UV Transilluminator Research Articles

Molecular Fish Sexing on Taisho Sanshoku Koi (Cyprinus carpio) Based on ArS.9-15 Gene Amplification using PCR Method

Taisho Sanshoku is a variant of Koi fish (Cyprinus carpio) that has high demand due to its high economic value and relatively expensive price. This study aimed to determine the sex of the Taisho Sanshoku Koi fish by molecular sexing using the PCR method to amplify the ArS.9-15 gene. This study was initiated by rearing a 4–6 month-old of 10 Taisho Sanshoku Koi fish in a fish tank with a filter and oxygen aeration. The fish were fed with fish pellets for 1–3 days. The Koi fishes were then anesthetized using Koi anesthesia containing β-hydroxyethyl phenyl ether. Each fish's peripheral blood was collected as much as 0.5 mL per fish and then stored in tubes containing Ca-EDTA anticoagulant. The genomic DNA was extracted from peripheral blood samples and used as a template DNA for PCR amplification targeting ArS.9-15 gene. Agarose with 1.5% concentration and CybrSafe staining was used in electrophoresis for visualization of the PCR results then visualized in a dark chamber using a UV transilluminator. The Taisho Sanshoku Koi fish's sex was determined using descriptive analysis based on the electrophoresis results. According to the PCR results, the female Taisho Sanshoku Koi fish only produced one 800 bp DNA band, whereas the male fish produced two 800 bp and 1,100 bp DNA bands. The outcome of molecular fish sexing of the 10 Taisho Sanshoku Koi fish reported that 60% were male and 40% were female.

Development of point-of-need colourimetric, isothermal diagnostic assays for specific detection of Bacillus subtilis using shikimate dehydrogenase gene.

The largest obstacle in the promotion of biopesticides is the existence of counterfeit products available in the market. Identification and quantification of antagonistic organisms in biopesticide products are the key to the reduction of spurious microbial pesticides. In this study, we have developed a simple, sensitive, isothermal-based colourimetric assay for specific detection of Bacillus subtilis from the biopesticide formulations and soil samples. A region specific to B. subtilis which codes for shikimate dehydrogenase was identified through in silico analysis. We employed conventional PCR, loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and qPCR for specific detection of B. subtilis in soil samples and biopesticide formulations. Specificity tests showed that the PCR primers amplified an amplicon of 521bp in four strains of B. subtilis only, and no amplification was found in negative control samples. Similarly, the LAMP assay showed sky blue colour in all four strains of B. subtilis and violet colour in negative control samples. Whereas in the RPA assay, upon the addition of SYBR Green dye, a bright green colour was seen in B. subtilis strains, while a brick-red colour was observed in negative control samples by visualizing under a UV transilluminator. The qPCR assay showed specific amplifications with a Ct value of 12 for B. subtilis strains and no amplification in negative control samples. In the sensitivity test, PCR could amplify DNA of B. subtilis up to 500pg/µL. DNA concentration as low as 10pg/µL was enough to show the colour change in the LAMP as well as the RPA assays, whereas the qPCR assay showed sensitivity till 100pg/µL. All four diagnostic assays developed in the study have been validated in soil samples and B. subtilis-based biopesticides. Compared to conventional PCR, the qPCR assay has the advantage of quantification and visualizing the result in real-time, whereas LAMP and RPA assays have the benefits of being colourimetric and less time-consuming. The other advantages are that the results can be visualized with the naked eye, and these assays do not require a costly thermal cycler and gel documentation system. Hence, LAMP and RPA assays are highly suitable for developing point-of-need diagnostic kits and, in turn, help regulators assess the quality of biopesticides in the market.

Molecular Identification of Ascaridia galli in Chickens from Traditional Markets in Surabaya, Indonesia

Background: Ascaridia galli is pathogenic in chickens and causes substantially reduced growth rates, weight loss, low egg production and death in infected chickens. It is necessary to carry out research on the molecular identification of A. galli in chickens in traditional markets in Surabaya using the Polymerase Chain Reaction (PCR) method with the COX-1 mtDNA gene primer. Methods: This research was carried out from April to July 2023. The number of samples used in this research was 100 chicken small intestines taken from traditional markets in Surabaya, Indonesia. A. galli worms obtained from worm collections were identified according to the morphology of the A. galli worms. DNA extraction was carried out followed by PCR laboratory procedure. The specific primers utilized were designed to target the COX-1 gene and the amplified products were visualized in 1.5% agarose. Result: The A. galli worm has a long body posture. Male A. galli worms measure 30-50 mm long; while, adult female A. galli worms measure 65-80 mm long. The results of electrophoresis with a 2% agarose gel run for 30 minutes at 100 Volts and visualized under ultraviolet light using a UV Transilluminator (Geldoc) showed the presence of a single band of the COX-1 mtDNA gene in the 533 base pair. Infection with A. galli worms often causes reduced growth rates and decreased body weight, which is associated with increased worm weight.

Screening and Evaluation of Biodegradation Potential of Bacterial Isolates Against Ethidium Bromide

Ethidium bromide (EtBr), an intercalating agent that is often employed in molecular biology procedures can bind to the DNA's minor groove, which can result in a variety of undesirable repercussions. EtBr is classified as one of the most lethal carcinogens, which makes its disposal extremely challenging and expensive. Reckless and irresponsible disposal of hazardous items can have severe impacts on the ecosystem and cause the environment's natural resources to wither away. Therefore, our study focuses on the isolation of bacterial isolates from different sources that have biodegradation potential against EtBr. Different bacterial isolates obtained from sewage water, tap water, and soil were grown in Luria Bertani (LB) broth and Nutrient agar (NA), followed by their screening and identification by performing various biochemical tests. All the isolates were grown in two different concentrations of EtBr (i.e., 30 g/ml and 60 g/ml) to determine their ability to degrade EtBr. For the current investigation, bacterial isolates obtained from the tap water (IS1, IS2, IS3, IS4, IS5, IS6) and sewage water (IS7, IS8, IS9, IS10, IS11, IS12, IS13) have shown degrading potential against EtBr at the concentration of 30µg/ml after 2 and 5 days, respectively, whereas, the bacterial isolates obtained from tap water (IS1, IS2, IS3, IS4, IS5, IS6) and sewage water (IS7, IS8, IS9, IS10, IS11, IS12, IS13) have shown degradation potential against EtBr at the concentration of 60µg/ml after 5 days and 8 days, respectively. All the isolates demonstrated EtBr bioaccumulation and were visible as vivid orange colonies under a UV transilluminator. None of the isolates obtained from the soil sample were able to degrade EtBr. The outcomes of the current investigation suggest that several bacterial isolates which were isolated from tap water and sewage water had remarkable biodegradation capacity against EtBr. The unique ability of bacterial isolates to biodegrade and accumulate EtBr can contribute to the improvement of the quality and safety of our environment. Further research into these isolates' potential for biodegrading various xenobiotics and dangerous substances could be very helpful in reducing the environment's rising toxicant concentrations.

Photoactivatable Blue Fluorescent Protein.

Photoactivatable and photoswitchable fluorescent proteins (FPs) are sophisticated molecular tools that in combination with super-resolution microscopy are helping to elucidate many biological processes. Through the Y66H mutation in the chromophore of the violet fluorescent protein SumireF, we created the first photoactivatable blue fluorescent protein (PA-BFP). This protein is rapidly activated over ordinary UV transilluminators at 302 or 365 nm in irreversible mode and by direct exposition to sunlight. The maximum excitation and emission wavelengths of this protein, centered at 358 and 445 nm, respectively, resemble the values of DAPI-the blue stain widely used in fluorescence microscopy to visualize nucleic acids in cells. Therefore, the immediate use of PA-BFP in cellular biology is clear because the technology required to follow this new genetically encoded reporter at the microscopic level has already been established. PA-BFP can potentially be used together with other photoactivatable fluorescent proteins of different colors to label multiple proteins, which can be simultaneously tracked by advanced microscopic techniques.

Modeling of PCR-RFLP genotyping of cattle by polymorphic markers of iNOS gene

Relevance. The study of the Bos taurus iNOS gene polymorphism and its association with resistance to bovine leukemia, as well as with breeding value in terms of milk productivity, is a topical subject of genetic selection research.The purpose of this study was to identify and map polymorphic restriction sites in 4 SNP markers (AH13-1, AH13-2, AH13-3, AH13-4) of the Bos taurus iNOS gene, followed by PCR-RFLP profiling of the encountered genotypes and modeling of the method of gene testing of cattle by the listed polymorphic markers of the analyzed locus.Methods. An effective tool for visualizing the result of computer simulation of the experiment, which contributed to predicting the applicability of the developed method of gene testing, was in silico modeling of the generated PCR-RFLP profiles of the encountered genotypes of polymorphic markers of the Bos taurus iNOS gene. At the same time, the very result of mapping the identified polymorphic restriction sites and subsequent profiling of the encountered genotypes indicated the technical feasibility of detecting polymorphic positions of 4 SNP markers of the Bos taurus iNOS gene by PCR-RFLP analysis. This, in turn, initiated the modeling of the method of gene testing of cattle by the listed polymorphic markers with the provision of successive stages of DNA isolation from whole preserved blood of cattle, PCR with oligonucleotide primers iNOS-F and iNOS-R, RFLP with selected restriction endonucleases (HinfI, Sau96I (AspS9I isoschizomer), HpyAV and MluCI (Sse9I isoschizomer), as well as performing gel electrophoresis detection with visualization of the obtained electrophoregrams in a UV transilluminator.

Association of Alu APO Gene Marker with Type 2 Diabetic patients of Dimapur District, Nagaland India

ABSTRACT: With Type 2 Diabetes, the body either produces insufficient insulin or rejects it, which leads to an accumulation of glucose in the blood. Type 2 diabetes affects 90% of the population.. Examining the relationship between Type 2 diabetes patients from the tribal tribes of Dimapur District and the Alu APO gene marker was the main goal of this study. Using the use of random sampling, blood samples were taken from 31 Type 2 Diabetes Mellitus patients at Eden Medical Centre in Dimapur and 31 control samples were collected from healthy individuals . Depending on the patient availability, blood samples were taken from both male and female patients. Within a day of collecting the blood, DNA was extracted. PCR and an allele-specific marker (APO) were used for genotyping. After genotyping, results were recorded by visualizing the PCR products in UV transilluminator. With this investigation, we discovered that the female exhibits a high statistical significance in the DD genotype (0.00*). On the other hand, neither genotype is statistically significant in males.

Association of Alu APO Gene Marker with Type 2 Diabetic patients of Dimapur District, Nagaland India

ABSTRACT: With Type 2 Diabetes, the body either produces insufficient insulin or rejects it, which leads to an accumulation of glucose in the blood. Type 2 diabetes affects 90% of the population.. Examining the relationship between Type 2 diabetes patients from the tribal tribes of Dimapur District and the Alu APO gene marker was the main goal of this study. Using the use of random sampling, blood samples were taken from 31 Type 2 Diabetes Mellitus patients at Eden Medical Centre in Dimapur and 31 control samples were collected from healthy individuals . Depending on the patient availability, blood samples were taken from both male and female patients. Within a day of collecting the blood, DNA was extracted. PCR and an allele-specific marker (APO) were used for genotyping. After genotyping, results were recorded by visualizing the PCR products in UV transilluminator. With this investigation, we discovered that the female exhibits a high statistical significance in the DD genotype (0.00*). On the other hand, neither genotype is statistically significant in males.

Highly Sensitive Detection of DNA Fragments by an Inexpensive and Simple Method Fabricated In-House Blue LED Transilluminator

Agarose gel electrophoresis is a routinely employed experimental technique for visualizing nucleic acids, typically combined with ethidium bromide or some other molecular dye staining and a UV transilluminator. However, the utilization of a UV transilluminator is restricted due to high cost and safety considerations. In this study, an inexpensive and simple in-house blue Light Emitting Diode (LED) transilluminator was developed using a high-brightness blue LED, carbon resistor, and smartphone charging cable. Its sensitivity for DNA detection was compared with conventional methods. A detection limit of 4 ng/band was confirmed by observing a 100 bp DNA fragment, which is comparable to ethidium bromide staining and UV transilluminator. Additionally, the in-house blue LED transilluminator can be attached to existing electrophoresis chambers, allowing real-time observation of electrophoresis and offering a superior alternative to conventional methods. In conclusion, the developed in-house blue LED transilluminator demonstrates high sensitivity in detecting DNA fragments and holds potential for advancing low-cost research facilities.

Virulence and Molecular Detection of Cassava Mosaic and Brown Streak Virus Genes on Elite Mutant Cassava Lines

Unavailability of germplasm with Cassava Brown Streak Disease (CBSD) and Cassava Mosaic Disease (CMD) tolerance genes remains the main bottleneck towards management of the two diseases in tropic and sub-tropic regions in Africa. We assessed the pathogenicity of two viruses and established the presence of genes linked to CBSD and CMD among some elite cassava lines using field techniques and PCR methods. The screen-house experiment was arranged in split plot in completely randomized block design replicated thrice. Severity data was done on a scale of 1 - 5 at an interval of 14 days post inoculation. Symptomatic fresh leaves were used in the extraction of genomic deoxyribonucleic acid and amplification done using markers linked to African cassava mosaic virus (ACMV), separated bands scored as present (+) or absent (-) and visualized under UV trans-illuminator. Virulence and pathogenicity results showed higher tolerance to CBSD and CMD under single inoculation. In contrast, dual inoculation synergistically increased severity expression leading defoliation at early stages of growth. DNA amplification found no CBSD and CMD alleles in CAS1. However, some traces of the alleles were amplified on CAS2, CAS3, KME3 and KME4. Mutant cassava lines exhibited low severity compared to parental lines which were genetically different in terms of viral genes detected by the ACMV linked markers.

Identification of <i>UFGT</i>-gene-associated groups of <i>Vitis vinifera</i> L. by the developed method of PCR-RFLP genotyping of grape

The Vitis vinifera L. UFGT gene is one of the diagnostically significant genes for genetic testing of technical grape varieties as well as wine materials and wines produced from them. The strategy for genetic identification of grape varieties and DNA authentication of wine materials that was previously developed by us and is based on direct sequencing of the specific PCR product with a length of 99 bp gave an impulse to prognostic assessment of feasibility of PCR-RFLP analysis for detection of five diagnostically significant polymorphic positions and the following identification of 13 UFGT gene-associated groups of Vitis vinifera L. The aim of this work consisted in identification of UFGT gene-associated groups of Vitis vinifera L. by detection of diagnostically significant polymorphic positions using the developed PCR-RFLP method for genotyping of grape. Objects of research were 24 samples of technical grape varieties. Their sample preparation was carried out by extracting 50–100 mg of mature grape pulp or stone with its mechanical comminution in a mortar and transfer to an Eppendorf-type tube. Then, nucleic acids were extracted using a commercial innuPREP Plant DNA Kit or DiamondDNA Plant kit. PCR-RFLP with the extracted grape DNA was performed with Phire Plant Direct PCR Master Mix and four selected restrictases (PstI, BsaXI, BtsIMutI and HinfI) according to the protocols presented in the paper. The detection of the PCR-RFLP fragments was performed by visualization of electropherograms in a UV transilluminator after horizontal electrophoresis in 2.5% agarose gel with stained TAE buffer. The method for PCR-RFLP genotyping of grapes developed specially for identification of UFGT gene-associated groups of Vitis vinifera L. by detecting diagnostically significant polymorphic positions demonstrated its feasibility when testing 24 samples of technical grape varieties. With that, the positive result was achieved due to the practical ability of each of four selected restrictases to discriminate the strictly specified polymorphic position generating characteristic PCR-RFLP profiles of 13 UFGT gene-associated groups of Vitis vinifera L., seven of which were revealed during this study. Therefore, as a result of the performed study, the genotypic affiliation of several tested grape varieties was established: six samples were identified as representatives of the UFGT gene-associated group No.1; one sample was assigned to gene-associated group No.2; two samples were characterized by the trait of associated group No.3; four samples belonged to group No. 4; one sample to group No. 5; six samples to group No.13.

Rapid detection of Staphylococcus aureus in food safety using an RPA-CRISPR-Cas12a assay

At present, the main challenge for Staphylococcus aureus ( S. aureus ) in food safety is still a lack of rapid, convenient detection methods. This study developed a combination of recombinase polymerase amplification (RPA) and the CRISPR-Cas12a technology (known as the RPA-CRISPR-Cas12a assay) for the detection of S. aureus in food. This novel assay could detect samples with a fluorescence reader, lateral flow dipstick (LFD) or the naked eye under a UV transilluminator. After optimization, the RPA-CRISPR-Cas12a assay could specifically and stably detect S. aureus as low as 1–10 copies/μL within 40 min at 37 °C. This assay had high sensitivity to the detection of S. aureus in both laboratory standard samples and field samples, which indicated the feasibility of this assay. To sum up, this accurate, portable detection method for S. aureus has great potential applications in food safety, especially in the field, without requiring sophisticated equipment or high level of technical expertise.

Spirulina carbon dots: a promising biomaterial for photocatalytic textile industry Reactive Red M8B dye degradation.

The colorful reactive dyes are toxic, carcinogenic to living organisms and pollute the water environment. We, for the first time, have studied the lab-scale synthesis of novel and eco-friendly carbon quantum dots (CQDs) from Spirulina platensis by microwave-assisted technology. Fluorescence, absorbance, emission, and excitation spectra of biosynthesized CQDs were recorded by UV transilluminator, UV, and photoluminescence spectrophotometer. Elemental analysis of CQDs were carried out by using energy-dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS). Morphological properties of CQDs were studied by using transmission electron microscope (TEM), scanning electron microscope (SEM), and particle size analyzer. Up to 95.5% of Reactive Red M8B was degraded by CQDs within 6h under sunlight. Dye degradation was facilitated by optimized parameters such as concentration of dyes, catalyst, and pH. Photocatalytic activity of CQDs were studied by gas chromatography mass spectrometry (GC/MS). It proved that the complex molecules were degraded to simpler and easily degradable molecules. Dye degradation reaction follows first-order kinetics, and the synthesized CQDs contain 89% of scavenging activity. MTT assay proved that the treated water was toxic free and charcoal was used to remove the CQDs from treated water in order to standardize the permitted level of physico-chemical parameters such as biological oxygen demand (BOD), chemical oxygen demand (COD), total organic carbon (TOC), and total inorganic carbon (TIC); chemical, metal, and toxic-free dye treated water was suitable to recycle for algae cultivation.

Biogenic Synthesis of Fluorescent Carbon Dots (CDs) and Their Application in Bioimaging of Agricultural Crops.

Fluorescent nanoparticles have a transformative potential for advanced sensors and devices for point-of-need diagnostics and bioimaging, bypassing the technical burden of meeting the assay performance requirements. Carbon dots (CDs) are rapidly emerging carbon-based nanomaterials. Regardless of their fate, they will find increasing applications. In this study, a simple approach for synthesizing CDs from fruit peels was developed. The CDs were fabricated from Annona squamosa (L.) peels using a carbonization technique through microwave-assisted hydrothermal digestion at temperatures around 200 °C. Synthesized CDs were detected using a UV transilluminator for the preliminary confirmation of the presence of fluorescence. UV-Vis spectrophotometry (absorbance at 505 nm) analysis, zeta potential measurement (-20.8 mV), nanoparticles tracking analysis (NTA) (average size: 15.4 nm and mode size: 9.26 nm), photoluminescence, and Fourier transform infrared (FT-IR) analysis were used to identify the capping functional groups on the CDs. The total quantum yield exhibited was 8.93%, and the field emission scanning electron microscopy (FESEM) showed the size range up to 40 nm. The germinating mung bean (Vigna radiata (L.)) seeds were incubated with biogenically synthesized CDs to check the absorption of CDs by them. The fluorescence was observed under a UV-transilluminator in the growing parts of seeds, indicating the absorption of CDs during the germination, development, and growth. These fluorescent CDs could be used as a bioimaging agent. This novel method of synthesizing CDs was found to be eco-friendly, rapid, and cost-effective.

A rapid method for DNA Isolation from blood, dried blood spots and rapid diagnosis test.

Malaria is a parasitic disease spread by Plasmodium parasite. Microscopy, lateral flow devices such as the Rapid Diagnostic Test (RDT), molecular methods such as Polymerase Chain Reaction (PCR), isothermal methods such as Loop-mediated isothermal amplification (LAMP), and other diagnostic methods are available for malaria. On the other hand, the accuracy of molecular diagnosis is dependent on genomic DNA isolation. A quick method for isolating and subjectively determining the presence of genomic DNA from blood, dried blood spot (DBS), and rapid diagnostic test (RDT), was identified. We have developed a protocol for isolating DNA from blood, DBS, and RDTs using the HUDSON Buffer (TCEP and EDTA). Isolated genomic DNA was seen with SYBR Safe DNA stain (1X) under a UV transilluminator without running in 0.8 percent gel electrophoresis or using a spectrophotometer. The technique for DNA isolation was accurate for the presence of malaria parasite genomic DNA from positive samples confirmed by microscopy with a sensitivity of 76% and specificity of 78.67% and RDT with a sensitivity of 88% and specificity of 66%. The requirements were minimal, and the process took 30 minutes for a hundred sample processing. Finding a fast and reliable method of separating nucleic acids from many samples is crucial. This approach extracts intact genomic DNA in under ten minutes, making it ideal for large-scale investigations.

Plasmid Profile and Enterobacterial Repetitive Intergenic Consensus-PCR Characterization of Salmonella Infantis Isolates Recovered From Poultry Sources

Background: Salmonella is known as one of the most important bacterial agents infecting both humans and animals. Salmonella Infantis has been reported as one of the 15 most prevalent serovars worldwide. Despite its clinical importance, there is little information on the molecular characteristics of S. Infantis in Iran. Objectives: This study was conducted to characterize S. Infantis isolates collected from poultry sources in the last decade. The isolates were typified by plasmid profile and enterobacterial repetitive intergenic consensus (ERIC-PCR). Methods: Forty S. Infantis isolates from poultry sources were subjected to plasmid profile and ERIC-PCR characterization. We used a commercial plasmid extraction kit to extract and purify plasmid DNA which then was separated by gel electrophoresis and viewed under a UV transilluminator. For ERIC-PCR, a commercial bacterial chromosomal DNA extraction kit was used. In this study, we chose ERIC2 primer for the ERIC-PCR test. Results: The plasmid profile revealed that 35% of isolates did not contain any plasmids, but the rest (65%) carried a variable number of plasmids with different molecular weights. Six plasmid profiles were found among 40 S. Infantis isolates. Using ERIC2 primer, 7 profiles were found among 40 S. Infantis isolates in ERIC-PCR. Bands with molecular weights ranging from 400 to 3000 bp were observed. Conclusion: This study provided some genetic data on S. Infantis isolates recovered from poultry sources, and these data can be used for a broader epidemiological study nationwide. These findings showed that although plasmid and ERIC profiles are valuable in epidemiological studies, they have some limitations, too.

Optimization of reduced Graphene oxide synthesis using central composite design analysis-A waste to value approach.

In recent times, reduced graphene oxide has gained more attention in various fields. In our study, a direct synthesis of reduced graphene oxide using a novel carbon-rich agro-waste from Pennisetum glaucum was used. Ferrocene acted as an oxidizing agent during thermal degradation at 300°C for 15 and 20min to promote graphene oxide and reduced graphene oxide formation. The X-ray diffraction peak at 2θ indicating a shift from 16.86 to 24.28°, presence of functional groups like -OH stretching, -C = C-, C = O, C-O, and C-OH by Fourier transmission infrared spectroscopy, prominent D and G bands at 1308cm-1 and 1578cm-1 by Raman spectra and UV-visible spectroscopy peak shift from 235 to 245nm (π-π*, C = C bonds) confirmed the reduction of graphene oxide to reduced graphene oxide. The average particle size values 233.3nm for graphene oxide and 63.57nm for reduced graphene oxide illustrate the nanoscale range of our synthesized material. The negative zeta potential values in the range - 45.5mV and - 29.5mV for graphene oxide and its reduced forms infer the dispersion stability along with surface oxygen group presence. We have also highlighted the formation of graphene oxide quantum dots by magnetic stirring and confirmed by UV transilluminator and photoluminescence spectra. The photodegradation efficiency was optimized using central composite design for dosage, dye concentration, pH, and time for both malachite green and reactive blue dye. The kinetic studies report pseudo-first-order kinetic model for catalytic degradation and statistical Analysis of variance proved the significance of the process for p value < 0.05. Thus, the synthesized graphene materials could be used as a potential candidate for environmental applications.

Melinjo (Gnetum Gnemon) Seed Protein Activity Against pBSKS DNA Cleavage and Its Cytotoxicity in T47D and 4T1 Cells

The plant protein of melinjo (Gnetum gnemon) in seeds is known to have antioxidant properties. Apart from being an antioxidant, melinjo seed protein also has the potential to be developed as an anticancer compound. One of the potential anticancer compounds has been screened based on supercoiled DNA cleavage activity (pBSKS). The purpose of this study was to determine the activity of the protein fraction isolated with DEAE and BUTYL matrices against supercoiled DNA cleavage of pBSKS. Supercoiled DNA cleavage test activity of pBSKS was determined using the electrophoresis method and visualized with a UV transilluminator at a wavelength of 312 nm. The cytotoxic activity of melinjo seeds was tested through the MTT assay method and was read on an ELISA reader with a wavelength of 550 and 595 nm seen from the IC50 value. The protein fraction of melinjo seeds isolated with DEAE and BUTYL matrices had concentrations of 470.1 and 81.02 g/mL, respectively. The visualization results showed that the DEAE and BUTYL protein fractions resulted in depletion of supercoiled DNA bands of pBSKS. The concentration of active protein in the melinjo seeds fractionated using the DEAE-650M matrix was higher than that of the BUTYL-650M matrix. In this study, after several cytotoxic tests were carried out, various results were obtained. The activity of protein isolates from melinjo seeds fractionated using DEAE-650M and BUTYL-650M against 4T1 and T47D cells could be categorized as non-toxic because the IC50 value was &gt; 1000 g/mL and the average percentage of viable cells was more than 50%, except for the seed protein fraction. Melinjo fractionated using DEAE-650M against T47D cells which had an IC50 value of 127.62 g/mL could be categorized as quite active and cytotoxic.&#x0D; Keywords: Gnetum gnemon L., protein isolation, ribosome-inactivating proteins (RIPs), DNA cleavage, cytotoxic, T47D, 4T1

Study of Meat Species Adulteration in Indonesian Commercial Beef Meatballs Related to Halal Law Implementation

Meatballs are processed meat products that are susceptible to adulteration. Indonesia enforces a halal product guarantee regulation so that all products in circulation are free from non-halal ingredients. This study aimed to detect with the PCR method the contamination by pork and chicken of commercial beef meatballs. The samples of commercial meatballs were obtained from 36 meatball shops in Bojonegoro Regency, East Java, and Boyolali Regency, Central Java Province, Indonesia. Reference meatballs as a positive control were prepared in the laboratory. DNA isolation was performed on commercial sample meatballs, reference meatballs, and 13 species of fresh meat for specificity tests. DNA concentrations were measured using a spectrophotometer, and visualization of the isolation and PCR results was conducted using agarose gel electrophoresis and a UV transilluminator. DNA isolates were amplified by PCR using gene targets 1.711B bovine repeat for identifying bovine species, CR1 SINE repeat element for identifying chicken species, and mitochondrial gene subunit ND1 for identifying pork species. The results showed that there was contamination by chicken meat in meatballs labeled as beef meatballs in 30 of 36 samples in Bojonegoro Regency and 33 of 36 samples in Boyolali Regency. The results also showed that 22 samples of commercial beef meatballs in the Boyolali Regency contained pork. The implementation of the PCR method using the three specific primers proved the presence of meat contamination that did not match the label on the meatballs circulating in the Indonesian market. The contamination by pork if beef meatballs has violated the regulation on halal product guarantees.

A Novel and Rapid Isothermal Nucleic Acid Based Detection Assay of Vibrio cholerae by Polymerase Spiral Reaction (PSR) in Emergency Situations.

The purpose of this survey was to develop a novel and rapid isothermal nucleic acid based detection assay of Vibrio cholerae by polymerase spiral reaction (PSR) in emergency situations. The current study was conducted in Baqiyatallah University of Medical Sciences, Tehran, Iran in 2021. The conserved ctxA gene sequence of V. cholerae was used as a target of designed two pairs of primers. Amplification of nucleic acids performed under isothermal temperature of 65 °C in 55 min by using Bst DNA polymerase. PSR amplified products were real-time visualized under UV transilluminator and also on agarose gel electrophoresis. Seven non- V. cholerae bacteria were negative for detection, which indicated the specificity of PSR assay was 100%. A 10- fold serial dilution of V. cholerae genomic DNA was subjected to conventional polymerase chain reaction (PCR) and real-time PCR to compare their sensitivities with PSR. The detection limit of PSR was 3 × 10-5 ng/μL within 60 min, which 100-fold higher than that of PCR (3 × 10-3 ng/μL), but the sensitivity of real-time PCR was found as same as it. The PSR assay developed in this study can provide a simple, cost-effective, rapid, and precise diagnosis technique in endemic cholera outbreaks, especially in low-income with limited access provinces.