Comparison of Time Course Detection of Human Male DNA from Blood Stains on Various Objects on Surface in a Natural Environment and in a Laboratory Using Loop-Mediated Isothermal Amplification (LAMP)

In forensic study, the biological evidence can easily degrade, especially DNA. Degraded and environmentally challenged samples can produce numerous problems in forensic DNA analysis including loss of band product. Loop-mediated isothermal amplification or LAMP is one of the DNA analysis techniques used in forensic study. This study explores the limitations of the efficiency of the LAMP technique on abandoned DNA. For the DNA template, 8 male and 2 female blood-stained samples were taken from the surfaces, namely, brick, cloth, and tile from inside, and buried outside the laboratory. The LAMP reaction was used to amplify the SRY gene for detecting male DNA. All the blood-stained samples were stored for 1, 7, 15, 30, and 45 day (s). The LAMP product from the blood-stained samples on all the surfaces that were kept in a laboratory was detected using the gel electrophoresis technique from day 1 until day 45. However, the LAMP product on day 30 and 45 was smear and dim. The LAMP product from the blood-stained samples buried outside the laboratory was observed using the gel electrophoresis technique within day 30 (smear and dim). To increase the efficiency of detection, the qLAMP technique detected product on all the male samples from all the surfaces buried outside the laboratory for 45 days. The results indicate that this LAMP condition was possible detecting male DNA and the environmental factors are the main influence on the sensitivity of the LAMP technique. In addition, the qLAMP technique can increase the capacity and sensitivity of the detection.

Ultra-rapid somatic variant detection via real-time threshold sequencing

Molecular markers are becoming increasingly important for cancer diagnosis, proper clinical trial enrollment, and even surgical decision making, motivating ultra-rapid, intraoperative variant detection. Sequencing-based detection is considered the gold standard approach, but typically takes hours to perform. In this work, we present Threshold Sequencing, a methodology for designing protocols for targeted variant detection on real-time sequencers with a minimal time to result. Threshold Sequencing analytically identifies a time-optimal threshold to stop target amplification and begin sequencing. To further reduce diagnostic time, we explore targeted Loop-mediated Isothermal Amplification (LAMP) and design a LAMP-specific bioinformatics tool--LAMPrey--to process sequenced LAMP product. LAMPrey's concatemer aware alignment algorithm is designed to maximize recovery of diagnostically relevant information leading to a more rapid detection versus standard read alignment approaches. Coupled with time-optimized DNA extraction and library preparation, we demonstrate confirmation of a hot-spot mutation (250x support) from tumor tissue in less than 30 minutes.

Detection of Globodera pallida directly from soil sample using mt-COI region based LAMP assay

Potato cyst nematodes (PCN), Globodera rostochiensis (Golden/yellow) and G. pallida (White), are economically important and relatively specialized pest of potato (Solanum tuberosum L.). Both the species are being identified based on cyst colour after 55-60 days after planting (DAP) however, after 65 DAP, we cannot differentiate based on cyst colour as both species turns brown. Moreover, the molecular techniques available to detect the PCN at species level is laborious, time consuming and costly. Therefore, development of rapid, accurate and economically cheap technique for detection of PCN at species level from the field is important to device effective management strategies for sustainable potato production. Accordingly, in the first instance, loop-mediated isothermal amplification (LAMP) assay was developed to detect G. pallida directly from soil by using the mitochondrial (mt-COI) gene specific primer. The LAMP assay was completed within 60 min at 60 °C isothermal conditions and the primer, efficiently detects the G. pallida without any cross reaction with G. rostochiensis, Meloidogyne incognita, M. javanica, Heterodera avenae, H. carotae, and Cactodera spp. In analytical sensitivity tests, the assay was able to detect G. pallida with 1000 times less DNA concentration (10 fg/µl) as compared to conventional PCR (10 pg/µl) and the LAMP product was visualized by using SYBR Gold nucleic acid dye and the assay can be highly useful in detection of G. pallida.

Philips Lamp Product Innovation into UV-C Disinfectant Desk Lamp to Improve The Company's Competitiveness during The Covid-19 Pandemic

The pandemic period is the reason for the company to set up an innovating strategy after conducting a series of research and product development. The purpose of Philips Company is to innovate not only to maintain the sustainability of product livecycle, but also to meet consumer needs for disinfectant tools by not leaving brand image in the pioneer of lighting products. Product innovation is done by designing products that effectively disable viruses, bacteria, fungi, and spores, in minutes. Consumer objectivity in deciding a product purchase due to the advantages of modern product design, energy saving, simple and put forward the ethics of environmentally friendly products.

Dry loop mediated isothermal amplification assay for detection of SARS-CoV-2 from clinical specimens

Coronavirus disease 2019 (COVID-19) has had a major disease burden on many countries around the world. The spread of COVID-19 is anticipated to have a major impact on developing countries including African nations. To establish a point-of-care test for COVID-19, we developed a dry loop mediated isothermal amplification (LAMP) method to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. We carried out reverse transcription (RT)-LAMP using the Loopamp SARS-CoV-2 Detection kit (Eiken Chemical, Tokyo, Japan). The entire mixture except for the primers is dried and immobilized inside the tube lid. To determine the specificity of the kit, 22 viral genomes associated with respiratory infections, including the SARS coronavirus, were tested. No LAMP product was detected in reactions performed with RNA from these pathogens. The sensitivity of this assay, determined by either a real-time turbidity assay or colorimetric change of the reaction mixture, as evaluated by the naked eye or under illumination with ultraviolet light, was 10 copies/reaction. After the initial validation analysis, we analyzed 24 nasopharyngeal swab specimens collected from patients suspected to have COVID-19. Nineteen (79.2%) of the 24 samples were positive for SARS-CoV-2 RNA, as determined by real-time RT-PCR analysis. Using the Loopamp SARS-CoV-2 Detection kit, we detected SARS-CoV-2 RNA in 15 (62.5%) of the 24 samples. Thus, the sensitivity, specificity, positive predictive value, and negative predictive value of the Loopamp 2019-CoV-2 detection reagent kit were 94.0%, 96.0%, 95.9%, and 94.1%, respectively. The dry LAMP method for detection of SARS-CoV-2 RNA was fast and easy to use, solves the cold chain problem, and therefore represents a promising tool for diagnosis of COVID-19 in developing countries.

Monitoring Methyl Benzimidazole Carbamate-Resistant Isolates of the Cucurbit Powdery Mildew Pathogen, Podosphaera xanthii, Using Loop-Mediated Isothermal Amplification

Powdery mildew, caused by the fungus Podosphaera xanthii, is one of the most economically important diseases affecting cucurbit crops in Spain. Currently, chemical control offers the most efficient management of the disease; however, P. xanthii isolates resistant to multiple classes of site-specific fungicides have been reported in the Spanish cucurbit powdery mildew population. In previous studies, resistance to the fungicides known as methyl benzimidazole carbamates (MBCs) was found to be caused by the amino acid substitution E198A on β-tubulin. To detect MBC-resistant isolates in a faster, more efficient, and more specific way than the traditional methods used to date, a loop-mediated isothermal amplification (LAMP) system was developed. In this study, three sets of LAMP primers were designed. One set was designed for the detection of the wild-type allele and two sets were designed for the E198A amino acid change. Positive results were only obtained with both mutant sets; however, LAMP reaction conditions were only optimized with primer set 2, which was selected for optimal detection of the E198A amino acid change in P. xanthii-resistant isolates, along with the optimal temperature and duration parameters of 65°C for 75 min, respectively. The hydroxynaphthol blue (HNB) metal indicator was used for quick visualization of results through the color change from violet to sky blue when the amplification was positive. HNB was added before the amplification to avoid opening the lids, thus decreasing the probability of contamination. To confirm that the amplified product corresponded to the β-tubulin gene, the LAMP product was digested with the enzyme LweI and sequenced. Our results show that the LAMP technique is a specific and reproducible method that could be used for monitoring MBC resistance of P. xanthii directly in the field.

Specific and rapid identification of the Pheretima aspergillum by loop-mediated isothermal amplification

Guang-dilong (Pheretima aspergillum) is a traditional Chinese animal medicine that has been used for thousands of years in China. In the present study, we purposed to establish a new rapid identification method for Guang-dilong. We provided a useful technique, loop-mediated isothermal amplification (LAMP), to differentiate Guang-dilong from other species. Four specific LAMP primers were designed based on mitochondrial cytochrome c oxidase I (COI) gene sequences of Guang-dilong. LAMP reaction, containing DNA template, four primers, 10× Bst DNA polymerase reaction buffer, dNTPs, MgSO4, and Bst DNA polymerase, was completed within 60 min at 63°C. The LAMP product can be visualized by adding SYBR Green I or detected by 2% gel electrophoresis. LAMP technology was successfully established for rapid identification of Guang-dilong. In addition, DNA template concentration of 675 fg/μl was the detection limit of LAMP in Guang-dilong, which was 1000-times higher than conventional PCR. The simple, sensitive, and convenient LAMP technique is really suited for on-site identification of Guang-dilong in herbal markets.

The Integration of the Pottery Handicraft Together with the Basketwork Handicraft Promoting Economic Community Strength

A study of the integration of the pottery handicraft together with the basketwork promoting economic community strength aims to 1) to study the basketwork identity to apply with the pottery handicraft 2) to examine the experimental design and to develop the pottery handicraft product combined with the basketwork in order to beautiful identical according to market qualifications and 3) to evaluate the market testing of the handicraft product developed conduce to the guidelines for producers apply to use. The samples were 400 people who interested in handicraft wherewith purposive sampling at the handicraft shop by used the accidental sampling, inquired for collect the data before as well as after design, evaluated by 3 groups of community product producer as 3 stars level which experience 10 years at least and 3 product design experts in order to evaluate the optimal pattern of product modified, therefrom, created the prototype as well as evaluated the opinion comments of consumer towards product designs. The results revealed that the most wanted of consumer that were the lamp product, basket product and decorated item products, the container rim of the 3rd pattern by weaving method was appropriate for forming pattern which were combined method with easy, strong and stable pattern, easy to fix when being ruined, the product pattern have been tested marketing by consumer found the satisfaction overall was the highest level, furthermore they satisfied in term of beauty than in term of usability.

Usulan Perbaikan Peracangan Produk Smart Light Menggunakan Metode Design for Assembly Boothroyd-Dewhurst

ABSTRACTSmart Lamp product is a street lighting product which developed by PT X with LED lights concept. The design of Smart Light product is found to be slightly violate the terms of good design to the assembly process proposed by Boothroyd-Dewhurst as there is a component with sharp side, too much for using fastener, difficult fastener installation because the component is blocked and so forth. Existing design efficiency of Smart Light product is based on a calculation using the Boothroyd-Dewhurst table is 7.63% with total assembly time for 1149.1 seconds while the proposed design efficiency is 15.52% with total assembly time is 539.84 seconds. The changes of the design result reduction of the estimated product cost from Rp1.831.721, - and the BEP in 1482 products on existing product to Rp1.732.609, - and the BEP in 1283 products on proposed product.Kata kunci: design efficiency, assembly time, estimated cost, break event point (BEP).ABSTRAKProduk Smart Light adalah merupakan sebuah produk lampu penerangan jalan yang dikembangkan oleh PT X dengan konsep lampu LED. Rancangan produk Smart Light ini ternyata tidak sedikit melanggar ketentuan-ketentuan perancangan yang baik untuk proses perakitan yang dikemukakan oleh Boothroyd-Dewhurst seperti terdapat komponen yang memiliki bagian yang tajam, penggunaan fastener yang terlalu banyak, pemasangan fastener yang sulit karena komponen terhalang dan sebagainya. Efisiensi desain existing produk Smart Light ini berdasarkan pada perhitungan menggunakan tabel Boothroyd-Dewhurst adalah 7,63% dengan waktu perakitan total selama 1149,1 detik sedangkan efisiensi desain usulan adalah 15,52% dengan waktu perakitan total selama 539,84 detik. Perubahan rancangan desain mengakibatkan pengurangan pada estimasi biaya produk dari Rp1.831.721,- dan break event point (BEP) pada produk ke 1482 untuk produk existing menjadi Rp1.732.609,- dan BEP pada produk ke 1283 untuk produk usulan..Keywords: efisiensi desain, waktu perakitan, estimasi biaya, break event point

Time Course of Detection of Human Male DNA from Stained Blood Sample on Various Surfaces by Loop Mediated Isothermal Amplification and Polymerase Chain Reaction

This study explores determining the sex of humans from blood stains taken from different surfaces and compares the time course of detection with the conventional PCR, Conventional Loop Mediated Isothermal Amplification (LAMP), and LAMP-Lateral Flow Dipstick (LFD). For the DNA templates, 7 male and 7 female blood stained samples were extracted and added to LAMP and PCR reaction solution to amplify the SRY gene. The DNA samples were extracted from the following blood stained materials: cloth, wood, clay, and tile. Then, the samples were stored at room temperature for 1, 7, 30, and 60 day(s). After the DNA amplification, the gel electrophoresis process was applied to detect LAMP product. The LFD was combined with the LAMP to detect LAMP product on the male cloth samples. For the male samples, the time course of detection on the first and seventh days indicated positive for both LAMP and PCR products on all the surfaces while no DNA amplification was found on any of the female samples. On day 30, positive LAMP product was still found on all the male samples. However, it had faded on the tiles. Moreover, all the male samples, which had tested positive for PCR product, were blurred and unclear. On day 60, LAMP product was still found on all the male samples. Conversely, the PCR method resulted in no bands showing for any of the male samples. However, the LAMP-LFD method detected product on all the male samples of cloth. The results show that the LAMP is an effective, practical, and reliable molecular-biological method. Moreover, the LFD can increase the efficiency and sensitivity of the LAMP, making it more suitable for field studies because gel electrophoresis apparatus is not required.