Abstract
Background:
Indonesia, home to the world’s largest Muslim population, enforces Law No. 33 of 2014, mandating halal certification for all products in the country. However, meatballs, a popular Indonesian dish, frequently fall victim to adulteration with non-halal meats, such as pork and wild boar due to economic incentives.

Aim:
This study aims to develop an analytical technique employing real-time polymerase chain reaction (PCR) with specific primers to detect pork and wild boar meat within beef meatballs.

Method:
The research commenced with the design of specific primers for pork and wild boar DNA via IDT software. Subsequent phases encompassed DNA isolation, specificity, linearity, limit of detection, efficiency, and repeatability assessments to evaluate the method.

Results:
The D-Loop 539 and D-Loop 409 primers successfully detected the pork and wild boar DNA in both raw meat and meatballs, with optimal annealing temperatures of 61.2°C and 60.6°C, respectively. The D-loop 539 primer amplified pork DNA and pork-infused meatballs, boasting a detection threshold of 0.1 ng, with a coefficient of variation (CV) of 0.25%. Similarly, the D-loop 409 primer successfully amplified wild boar DNA and meatball sample, showcasing detection limits of 1 and 0.1 ng, accompanied by CV values of 0.37% and 0.44%. All primers passed the critical PCR validation tests, making them suitable for analysis of meatball samples across 12 sub-districts in Sleman-Yogyakarta. The observed results showed negative amplification signals for both pork and wild boar components.

Conclusion:
Using the D-Loop 539 and D-Loop 409 primers, this method is capable to detect the presence of pork and wild boar meat within beef meatballs This research contributes to the authentication of halal products in alignment with the provisions of Law No. 33 of 2014, thereby endorsing consumer confidence in product integrity.

Key words: D-loop mitochondria, Real-time PCR, Halal, Adulteration, Meatballs