The Use of Primer-Specific Targeting on Mitochondrial Cytochrome b Combined with Real-Time Polymerase Chain Reaction for the Analysis of Dog Meat in Meatballs

Manalu, Henny Yulisa; Sismindari, Sismindari; Rohman, Abdul

doi:10.21315/tlsr2019.30.3.1

Cited by 6 publications

(5 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Therefore, it could be stated that the LoD value of real-time PCR for analysis of dog DNA was 5 pg. Manalu et al [26] reported that Real-PCR using primers targeting on Cyt-b gene primer could detect the presence of DNA at a concentration as low as 0.25 ng/mL, corresponding to 1% of dog meat in beef meatballs. Rahman et al [4] reported LoD values of 0.02 ng dog DNA corresponding to 0.1% of dog meat in meatball samples, and this LoD value was similar to that reported by Ali et al [25] in frankfurters using chicken and beef meat.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…Rahman et al [4] have used primers targeting the cytochrome b gene (Cyt-b) in meatball samples. Primers targeting on Cyt-b gene were used for the analysis of DNA dog meat in commercial frankfurters [25] and in meatball samples [26]. However, the application of PCR using primers targeting mitochondrial cytochrome c oxidase subunit 1 (COI) was limited.…”

Section: ■ Introductionmentioning

confidence: 99%

See 1 more Smart Citation

The Use of Real-Time Polymerase Chain Reaction Combined with Specific-Species Primer for Analysis of Dog Meat DNA in Meatball

et al. 2020

Self Cite

View full text Add to dashboard Cite

The presence of dog meat is a crucial issue because dog meat is non-halal meat for Muslims. The objective of this study was to design and validate species-specific primer for the identification of dog meat DNA in meatball using real-time polymerase chain reaction (real-time PCR). The specific primer targeting mitochondrial cytochrome c oxidase subunit 1 (CO1) was validated. The specific primers used were designed using Integrated DNA Technologies (IDT) software and subjected to NCBI BLAST procedure. The candidate primers were tested for specificity study using several DNAs from fresh meat of pork, chicken, beef, lamb, and rat. The method was also validated by determining several parameters of linearity, sensitivity, precision, and efficiency. The results showed that primer could amplify specifically DNA target at an optimized annealing temperature of 56.6 °C. The limit of detection (LoD) obtained was 5 ng DNA, corresponding to 2.5% of dog meat in a meatball. The repeatability evaluation, expressed with relative standard deviation (RSD), and efficiency value was in the acceptable range (RSD < 25% and efficiency (90–105%). This method was successfully used for the analysis of marketed samples. Real-time PCR can be used as a standard method in halal authentication analysis through DNA analysis.

show abstract

Section: ■ Results and Discussionmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

The Use of Real-Time Polymerase Chain Reaction Combined with Specific-Species Primer for Analysis of Dog Meat DNA in Meatball

et al. 2020

Self Cite

View full text Add to dashboard Cite

show abstract

“…In addition, many molecular-based analysis methods can be used because of the fast process and results in research, one of which is the DNA-based method which has been widely developed, such as fingerprinting using PCR, multiplex PCR assay, and real-time PCR; this method has been widely used for several last decades ( Manalu and Rohman, 2019 ). The results of DNA amplification of the chuA gene of the invasive enterohemorrhagic species E. coli 0157:H7 using qPCR can be used as an analysis to detect the presence of DNA other than in horse feces.…”

Section: Discussionmentioning

confidence: 99%

Detection of the chuA gene encoding the invasive enterohemorrhagic species Escherichia coli 0157:H7 using qPCR in horse feces samples on Sumbawa Island, Indonesia

Kholik,

Sukri,

Riwu

et al. 2024

Open Vet J

View full text Add to dashboard Cite

Background: Bacterial identification can be done using various testing techniques. Molecular techniques are often used to research dangerous diseases, an approach using genetic information on the pathogenic agent. The enterohemorrhagic invasive species Escherichia coli 0157:H7 was identified from the feces of working horses on the island of Sumbawa. Another advance in molecular technology is genome amplification with qPCR which is the gold standard for detecting E. coli. Aim: This study aims to detect and identify the invasive species Escherichia coli 0157:H7 using the gene encoding chuA with the qPCR method sourced from horse feces. Methods: Fresh fecal samples from horses on Sumbawa Island were isolated and identified, then continued with molecular examination using the gene encoding chuA using the qPCR method. Results: qPCR testing in this study showed that 6 sample isolates that were positive for Escherichia coli 0157:H7 were detected for the presence of the chuA gene, which is a gene coding for an invasive species of E. coli bacteria. The highest to lowest Cq values and Tm from the qPCR results of the sample isolates were 15.98 (4KJ), 14.90 (19KG), 14.6 (3KJ), 13.77 (20KG), 12.56 (5KGB), 12.20 (6KJ). Tm values are 86.7 (4KJ), 86.69 (3KJ), 86.56 (5KGB), 85.88 (20KGB), 85.81 (19KG), 85.74 (6KJ). Conclusion: Validation, standardization of the development, and modification of qPCR technology must be carried out to harmonize testing throughout to avoid wrong interpretation of the test results so that the determination of actions to eradicate and control diseases originating from animals in the field does not occur.

show abstract

“…Previous research has used a variety of primer sources of DNA extracted from meat samples. The species-specific primers (SSPs) focusing on Mitochondrial Cytochrome b have been applied in the studies for the identification of CMballs [ 24 ], dog meatballs [ 4 ], and wild boar meatballs [ 25 , 26 ], respectively. The use of SSP that targets D-loop mitochondria for authentication of wild boar in meatballs was done by Arini et al [ 25 ].…”

Section: Introductionmentioning

confidence: 99%

The employment of real-time polymerase chain reaction for analysis of canine meat in meatball products for halal authentication analysis

Rumiyati,

Arini,

Purwanto

et al. 2024

J Adv Vet Anim Res

View full text Add to dashboard Cite

Objective: Meatballs are a popular meat-based food consumed widely in Indonesian society. However, the issue of unethical substitution of halal meatballs with non-halal meats, particularly pork and canine meat (CM), has emerged. The existence of non-halal meats, including CM, in food products is prohibited in Islam, necessitating the development of reliable analytical techniques for their identification. In this study, we designed species-specific primers (SSPs) targeting the D-loop region of mitochondrial DNA for CM meatball product identification. Materials and Methods: The study was commenced by creating specific primers for canine DNA using Integrated DNA Technologies software and subsequently performing DNA isolation. The designed primers were then subjected to comprehensive evaluation using RT-PCR, including spec¬ification, linearity, limit of detection, efficiency, and repeatability. Results: The results indicated that the primer D-Loop 443 (forward: 5¢-GGG ACA TCT CGA TGG ACTA ATG-3’, reverse: 5’-GCG GTC ATA GAT GAG TGA TAG C-3’) designed and validated in silico using primer-basic local alignment search tool nucleotide (BLAST) program from NCBI accurately identified canine DNA when the optimal annealing temperature was set at 57.5oC. The real-time PCR technique utilizing the D-loop 443 primer exhibited the ability to amplify canine DNA down to a minimum quantity of 100 pg, with an efficiency value of 91.8%, a correlation coefficient (R) of 0.990, and a precision value (RSD) of 0.30%. Conclusion: The SSP-based RT-PCR method developed is a versatile and efficient tool for detect¬ing CM in meatballs. Its implementation helps maintain consumer trust and addresses concerns regarding the substitution of halal meats with non-halal alternatives.

show abstract

The Use of Primer-Specific Targeting on Mitochondrial Cytochrome b Combined with Real-Time Polymerase Chain Reaction for the Analysis of Dog Meat in Meatballs

Cited by 6 publications

References 25 publications

The Use of Real-Time Polymerase Chain Reaction Combined with Specific-Species Primer for Analysis of Dog Meat DNA in Meatball

The Use of Real-Time Polymerase Chain Reaction Combined with Specific-Species Primer for Analysis of Dog Meat DNA in Meatball

Detection of the chuA gene encoding the invasive enterohemorrhagic species Escherichia coli 0157:H7 using qPCR in horse feces samples on Sumbawa Island, Indonesia

The employment of real-time polymerase chain reaction for analysis of canine meat in meatball products for halal authentication analysis

Contact Info

Product

Resources

About