products: Focusing on Food Safety and Detection of Meat Adulterations

Thrita ◽  
2021 ◽  
Vol In Press (In Press) ◽  
Author(s):  
Peyman Gholamnezhad ◽  
Hamed Ahari ◽  
Gholamreza Nikbakht Brujeni ◽  
Seyed Amir Ali Anvar ◽  
Abbas Ali Motalebi
Keyword(s):  
High Resolution ◽  
Real Time ◽  
Species Identification ◽  
Real Time Pcr ◽  
High Resolution Melting ◽  
Meat Products ◽  
Hrm Analysis ◽  
Melting Analysis ◽  
Meat Species ◽  
Meat Samples

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.

Duplex real-time PCR assay for rapid identification ofStaphylococcus aureusisolates from dairy cow milk

2013 ◽  
Vol 80 (2) ◽  
pp. 223-226 ◽  
Author(s):  
Rachel Pilla ◽  
Gustavo G M Snel ◽  
Michela Malvisi ◽  
Renata Piccinini
Keyword(s):  
High Resolution ◽  
Real Time ◽  
Dairy Cow ◽  
Real Time Pcr ◽  
High Resolution Melting ◽  
Rapid Identification ◽  
Pcr Assay ◽  
Staph Aureus ◽  
Bacteriological Analysis ◽  
Milk Samples

Staphylococcus aureusisolates from dairy cow mastitis are not always consistent with the characteristic morphology described, and molecular investigation is often needed. The aim of the study was to develop a duplex real-time PCR assay for rapid identification ofStaph. aureusisolates, targeting bothnucandSa442. Overall, 140 isolates collected from dairy cow mastitis in 90 different herds, were tested. All strains had been identified using morphological and biochemical characteristics. DNA from each strain was amplified in real-time PCR assay, to detectnucorSa442. Thereafter, a duplex real-time PCR assay was performed, and specificity of the amplified products was assessed by high resolution melting curve analysis. Out of 124Staph. aureusisolates, 33 did not show the typical morphology or enzymic activity; in 118 strains, the two melt-curve peaks consistent withnucandSa442were revealed, while 2 isolates showed only the peak consistent withSa442. Four isolates bacteriologically identified asStaph. aureus, were PCR-negative and were further identified asStaph. pseudintermediusby sequencing.Staph. pseudintermediusand coagulase-negative staphylococci did not carrynucorSa442. The results showed the correct identification of all isolates, comprehending also coagulase—or nuc-negativeStaph. aureus, while other coagulase-positive Staphylococci were correctly identified as non-Staph. aureus. Both sensitivity and specificity were 100%. High resolution melting analysis allowed easy detection of unspecific products. Finally, the duplex real-time PCR was applied directly to 40 milk samples, to detect infected mammary quarters. The assay confirmed the results of bacteriological analysis, onStaph. aureus-positive or—negative samples. Therefore, the proposed duplex real-time PCR could be used in laboratory routine as a cost-effective and powerful tool for high-throughput identification of atypicalStaph. aureusisolates causing dairy cow mastitis. Also, it could be applied directly to milk samples, to detectStaph. aureusmammary infections avoiding bacteriological analysis.

scholarly journals High Resolution Meltcurve PCR assay for detection of Salmonella and Escherichia coli o157:h7 in foods

2017 ◽  
Author(s):  
◽  
Yuejiao Liu
Keyword(s):  
Escherichia Coli ◽  
High Resolution ◽  
Real Time ◽  
Real Time Pcr ◽  
Escherichia Coli O157 ◽  
Single Mutation ◽  
Pcr Assay ◽  
E Coli ◽  
Hrm Analysis ◽  
Pcr Targeting

Foodborne illnesses associated with Salmonella and Escherichia coli O157:H7 have become world-wide public-health problems. Conventional methods for the identification of foodborne pathogens are tedious, expensive, and time-consuming. Alternatively, real-time PCR (RT-PCR) as a promising method to detect pathogens in food samples, has recently been widely applied in food safety areas. High Resolution Meltcurve (HRM) analysis, performed immediately at the end of a real-time PCR, is able to yield a higher resolution plot compared with SYBR Green I PCR. HRM dyes completely saturate all amplicons without showing preferential bindings, making the results more clear and distinct. In this research, a multiplex real-time PCR targeting the invA, fimA and stn genes were developed to efficiently detect Salmonella in foods. Furthermore, HRM analysis is sensitive to any single mutation in PCR products, thus it was also applied in this study to distinguish E. coli O157 from other serogroups of E. coli by targeting the uidA gene. The specificity of primers used in this study was checked using many different strains. Results of artificially contaminated foods presented a high sensitivity of the HRM detection methods. Due to its low cost, simplicity of the approach and rapidness, HRM technology is highly competitive with relaxed-condition PCR and probe-based PCR. Besides, an HRM assay can be performed on generic real-time PCR instrumentations found in many laboratories. In conclusion, HRM-based PCR assay are proved to be efficient methods in foodborne pathogen detections.

scholarly journals High resolution melting analysis for the differentiation of Mycobacterium species

2014 ◽  
Vol 63 (10) ◽  
pp. 1284-1287 ◽  
Author(s):  
Rahizan Issa ◽  
Hatijah Abdul ◽  
Siti Hasmah Hashim ◽  
Valentinus H. Seradja ◽  
Nurul ‘Aishah Shaili ◽  
...  
Keyword(s):  
High Resolution ◽  
16S Rrna ◽  
Real Time ◽  
Real Time Pcr ◽  
High Resolution Melting ◽  
Rrna Gene ◽  
Hrm Analysis ◽  
Suitable Target ◽  
Effective Diagnosis ◽  
Dna Template

A quantitative real-time PCR (qPCR) followed by high resolution melting (HRM) analysis was developed for the differentiation of Mycobacterium species. Rapid differentiation of Mycobacterium species is necessary for the effective diagnosis and management of tuberculosis. In this study, the 16S rRNA gene was tested as the target since this has been identified as a suitable target for the identification of mycobacteria species. During the temperature gradient and primer optimization process, the melting peak (Tm) analysis was determined at a concentration of 50 ng DNA template and 0.3, 0.4 and 0.5 µM primer. The qPCR assay for the detection of other mycobacterial species was done at the Tm and primer concentration of 62 °C and 0.4 µM, respectively. The HRM analysis generated cluster patterns that were specific and sensitive to distinguished small sequence differences of the Mycobacterium species. This study suggests that the 16S rRNA-based real-time PCR followed by HRM analysis produced unique cluster patterns for species of Mycobacterium and could differentiate the closely related mycobacteria species.

scholarly journals Rapid and specific detection of porcine parvovirus using real-time PCR and High Resolution Melting (HRM) analysis

2015 ◽  
Vol 11 (1) ◽  
pp. 46 ◽  
Author(s):  
Hai-Qiong Yu ◽  
Xian-Quan Cai ◽  
Zhi-Xiong Lin ◽  
Xiang-Li Li ◽  
Qiao-Yun Yue ◽  
...  
Keyword(s):  
High Resolution ◽  
Real Time ◽  
Real Time Pcr ◽  
High Resolution Melting ◽  
Porcine Parvovirus ◽  
Specific Detection ◽  
Hrm Analysis
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