Optimization of Various ITS rDNA Amplification Protocol of Yeast Isolated from Giant Honey Beehives (Apis dorsata)

Indonesia is a country with high variability of microorganisms, including bacteria, yeast, and fungi. Yeast isolates could be isolated from the honeycomb of Apis dorsata. Molecular approaches were used to identify yeast using ribosomal DNA gene sequences, called the ITS gene. The optimum condition for DNA extractions and amplifications are needed for the successfully of molecular identification. Therefore, it is necessary to optimize the DNA extraction and amplification of several protocols to obtain good identification results. This study aimed to compare the effects of DNA extraction with various temperatures and different amplification protocols. LIPI reference DNA extraction protocol with the boiling method and variations in incubation time of 10, 15, and 20 minutes at a temperature of 98° C. Meanwhile, for the amplification of yeast DNA using a variety of different amplification protocols. The results showed the optimal time of incubation was 10 minutes in K1 isolates with DNA purity of 1.896. meanwhile, for isolates K2, K3, and K4 each with a purity of 2.246, 2.335, and 1.748. optimal DNA amplification results were indicated by the presense of DNA bands for each sample K1, K2, K3, and K4, namely 503, 542, 492, and 526 bp. In this study, it can be concluded that the optimal incubation time for the extraction process is 10 minutes. In addition, the optimal amplification protocol was shown in the DNA bands in all sample.

EXTRACTION AND PURITY DNA OF Culex spp MOSQUITO IN KEMELAK VILLAGE, BINDUNG LANGIT, OGAN KOMERING ULU

Culex spp are mosquito vectors that have a very wide distribution capability and are carriers of pathogens that can interfere with human and animal health. The wide distribution makes Culex spp a dangerous threat. DNA extraction is one of the important steps in obtaining genetic information and genetic analysis. Good quality DNA is used for activities such as the use of molecular markers, genome library creation, and sequencing. This study aims to determine the quality, concentration and purity of Culex spp mosquito DNA in Kemelak Bindung Langit Village, OKU Regency. It is hoped that the sample can be used for further research analysis on Mitochondria D-Loop Sequences in Culex spp mosquitoes. Quantitative measurement of DNA in the form of concentration and purity of DNA using Nanodrop Thermo cycle while qualitative DNA using electrophoresis technique. The results of the isolation of the mosquito genome DNA, obtained clear DNA bands without any degradation (smear) and the concentration results for the four samples ranged from 10-100 ng/µL and the DNA purity was good, ranging from 1.8 to 2.00.

REAL TIME POLYMERASE CHAIN REACTION (RT-PCR) FOR DNA PIG CONTAMINATION IDENTIFICATION SAUSAGE IN SIX DISTRICT PANDEGLANG BANTEN

Sausage is a meat product processed that is popular food especially in Pandeglang, Banten Province. The importance of halal certificates or the existence of the MUI (Indonesian Ulama Council) halal logo for processed meat products makes Muslim people confident to consume them. The aim this research was to identify pig DNA contamination in sausage products in six districts in Pandeglang without the MUI halal labels using RT-PCR (Real Time-Polymerase Chain Reaction). RT PCR that can calculate to pig to fill these sample free from pig contamination. This research was divided into two stage, the first stage is extracted or carried out DNA and the second stage is RT PCR analysis. The results of the DNA purity test on sausage samples had DNA purity values ​​of 1.84-1.9 (A260 / A280) and resulted in sample concentrations ranging from 37.8 to 102.5 ng / µl. The only amplification on the FAM curve was in the positive control pig. the Cq value ranges from 30 - 31.29. The results of RT PCR on sausage samples in the district area in Pandeglang Banten did not detect the presence of pig DNA.

Optimasi Metode Ekstraksi DNA pada Melon (Cucumis melo L.) Berdasarkan Suhu, Lama Inkubasi, dan Kondisi Daun

Melon is a fruit commodity that has high economic value and is in demand by the community, so it has the potential to be developed. Therefore it is necessary to study various sciences, one of which is the molecular approach. DNA is an essential element in molecular research. The right extraction technique will determine the quality and quantity of DNA produced. The temperature and incubation time applied in the DNA extraction technique, as well as the quality of the leaves as a source of plant DNA, are among the determining factors for the quality and quantity of extracted DNA, so it is necessary to carry out an assessment and optimization. This study aims to assess the optimal temperature and incubation time in extracting DNA from material (melon leaves) under different conditions. Research activities include planting melon seeds, collecting leaf samples, DNA extraction and quantitative DNA testing. The results showed that the concentration and purity of DNA extracted from cold leaves was higher than that from fresh leaves. The highest DNA concentration was obtained from the 65 ° C incubation treatment for 20 minutes, namely 2707.6 ng / μl, and the highest DNA purity was obtained from the 70 ° C incubation treatment for 10 minutes, namely 1.94 from leaf material that had been cooled overnight at a temperature of -20 ° C . Abstrak Melon merupakan salah satu komoditas buah yang bernilai ekonomi tinggi dan diminati masyarakat, sehingga potensial untuk dikembangkan. Oleh karenanya perlu pengkajian dari berbagai ilmu salah satunya dengan pendekatan molekuler. DNA merupakan unsur yang cukup esensial dalam riset molekuler. Teknik ekstraksi yang tepat sangat menentukan kualitas dan kuantitas DNA yang dihasilkan. Suhu dan lama inkubasi yang diterapkan dalam teknik ekstraksi DNA, serta kualitas daun sebagai sumber DNA tanaman merupakan salah satu faktor penentu kualitas dan kuantitas DNA hasil ekstraksi, sehingga perlu dilakukan pengkajian dan optimasi. Penelitian ini bertujuan untuk mengkaji suhu dan lama inkubasi yang optimal dalam mengekstraksi DNA dari bahan (daun melon) dengan kondisi yang berbeda. Kegiatan penelitian meliputi penanaman benih melon, koleksi sampel daun, ekstraksi DNA dan uji kuantitatif DNA. Hasil penelitian menunjukkan bahwa konsentrasi dan kemurnian DNA hasil ekstraksi dari daun dingin lebih tinggi dibandingkan dari daun segar. Konsentrasi DNA tertinggi diperoleh dari perlakuan inkubasi 65°C selama 20 menit, yaitu 2707.6 ng/μl, dan kemurnian DNA tertinggi diperoleh dari perlakuan inkubasi 70°C selama 10 menit, yaitu 1.94 dari bahan daun yang sudah didinginkan semalaman pada suhu -20°C.

Comparative Evaluation of qnrA, qnrB, and qnrS Genes in Enterobacteriaceae Ciprofloxacin-Resistant Cases, in Swine Units and a Hospital from Western Romania

Excessive use of antimicrobials and inadequate infection control practices has turned antimicrobial resistance (AMR) into a global, public health peril. We studied the expression of qnrA, qnrB, and qnrS plasmid in ciprofloxacin (CIP)-resistant strains of Escherichia coli in swine and humans from Romania, using the Polymerase Chain Reaction (PCR) technique. Antibiotic Susceptibility Testing (AST) for human subjects (H) on 147 samples and 53 swine (S) was ascertained as well as the isolation of bacterial DNA (E. coli) as follows: bacteriolysis, DNA-binding, rinsing, elution, amplification, and nucleic acids’ migration and U.V. visualization stages. From 24 samples of E. coli resistant to CIP collected from H subjects and 15 from S, for PCR analysis, 15 H and 12 S were used, with DNA purity of 1.8. The statistically analyzed results using the Crosstabs function (IBM SPSS Statistics-Ver. 2.1.), revealed the qnrS (417 bp) gene in 13 human subjects (52.0%), as well as in all swine samples studied. The qnrB (526 bp) gene was exposed in 9 of the human patients (36.0%) and in all swine isolates, and the qnrA (516 bp) gene was observed only in 3 of the isolates obtained from human subjects (12.0%) and was not discovered in pigs (p > 0.05). The presence of plasmids qnrA, qnrB, and qnrS in the human samples and of qnrB and qnrS in swine, facilitates the survival of pathogens despite the CIP action. The long-term use of CIP could cause a boost in the prevalence of qnr resistance genes, and resistance in the pigs destined for slaughter, a perturbing fact for public health and the human consumer.

Evaluating the efficiency of ethanol precipitation method in purification of gDNA and PCR product

A numerous clean-up methods of nucleic acid were developed to achieve the requirements of downstream reactions like PCR and sequencing. The methods were varied in their mechanism, efficiency of purification and final product yield. The present study evaluated the efficiency of ethanol-sodium acetate (EOH-NaOAc3) precipitation method in purification of nucleic acid to satisfy downstream reactions requirements. The yield and purity of nucleic acid were considered as the main standard parameters to estimate the efficiency of method. Geneaid gel extraction kit DF100 was considered as a standard method for comparison. The results of methods comparison revealed that EOH-NaOAc3 method was significantly (P=0.000) surpassed the kit method in the yield of purified PCR product (93.24 and 18.37 ng/µl) with no significant differences (P=0.239) in quality (Absorbance (A260/280+) = 1.816 and 1.843) respectively. To determine the productivity of EOH-NaOAc3 method, a specific amount of genomic DNA (G-DNA) (187.93 ng/ µl) was processed and the results showed that EOH-NaOAc3 method was efficiently conserved 89.6% of total processed G-DNA (168.51 ng/µl) accompanied by significant (P=0.03) elevation of DNA purity (A260/280, 3.07 – 2.53).

Detection of Porcine DNA in Processed Beef Products Using Real Time – Polymerase Chain Reaction

Meat is one of food materials which has protein source and mostlyconsumed by non-vegetarian. Consuming halal food is an obligation for every Muslim. Meat processed products usually contaminated by pork. One of technique that is often chosen as an authentication process for proofing halalness of the product is PCR technique, one of PCR technique which most commonly used is RT-PCR. RT-PCR technique was chosen as identification method because it has high accuration for detection of porcine DNA in fresh meat and processed products. RT-PCR is the amplification technique in the specific regions that are restricted by two oligonucleotide with the help of polymerase enzymes. Annealing is the first process of RT-PCR analysis who was primary attachment to the DNA template that determines the specificity and amount of DNA produced. In this study, extraction kit and detection kit were used for analysis Porcine DNA in meatballs. The results obtained from this study were from whole DNA samples, which had DNA purity ranging from 1.82 to 1.93. From the all samples three of them containing porcine DNA. The positive samples shown from amplification curves who was specifically formed when probes reacts with porcine gene.

Improved Protocols of ITS1-Based Metabarcoding and Their Application in the Analysis of Plant-Containing Products

Plants are widely used for food and beverage preparation, most often in the form of complex mixtures of dried and ground parts, such as teas, spices or herbal medicines. Quality control of such products is important due to the potential health risks from the presence of unlabelled components or absence of claimed ones. A promising approach to analyse such products is DNA metabarcoding due to its high resolution and sensitivity. However, this method’s application in food analysis requires several methodology optimizations in DNA extraction, amplification and library preparation. In this study, we present such optimizations. The most important methodological outcomes are the following: 1) the DNA extraction method greatly influences amplification success; 2) the main problem for the application of metabarcoding is DNA purity, not integrity or quantity; and 3) the “non-amplifiable” samples can be amplified with polymerases resistant to inhibitors. Using this optimized workflow, we analysed a broad set of plant products (teas, spices and herbal remedies) using two NGS platforms. The analysis revealed the problem of both the presence of extraneous components and the absence of labelled ones. Notably, for teas, no correlation was found between the price and either the absence of labelled components or presence of unlabelled ones; for spices, a negative correlation was found between the price and presence of unlabelled components.

Purificación y amplificación de ADN genómico en escamas de la serpiente Bothrops atrox “jergón” (Ofidia: Viperidae)

Purificación y amplificación de ADN genómico en escamas de la serpiente Bothrops atrox “jergón” (Ofidia: Viperidae) Genomic DNA purification and amplification from scales to snake Bothrops atrox “lancehead” (Ofidia: Viperidae) Rommel Rojas, Roberson Ramirez, Marianela Cobos y Juan Castro Centro de Investigaciones de Recursos Naturales-CIRNAUniversidad Nacional de la Amazonia Peruana-UNAP. Apartado postal 496S Facultad de Ciencias Biológicas. DOI: https://doi.org/10.33017/RevECIPeru2011.0052/ RESUMEN Los estudios moleculares exigen la creación de nuevos protocolos que permitan obtener un ADN de alta calidad en la mayor cantidad de especies posibles y garantizar el conocimiento de la diversidad genética, por tal motivo el objetivo del presente trabajo fue purificar y amplificar de ADN genómico de las escamas de la serpiente Bothrops atrox “jergón”. Las escamas fueron recolectadas en el campo utilizando la técnica de revelamiento por encuentros casuales [1], para la purificación del ADN genómico se modificó el protocolo de [2], verificándose su pureza por el método electroforético utilizando agarosa 2% y su calidad y concentración por el método espectrofotométrico, la amplificación fue realizada por la reacción en cadena de polimerasaPCR utilizando iniciadores aleatorios. Los resultados muestran la obtención de ADN en excelente estado con un alto ratio de calidad (2) y concentración (585.85 µg/mL), asimismo se logró la amplificación del ADN, demostrándose que el protocolo utilizado es útil para estudios moleculares y genéticos. Se concluye mencionado que el método empleado es efectivo para obtener ADN de buena calidad en escamas de serpientes. Descriptores: Serpiente, escama, ADN, reacción en cadena de polimerasa-PCR. ABSTRACT Molecular research demand the creation of new protocols and permit the obtain a high quality DNA in the most quantity of species and know the genetic diversity, for that objective of the present research was obtained the purification and amplification of scales DNA in the snake Bothrops atrox. the scales were recollected in the field using the visual encounter survey [5] The For the purification of the DNA was modification the protocol [4], the DNA purity was verifiable for the electrophoretic method and it quality and concentration for spectrophotometry, the amplification was carried for Polymerase chain reaction-PCR using random primers. The results show the successful DNA purification, showing an acceptable quality ratio (2) and concentration (585.85 µg/mL), at the same time was made the amplification of DNA for PCR. We conclude indicating the useful of this technique for the purification of high quality DNA from snake´s scales for molecular and genetic research. Keywords: snake, scale, DNA, polymerase chain reaction-PCR.