Development of a Duplex qPCR Assay for Detecting Porcine Adulteration in Gudeg Krecek: A Complex Traditional Indonesian Food Matrix

Ensuring halal authenticity in traditional bovine hide-based products, such as Krecek (rambakcracker in Gudeg), is challenging because of the visual similarity between bovine hides andporcine skin and the severe DNA degradation caused by the intensive processing (fryingand boiling in coconut milk) of these products. This study developed and validated a DuplexSYBR Green qPCR assay targeting the porcine-specific mitochondrial ND4L gene andeukaryotic 18S rRNA gene as an internal amplification control (IAC). Optimization of theprimer concentrations revealed that a 2:1 ratio (ND4L:18S rRNA) provided the most balancedamplification of the target genes. The assay demonstrated high specificity, distinguishing theporcine target from the IAC by distinct melting temperatures (Tm): ND4L at 78.0°C and 18SrRNA at 85.0°C. The method was validated with a limit of detection (LOD) of 500 pg/μL, aCV of 0.60%, and specificity against 18 non-target species. An analysis of 42 commercialKrecek samples (labelled as beef) collected from traditional Gudeg vendors in Yogyakartashowed no porcine-specific melting peaks. The 100% IAC melting peak (Tm 85.0°C) wasdetected in all samples, confirming the successful recovery of amplifiable DNA despite thecomplex sample matrix. These results confirm the absence of porcine adulteration in thesurveyed products and establish the proposed duplex qPCR as a robust tool for routine halalverification of highly processed, high-fat food products.

• Publication year

  2026

• Venue

  Buletin Peternakan

• Publication date

  2026-02-28

• Fields of study

  Not labeled

• Identifiers
  DOI 10.21059/buletinpeternak.v50i1.114431
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar

• No claims are published for this paper.

• No concepts are published for this paper.

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