A Combined Rapid DNA Extraction and Multiplex PCR for the Detection of Porcine DNA in Raw and Processed Food

Ahmed, Siti Aminah; Pin, Lee Li; Raabe, Carsten A.; Rozhdestvensky, Timofey S.; Hock, Tang Thean

doi:10.1007/978-981-10-1452-9_18

Cited by 2 publications

(2 citation statements)

References 15 publications

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“…Metode analisis berbasis DNA memiliki beberapa keuntungan, yaitu sifat DNA yang tahan terhadap panas sehingga memungkinkan untuk menganalisis produk makanan yang dimasak dengan suhu tinggi dan DNA merupakan molekul yang stabil pada proses ekstraksi (Piskata et al, 2019). Selain itu, DNA dapat ditemukan pada semua tipe sel dengan informasi genetik yang lestari sehingga analisis DNA sangat mungkin dikerjakan dari beberapa tipe sampel yang berbeda (Ahmed et al, 2017).…”

Section: Pendahuluanunclassified

Biosensor DNA Elektrokimia untuk Deteksi Makanan Mengandung Babi (Sus scrofa) Menggunakan Screen Printed Carbon Electrode Termodifikasi Emas

et al. 2022

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Metode deteksi berbasis deoxyribonucleic acid (DNA) merupakan metode yang paling akurat, spesifik, dan sensitif untuk mengidentifikasi adanya campuran komponen babi dalam produk pangan. Penelitian ini bertujuan untuk mengembangkan biosensor DNA secara elektrokimia menggunakan Screen Printed Carbon Elektrode termodifikasi emas (SPCE-Au) untuk mendeteksi DNA babi dalam makanan olahan. Permukaan SPCE dimodifikasi dengan emas menggunakan metode adsorbsi pasif, kemudian dikarakterisasi dengan Scanning Electron Microscopy (SEM) dan Differential Pulse Voltammetry (DPV). DNA probe yang spesifik terhadap DNA mitokondria babi diimobilisasi ke permukaan SPCE-Au melalui perantara gugus tiol. Proses hibridisasi DNA probe-DNA komplemen dikarakterisasi menggunakan DPV berdasarkan sinyal oksidasi guanin. Kondisi percobaan dioptimasi menggunakan desain eksperimen Box-Behnken, yaitu konsentrasi DNA probe (0,5; 1,0; 1,5 µg/mL), waktu imobilisasi DNA probe (10, 20, 30 menit), dan waktu hibridisasi DNA probe-DNA komplemen (5, 10, 15 menit). Kondisi optimum digunakan untuk menentukan respons arus oksidasi guanin menggunakan DPV terhadap variasi konsentrasi DNA komplemen. Selanjutnya, biosensor DNA diaplikasikan terhadap sampel bakso yang mengandung campuran daging babi, ayam, dan sapi. Hasil penelitian menunjukkan modifikasi SPCE dengan Au menghasilkan peningkatan arus yang diuji menggunakan sistem redoks K3[Fe(CN)6] secara DPV. Kondisi optimum percobaan adalah: konsentrasi DNA probe 1 µg/mL, waktu imobilisasi DNA probe 20 menit, dan waktu hibridisasi DNA probe-DNA komplemen 10 menit. Biosensor DNA ini memiliki batas deteksi 0,31 µg/mL, batas kuantifikasi 1,06 µg/mL dan recovery 99,2% untuk rentang konsentrasi 0,1 - 2 µg/mL. Deteksi sampel bakso menggunakan biosensor DNA menunjukkan peningkatan respons arus yang signifikan pada sampel yang mengandung 100% daging babi (3,417 µA) dan masih dapat mendeteksi adanya kandungan daging babi sampai 1%. Metoda biosensor DNA babi menggunakan SPCE-Au tanpa indikator dan biokonjugat yang dikembangkan lebih sederhana dan cepat dan mudah untuk diaplikasikan ke sampel nyata. Electrochemical DNA Biosensor for Detection of Pork (Sus scrofa) Using Gold Modified Screen Printed Carbon Electrode. The DNA-based detection method is the most accurate, specific, and sensitive method for identifying the presence of a mixture of pork components in food products. This study aims to develop an electrochemical DNA biosensor using a gold-modified Screen Printed Carbon Electrode (SPCE-Au) to detect pork DNA in processed food. The surface of the SPCE was modified with gold using a passive adsorption method, then characterized by scanning electron microscopy (SEM) and differential pulse voltammetry (DPV). The probe DNA specific to porcine mitochondrial DNA was immobilized to SPCE-Au surface via an intermediate thiol group. The hybridization of probe DNA-complement DNA was characterized using DPV based on the guanine oxidation signal. The experimental conditions were optimized using the Box-Behnken experimental design by applying probe DNA concentration (0.5; 1.0; 1.5 µg/mL), the immobilization of the probe DNA (10, 20, 30 minutes), and the hybridization of the probe DNA-complement DNA (5, 10, 15 minutes). The optimum conditions were used to determine the DPV current response vs. complementary DNA concentrations. Furthermore, the DNA biosensors were applied to meatball samples containing a mixture of pork, chicken, and beef. The results showed that modification of SPCE with Au produced an increase in current responses tested using K3[Fe(CN)6] redox system by DPV. The optimum conditions of the experiment were: probe DNA concentration was 1 µg/mL, time to immobilize probe DNA was 20 minutes, and time to hybridize probe DNA-complement DNA was 10 minutes. The limit of detection, limit of quantification and percent recovery of DNA biosensor was 0.31 µg/mL, 1.06 µg/mL and 99.2%, respectively. Detection of meatball samples by DNA biosensor showed a significant increase of current response for sample that contains 100% swine DNA (3.417 µA). The biosensor is still able to detect the pork content until 1%. The pork DNA biosensor using SPCE-Au without indicator and bioconjugate developed in this study is simpler and applicable to real samples.

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Section: Pendahuluanunclassified

Biosensor DNA Elektrokimia untuk Deteksi Makanan Mengandung Babi (Sus scrofa) Menggunakan Screen Printed Carbon Electrode Termodifikasi Emas

et al. 2022

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“…This concern has led to the importance of the development of an analytical method that would be able to identify different meat types in food products. Presently, there are various analytical methods available including the microscopic methods (Balizs et al 2011), lipid-based methods with analysis of TAG and 2-MAG (Szabo et al 2007), Fourier transform infrared (FTIR) spectroscopy (Rohman et al 2011), proteinbased methods (ELISA) (Giovannacci et al 2004), and DNAbased methods (polymerase chain reaction/PCR) (Ali et al 2012;Erwanto et al 2011;Maryam et al 2016;Ahmed et al 2017). DNA-based analysis methods are advantageous given the stability of DNA reaction against heat, allowing the analysis of processed food products under high temperature.…”

Section: Introductionmentioning

confidence: 99%

A Gold Nanoparticle–DNA Bioconjugate–Based Electrochemical Biosensor for Detection of Sus scrofa mtDNA in Raw and Processed Meat

et al. 2019

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The presence of pork content in food products can be a serious issue over an adulteration of food, particularly for consumers who pay attention to halal certification or other religious beliefs concerning the consumption of pork. Due to this fact, finding a method for the detection of pork content in food products that is sensitive, fast, and accurate is of interest. In this study, an electrochemical DNA biosensor with gold nanoparticle-DNA probe bioconjugates was developed to detect Sus scrofa mitochondrial DNA (mtDNA) using a gold-modified screen-printed carbon electrode (SPCE-Gold). The bioconjugates were formed by attaching the DNA probe with gold nanoparticles (AuNPs). Detection was carried out based on the optimum conditions resulting from the Box-Behnken experimental design that were 40 μL of 153 μg/mL bioconjugates; bioconjugate immobilization time, 20 min; and DNA hybridization time, 60 min. The hybridization of target synthetic DNA to the DNA probe was characterized by voltammetry based on the methylene blue indicator signal at a potential of about − 0.35 V. The results showed that the detection limit was 0.58 μg/mL and the recovery was 101.74%. mtDNA samples from various raw and processed meat were isolated and cut with restriction enzyme Sal1. Hybridization of mtDNA samples to the gold nanoparticle-DNA probe bioconjugates anchored to the SPCE-Gold surface was characterized based on the current response generated by methylene blue as an indicator. The result shows that there is an increasing trend in the response of raw and processed meat samples containing pork DNA. The development of an electrochemical DNA biosensor, based on gold nanoparticle-DNA probe bioconjugates, has not been reported previously. This biosensor is selective towards 10% of the pork DNA content in the mixture. Therefore, this biosensor method can be used as an alternative method to distinguish samples containing pork, according to the detection limit.

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A Combined Rapid DNA Extraction and Multiplex PCR for the Detection of Porcine DNA in Raw and Processed Food

Cited by 2 publications

References 15 publications

Biosensor DNA Elektrokimia untuk Deteksi Makanan Mengandung Babi (Sus scrofa) Menggunakan Screen Printed Carbon Electrode Termodifikasi Emas

Biosensor DNA Elektrokimia untuk Deteksi Makanan Mengandung Babi (Sus scrofa) Menggunakan Screen Printed Carbon Electrode Termodifikasi Emas

A Gold Nanoparticle–DNA Bioconjugate–Based Electrochemical Biosensor for Detection of Sus scrofa mtDNA in Raw and Processed Meat

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