DNA extraction is needed in the analysis using the Loop-mediated isothermal amplification (LAMP) method because this method identifies nucleic acids. Some extraction methods that can be selected including commercial kits extraction method and phenol-chloroform extraction method. The purpose of this study was to obtain the best quality DNA extract between the two extraction methods. The DNA extraction process produced DNA concentrations between 31.06 - 410.18 ng / ml for the commercial kit DNA extract and 212.60 - 1502.30 ng / ml for the phenol-choroform DNA extract, while the purity of DNA were 1.82-2.02 for commercial kit DNA extract and 1.93-2.02 for phenol-chloroform DNA extract. The concentration and purity of extracts produced from both methods meet the requirements for molecular analysis. The purity and visualization results of commercial kit DNA extract are better than those produced from extraction from the phenol-chloroform method. DNA extract obtained from the commercial kit method was chosen to be used in the amplification stage of the method (LAMP).
Dalam teknik deteksi molekuler seperti Loop-Amplification Mediated Polymorphism (LAMP) dan Polymerase Chain Reaction (PCR), pembuatan hulu asam deoksiribonukleat (DNA) sangat penting. Ekstraksi fase cair dan ekstraksi fasa padat merupakan beberapa metode ekstraksi DNA yang tersedia. Tujuan dari penelitian ini adalah untuk mengkarakterisasi metode dan produk ekstraksi DNA berdasarkan kemurnian DNA, visualisasi DNA, konsentrasi DNA, dan waktu pemrosesan metode ekstraksi DNA. Metode ekstraksi yang dievaluasi meliputi metode fenol-kloroform (Metode A) sebagai ekstraksi fasa cair dan metode Ekstraksi Surefood kit (Metode B) sebagai ekstraksi fasa padat. Hasil penelitian menunjukkan bahwa Metode A dapat dilakukan pada sampel dengan konsentrasi DNA sangat rendah berkisar antara 7,00 sampai 9,45 ng / μl dengan kemurnian yang baik (1,80-2,10). Meski tidak menunjukkan DNA isolat band pada gel agarosa 1% dan membutuhkan waktu pemrosesan ± 30 jam. Metode B memiliki performa yang baik dalam mengekstraksi sampel dengan DNA dengan konsentrasi tinggi (49,67 sampai 357,28 ng / μl) dengan kemurnian yang baik (1,93 sampai 2,07). Metode ini menunjukkan band untuk setiap sampel DNA pada gel agarose 1% dan membutuhkan waktu ±1 jam. Kedua metode tersebut dapat digunakan untuk preparasi sampel dalam analisis molekuler termasuk tujuan otentikasi halal.KATA KUNCI: fenol, kloroform, Surefood kit, LAMP DNA EXTRACTION METHOD FOR MOLECULAR DETECTIONABSTRACTIn molecular detection technique such as Loop-Amplification Mediated Polymorphism (LAMP) and Polymerase Chain Reaction (PCR), right upstream preparation of deoxyribonucleic acid (DNA) is very important. Liquid phase extraction and solid phase extraction are some of DNA extraction methods those are available. The purpose of this research was to characterizedthe method and product of DNA extraction based on DNA purity, DNA visualization, DNA concentration, and processing time of DNA extraction methods. Extraction methods evaluated included phenol-chloroform method (Method A)as liquid phase extraction and Surefood Extraction kit method (Method B) as solid phase extraction. Result showed that Method A could be performed on samples with very low DNA concentrations ranging from 7.00 to 9.45 ng/µl with a good purity (1.80 to 2.10). Although, it showed no DNA isolates bands on gel agarose 1% and need ± 30 hours processing time. Method B had a good performa in extracting sample with high concentration DNA (49.67 to 357.28 ng/µl) with a good purity (1.93 to 2.07). This method showed bands for each DNA samples on gel agarose 1% and need about ± 1 hour processing time. Both methods can be used for sample preparation in molecular analysis including halal authentication purposes.
Designing a simple, sensitive, and specific method to detect pork contamination in the food industry remains a major challenge. In the current study, we developed a sensitive thiol-bond AuNP-Probe biosensor that will change color when detecting pork DNA in the Cytochrome B region. The interaction of biosensor and DNA sample is measured by spectrophotometer at 540 nm. The biosensor is made by reducing gold with sodium citrate to produce gold nanoparticle with 39.05 nm diameter. The AuNP-Probe biosensor (gold nano probe) achieved 16.04 ng DNA/µl limit of detection and 53.48 ng DNA/µl limit of quantification. The linearity (R 2 ) between color absorbance changes and DNA concentration is 0.9916. The biosensor has a good specificity as it does not cross-react with DNA of chicken and beef. To verify specificity towards the target sequence, qPCR was tested to the target sequence and reacted to the PCR product with the biosensor. The PCR DNA isolate result 2.7 fold higher absorbance compared to pork-DNA isolate alone (without PCR). The sensitivity and specificity of the method show the promising application of the thiol-bond AuNP biosensor in pork-detection.
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