Validation of a Non-Specific Dye Real-Time PCR Assay for Porcine Adulteration in Meatball Using ND5 Primer

https://doi.org/10.22146/ijc.22646

Tri Joko Raharjo(1*), Ery Nourika Alfiraza(2), Esti Enjelina(3), Deni Pranowo(4),

(1) Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Gadjah Mada, Sekip Utara, PO BOX BLS 21, Yogyakarta 55281
(2) Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Gadjah Mada, Sekip Utara, PO BOX BLS 21, Yogyakarta 55281
(3) Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Gadjah Mada, Sekip Utara, PO BOX BLS 21, Yogyakarta 55281
(4) Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Gadjah Mada, Sekip Utara, PO BOX BLS 21, Yogyakarta 55281
(*) Corresponding Author

Abstract


Porcine adulteration in meatball samples were analyzed using real-time polymerase chain reaction (RT-PCR), based on the ND5 primer obtained by previous study. This work consisted of three stages which were annealing temperature optimization, method validation, and application. DNA template was extracted using phenol-CIAA (chloroform-iso amyl alcohol) method. The optimum annealing temperature for ND5 primers (forward primer 5'-CATTCGCCTCACTCACATTAACC-3' and reverse primer 5'-AAGAGAGAGTTCTACGGTCTGTAG-3') was 58.0 °C, obtained after testing annealing at 50.5 to 59.5 °C gradient temperature with 5 °C interval. Melting curve analysis was done at 65.0 to 95.0 °C, with increasing temperature for 0.5 °C per 2 sec. Method was validated for its specificity, precision and limit of detection. RT-PCR method with ND5 primers produced 227 bp DNA fragment with 78.50 °C Tm value. From eight commercial meatball samples, one was detected containing porcine. The methods showed high specificity and precision, with experimentally determined limits for porcine were no less than 1%.


Keywords


meatballs; porcine DNA; method validation; ND5 primer; real-time PCR

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DOI: https://doi.org/10.22146/ijc.22646

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