scholarly journals Detection of DNA pork in processed meat products with real-time polymerase chain reaction

Food Research ◽  
2020 ◽  
Vol 4 (5) ◽  
pp. 1719-1725
Author(s):  
W.P. Rahayu ◽  
A.R.W. Dianti ◽  
Nurjanah S. ◽  
N. Pusparini ◽  
W. Adhi
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Melting Curve ◽  
Meat Products ◽  
Processed Meat ◽  
Cyt B ◽  
Chain Reaction ◽  
Ct Value ◽  
Polymerase Chain ◽  
Cyt B Gene

Real-time polymerase chain reaction (qPCR) technique is a suitable method for identifying animal species in processed meat because of its ability to amplify a few fragments of DNA. A specific fragment of pork (mitochondrial cytochrome b gene (cytb)) was used as a DNA ladder. This study aimed to evaluate the use of a cyt-b gene generated primer for detecting the presence of pork in processed meat products by qPCR and determining the threshold cycle cut-off. The evaluation of the primer effectiveness was performed by threshold cycle (Ct) value, amplicon size by electrophoresis and melting curve. Two corned (A, B) and two jerkies (C, D) collected from the market were used as the sample. Genomic DNA from samples, fresh beef (as negative control) and fresh pork (as positive control) were extracted using Qiagen Kits. Amplification condition for 50 cycles of the cyt-b gene was performed as the initial step at 95°C for 10 mins, denaturation step at 95°C for 15 s, annealing step at 55°C for 60 s, extension step at 72°C for 30 s and post-PCR at 72°C for 3 mins. The threshold cycle (Ct) cut-off less than 30 confirmed as pork positive. The result obtained indicated that sample A and D were pork negative, with Ct value respectively 40.73 and 43.59. Melting temperatures of amplicon were ranged from 79.5 to 80.5°C, only differed by 1°C, and the amplicon electrophoresis resulting in a single band of the same size (149 bp). Hence, the melting curve analysis and electrophoresis of PCR products were not able to differentiate between pork and beef.

scholarly journals REAL TIME POLYMERASE CHAIN REACTION (RT-PCR) FOR DNA PIG CONTAMINATION IDENTIFICATION SAUSAGE IN SIX DISTRICT PANDEGLANG BANTEN

2021 ◽  
Vol 1 (1) ◽  
pp. 81-88
Author(s):  
Hadi Susilo
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Meat Product ◽  
Meat Products ◽  
Pcr Analysis ◽  
Processed Meat ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Dna Purity ◽  
Polymerase Chain

Sausage is a meat product processed that is popular food especially in Pandeglang, Banten Province. The importance of halal certificates or the existence of the MUI (Indonesian Ulama Council) halal logo for processed meat products makes Muslim people confident to consume them. The aim this research was to identify pig DNA contamination in sausage products in six  districts in Pandeglang without the MUI halal labels using RT-PCR (Real Time-Polymerase Chain Reaction). RT PCR that can calculate to pig to fill these sample free from pig contamination. This research was divided into two stage, the first stage is extracted or carried out DNA and the second stage is RT PCR analysis. The results of the DNA purity test on sausage samples had DNA purity values ​​of 1.84-1.9 (A260 / A280) and resulted in sample concentrations ranging from 37.8 to 102.5 ng / µl.  The only amplification on the FAM curve was in the positive control pig.  the Cq value ranges from 30 - 31.29. The results of RT PCR on sausage samples in the district area in Pandeglang Banten did not detect the presence of pig DNA.

scholarly journals Genotyping of Benzimidazole-Resistant and Dicarboximide-Resistant Mutations in Botrytis cinerea Using Real-Time Polymerase Chain Reaction Assays

2008 ◽  
Vol 98 (4) ◽  
pp. 397-404 ◽  
Author(s):  
Shinpei Banno ◽  
Fumiyasu Fukumori ◽  
Akihiko Ichiishi ◽  
Kiyotsugu Okada ◽  
Hidetoshi Uekusa ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Botrytis Cinerea ◽  
Melting Curve ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cross Resistance ◽  
Melting Curve Analysis ◽  
Chain Reaction ◽  
Polymerase Chain

Botrytis cinerea, an economically important gray mold pathogen, frequently exhibits multiple fungicide resistance. A fluorescence resonance energy transfer-based real-time polymerase chain reaction assay has been developed to detect benzimidazole- and dicarboximide-resistant mutations. Three benzimidazole-resistant mutations—198Glu to Ala (E198A), F200Y, and E198K—in β-tubulin BenA were detected using a single set of fluorescence-labeled sensor and anchor probes by melting curve analysis. Similarly, three dicarboximide-resistant mutations—I365S, V368F plus Q369H, and Q369P—in the histidine kinase BcOS1 were successfully distinguished. Unassigned melting profiles in BenA genotyping assay resulted in the identification of a new benzimidazole-resistant BenA E198V mutation. This mutation conferred resistance to carbendazim as do E198A and E198K mutations. The isolates with BenA E198V mutation showed a negative cross-resistance to diethofencarb, but to a lesser extent than the E198A mutants. A survey of 210 B. cinerea field isolates revealed that most of benzimidazole-resistant isolates possessed the E198V or E198A mutation in the BenA gene, and the I365S mutation in the BcOS1 gene was also frequently observed in Japanese isolates. However, benzimidazole-resistant isolates with BenA F200Y or E198K mutations, which confer the diethofencarb-insensitive phenotype, were rare. Our BenA and BcOS1 genotyping is a rapid and reliable method that is suitable for monitoring the fungicide-resistant field population.

scholarly journals Collaborative Trial for Validation of a Real-Time Reverse Transcriptase-Polymerase Chain Reaction Assay for Detection of Central Nervous System Tissues as Bovine Spongiform Encephalopathy RiskMaterial: Part 1

2006 ◽  
Vol 89 (5) ◽  
pp. 1335-1340
Author(s):  
Amir Abdulmawjood ◽  
Holger Schnenbrcher ◽  
Michael BÜlte
Keyword(s):  
Central Nervous System ◽  
Polymerase Chain Reaction ◽  
Real Time ◽  
Meat Products ◽  
Spongiform Encephalopathy ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Heat Treated ◽  
Polymerase Chain

Abstract A collaborative trial was conducted to evaluate a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay for detection of central nervous system (CNS) tissues in meat products (e.g., sausages). The method is based on the detection of ruminant glial fibrillary acidic protein (GFAP) mRNA by applying real-time RT-PCR. The assay was evaluated through a multicenter trial involving 12 participating laboratories that received coded cDNA obtained from 3 different types of sausages. The participants used 5 different real-time detection systems. The results obtained in this validation revealed that this real-time RT-PCR assay performed well in the different laboratories with a detection limit of at least 0.1% CNS in those test materials that contained strongly heat-treated samples (sausages cooked at 120C) and the medium heat-treated samples (sausages cooked at 80C). The detection limit of liver sausages was determined to be 0.2% of CNS. Neither the samples with no CNS additive nor the bovine DNA and the negative control containing 100% swine brain gave any positive signals. The presented results indicate that the real-time RT-PCR assay was just as reproducible between laboratories, as repeatable within a laboratory, could reliably be used for detection of bovine spongiform encephalopathy risk material in meat and meat products, and signify that it may be used with confidence in any laboratory.

scholarly journals Designing a time-effective TaqMan probe-based real-time polymerase chain reaction protocol for the identification of Yersinia enterocolitica in raw pork meat

2017 ◽  
Vol 86 (4) ◽  
pp. 317-323
Author(s):  
Milena Alicja Stachelska
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Yersinia Enterocolitica ◽  
Meat Products ◽  
Taqman Probe ◽  
Pork Meat ◽  
Chain Reaction ◽  
Microbial Risk ◽  
Polymerase Chain

The aim of this study was to design a time-effective method comprising a short pre-enrichment step in a non-selective broth in combination with the TaqMan probe applied in the real-time polymerase chain reaction to detectYersinia enterocoliticastrains in raw pork meat. The method enabled to detect 1 colony forming unit per 25 mg ofYersinia enterocoliticain pork meat. The specificity and reliability of the method was not diminished by the company of microflora naturally present in meat. The method was found successful to detect pathogenicYersinia enterocoliticastrains in pork meat. It is advised to be used for assessing the microbial risk and for controlling the microbial quality of meat and meat products.

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