Deoxyribonucleic Acid Amplification Research Articles

Bubble-free diatoms polymerase chain reaction

Polymerase chain reaction (PCR) in small fluidic systems not only improves speed and sensitivity of deoxyribonucleic acid (DNA) amplification but also achieves high-throughput quantitative analyses. However, air bubble trapping and growth during PCR has been considered as a critical problem since it causes the failure of DNA amplification. Here we report bubble-free diatom PCR by exploiting a hierarchically porous silica structure of single-celled algae. We show that femtoliters of PCR solution can be spontaneously loaded into the diatom interior without air bubble trapping due to the surface hydrophilicity and pore structure of the diatom. We discover that a large pressure gradient between air bubbles and nanopores rapidly removes residual air bubbles through the periodically arrayed nanopores during thermal cycling. We demonstrate the DNA amplification by diatom PCR without air bubble trapping and growth. Finally, we successfully detect DNA fragments of SARS-CoV-2 with as low as 10 copies/μl by devising a microfluidic device integrated with diatoms assembly. We believe that our work can be applied to many PCR applications for innovative molecular diagnostics and provides new opportunities for naturally abundant diatoms to create innovative biomaterials in real-world applications.

Performance Analysis of a Phase Changing Material Based Thermocycler for Nucleic Acid Amplification

Abstract Modeling, simulation, and thermal performance analysis of a thermocycler for the continuous-flow polymerase chain reaction (CF-PCR), with a phase changing material (PCM)-laden annealing flow path, is presented. The incessant threat of microorganisms such as viruses, bacteria, and fungi has fostered effective, quick, and miniature detection devices in order to curtail the wide-spreading of infections. Microfluidics-based CF-PCR systems are compact and ideal for faster response. The thermal cycling process involves a sequential exposure of a given liquid sample to various temperature conditions when it is taken through the continuous-flow path. As a result, a prescribed periodic change of temperature suitable for deoxyribonucleic acid (DNA) amplification is achieved. A rapid temperature reduction and maintenance of isothermal conditions to facilitate the annealing phase of CF-PCR process by a PCM-assisted cooling is envisaged in the present study. Unsteady, two-dimensional, incompressible fluid flow, and internal convection heat transfer in a microchannel annealing path with melting of tetracosane (C24H50) boundary has been simulated using semi-implicit method for pressure linked equations-consistent (SIMPLEC) algorithm based finite volume solver. Solver validation is carried out against the experimental data on internal convection heat transfer in a rectangular microchannel. A detailed numerical study has been performed to assess the spatiotemporal heat transfer characteristics of internal convection in the microfluidic path when the flow triggers the melting of encapsulated PCM. A minimum sample flowrate with PCM encapsulation of less than 600 μm is found to be ideal for achieving desired thermal performance. The present study evidences the swift temperature reduction and management of isothermal conditions congenial for the annealing process in the CF-PCR system for various sample flowrates and PCM masses. The study offers valuable design input for the development of a microfluidic thermocycler for CF-PCR applications.

Selective Translation of Maternal mRNA by eIF4E1B Controls Oocyte to Embryo Transition.

Maternal messenger ribonucleic acids (mRNAs) are driven by a highly orchestrated scheme of recruitment to polysomes and translational activation. However, selecting and regulating individual mRNAs for the translation from a competitive pool of mRNAs are little-known processes. This research shows that the maternal eukaryotic translation initiation factor 4e1b (Eif4e1b) expresses during the oocyte-to-embryo transition (OET), and maternal deletion of Eif4e1b leads to multiple defects concerning oogenesis and embryonic developmental competence during OET. The linear amplification of complementary deoxyribonucleic acid (cDNA) ends, and sequencing (LACE-seq) is used to identify the distinct subset of mRNA and its CG-rich binding sites within the 5' untranslated region (UTR) targeted by eIF4E1B. The proteomics analyses indicate that eIF4E1B-specific bound genes show stronger downregulation at the protein level, which further verify a group of proteins that plays a crucial role in oocyte maturation and embryonic developmental competence is insufficiently synthesized in Eif4e1b-cKO oocytes during OET. Moreover, the biochemical results in vitro are combined to further confirm the maternal-specific translation activation model assembled by eIF4E1B and 3'UTR-associated mRNA binding proteins. The findings demonstrate the indispensability of eIF4E1B for selective translation activation in mammalian oocytes and provide a potential network regulated by eIF4E1B in OET.

Label-free DNA quantification using isothermal amplification on an exposed core optical fiber microfluidic platform.

Isothermal amplification technology has triggered a surge in research due to its compatibility with small and portable equipment, simplicity, and high efficiency, especially in light of the COVID-19 pandemic where reliable widescale testing is critical to outbreak management. In this paper, a label-free isothermal deoxyribonucleic acid (DNA) amplification method based on refractive index (RI) quantification is proposed and demonstrated for the first time by combining optical fiber sensing, microfluidics, and isothermal amplification. A highly RI-sensitive Mach-Zehnder (MZ) interference is formed by splicing a short length of an exposed-core fiber between two lengths of a single-mode fiber while the microfluidic liquid channel on the exposed side of the ECF is filled with target DNA and the amplification solution. Real-time quantitative measurement of the target DNA is then realized by monitoring the change in RI of the solution during the isothermal DNA amplification process. The experimental results show that the platform successfully realizes real-time label-free monitoring of isothermal amplification of 0.16 aM DNA samples. This method is a breakthrough for applications in the fields of DNA detection and quantification where simple operation, rapid detection, portability, small size, high selectivity, and high sensitivity are required.

DNA technology for big data storage and error detection solutions: Hamming code vs Cyclic Redundancy Check (CRC)

There is an increasing need for high-capacity, highdensity storage media that can retain data for a long time, due to the exponential development in the capacity of information generated. The durability and high information density of synthetic deoxyribonucleic acid (DNA) make it an attractive and promising medium for data storage. DNA data storage technology is expected to revolutionize data storage in the coming years, replacing various Big Data storage technologies. As a medium that addresses the need for high-latency, immutable information storage, DNA has several potential advantages. One of the key advantages of DNA storage is its extraordinary density. Theoretically, a gram of DNA can encode 455 exabytes, or 2 bits per nucleotide. Unlike other digital storage media, synthetic DNA enables large quantities of data to be stored in a biological medium. This reduces the need for traditional storage media such as hard disks, which consume energy and require materials such as plastic or metals, and also often leads to the generation of electronic waste when they become obsolete or damaged. Additionally, although DNA degrades over thousands of years under non-ideal conditions, it is generally readable. Furthermore, as DNA possesses natural reading and writing enzymes as part of its biological functions, it is expected to remain the standard for data retrieval in the foreseeable future. However, the high error rate poses a significant challenge for DNA-based information coding strategies. Currently, it is impossible to execute DNA strand synthesis, amplification, or sequencing errors-free. In order to utilize synthetic DNA as a storage medium for digital data, specialized systems and solutions for direct error detection and correction must be implemented. The goal of this paper is to introduce DNA storage technology, outline the benefits and added value of this approach, and present an experiment comparing the effectiveness of two error detection and correction codes (Hamming and CRC) used in the DNA data storage strategy.

Advances in deoxyribonucleic acid extraction techniques and point-of-care molecular diagnosis of foodborne pathogens.

A conventional molecular assay-based point-of-care (POC) diagnostic test involves three major stages: deoxyribonucleic acid (DNA) extraction, amplification, and amplicon detection. Among these steps, DNA extraction is costly and time-consuming. Nevertheless, it is a crucial step for the identification of sensitive and specific diseases. This review summarizes the advantages and disadvantages of DNA extraction methods over the past 10 years to effectively implement POC pathogen testing in the future. The first section briefly explains the necessity of DNA extraction and molecular assays for food pathogen detection. The second section extensively discusses DNA extraction based on liquid-liquid extraction, solid-phase extraction, and electrophoretic techniques. Molecular assay-based methods and a few commercially available POC devices for the detection of foodborne pathogens are detailed in the third and fourth sections. Finally, present challenges and future perspectives for the fabrication of integrated POC devices are highlighted.

Occult hepatitis B virus infection in patients with chronic liver disease of different etiology in a Brazilian referral center: comparison of two different hepatitis B virus deoxyribonucleic acid amplification protocols: a cross-sectional study.

Occult hepatitis B virus infection (OBI) is defined as the presence of hepatitis B virus (HBV) deoxyribonucleic acid (DNA) in the liver of individuals with undetectable hepatitis B virus surface antigen (HBsAg) in the serum. The actual prevalence of OBI and its clinical relevance are not yet fully understood. To evaluate the prevalence of HBV DNA in liver biopsies of HBsAg-negative patients with chronic liver disease of different etiologies in a referral center in Brazil and compare two different HBV DNA amplification protocols to detect HBV. This cross-sectional observational study was conducted at the Liver Outpatient Clinic, Hospital das Clínicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil, between January 2016 and December 2019. HBV DNA was investigated in 104 liver biopsy samples from individuals with chronic liver disease of different etiologies, in whom HBsAg was undetectable in serum by nested-polymerase chain reaction (nested-PCR), using two different protocols. OBI, diagnosed by detecting HBV DNA using both protocols, was detected in 6.7% of the 104 individuals investigated. Both protocols showed a good reliability. In addition to the differences in the prevalence of HBV infection in different regions, variations in the polymerase chain reaction technique used for HBV DNA amplification may be responsible for the large variations in the prevalence of OBI identified in different studies. There is a need for better standardization of the diagnostic methods used to diagnose this entity.

Genetic diversity in populations of Girardinia diversifolia from Nepal Himalaya using ISSR markers

Girardinia diversifolia (Link) Friis from Urticaceae family is a perennial herb, commonly known as the Himalayan giant nettle. It has cultural, medicinal, and economic values among the indigenous peoples and local communities living in the mountains of Nepal and India. Prior knowledge of genetic diversity in plant species may contribute to conservation and sustainable utilization of important genotype. The aim of this study is to assess the genetic diversity within and among different populations of the 45 accessions of G. diversifolia collected from Far-western, Central, and Eastern regions of Nepal. The amplification of genomic deoxyribonucleic acid (DNA) with ten inter-simple sequence repeat (ISSR) primers yielded 131 clear DNA bands, of which 98.09% were found to be polymorphic. The mean effective number of alleles (ne), Nei's gene diversity (h) and Shannon's information index (I) were 1.598, 0.349, and 0.523, respectively. The total genetic diversity Ht (0.350 ± 0.04), low intrapopulation genetic diversity Hs (0.141 ± 0.03) and low estimated gene flow Nm (0.355 ± 0.11) reflected high genetic differentiation among population Gst (0.594 ± 0.08). The analysis of molecular variance among and within five populations of G. diversifolia showed that the value of genetic variation among the population was 60% whereas the value of genetic variation within the population was 40%. The results show that the ISSR markers are informative for the study of genetic diversity, which may help in the conservation and sustainable utilization of G. diversifolia and allied taxa.

Analysis of Cocoa Clonal Seedlings Purity Through Deoxyribonucleic Acid (DNA) Barcoding and Random Amplification of Polymorphic DNA (RAPD) Fingerprinting

The genetic purity of a plant indicates the similarity properties between seedlings in the field and the description of the plant in the database. Identifying plant purity through a morphological approach has several drawbacks, including time efficiency and environmental factors, and the diversity is limited and inconsistent. This condition encourages the development of detection methods using DNA molecular markers. Plant identification through fingerprinting or the use of molecular markers has not been widely carried out on a commercial scale, considering the investment costs for this analysis are still quite expensive. Another method used for plant identification through DNA Barcoding by comparing variations between DNA sequences. The primers used to identify barcodes on cocoa were derived from the chloroplast genome, including rbcL and matK. This research aims to see the consistency of the rbcL primer when applied to other cocoa clones, and the analysis of the polymorphic diversity of each cocoa clone using DNA fingerprinting RAPD. This method will be tested on clones of Sulawesi 1, Sulawesi 2, ICCRI 03, and ICCRI 09, which were propagated by SE and mother plants in the field using cocoa leaf samples. The stages include DNA extraction, sequencing, and analysis of the sequencing results. The results of seedlings uniformity analysis using DNA barcoding on cocoa plants produced from in vitro propagation showed that the multiplied seeds did not show any difference in sequence with the parent plant (DR2, Sulawesi 1, Sulawesi 2, and ICCRI 09). The analysis of the diversity of cocoa clones DR 2, MCC 2, Sulawesi 1, Sulawesi 2, and ICCRI 09 through DNA Fingerprinting RAPD showed that the OPA 15 primer produced a more apparent polymorphic band than the other three primers (OPP 08, OPW 11, and M 29).

Printed Circuit Boards: The Layers' Functions for Electronic and Biomedical Engineering.

This paper describes the fabrication opportunities that Printed Circuit Boards (PCBs) offer for electronic and biomedical engineering. Historically, PCB substrates have been used to support the components of the electronic devices, linking them using copper lines, and providing input and output pads to connect the rest of the system. In addition, this kind of substrate is an emerging material for biomedical engineering thanks to its many interesting characteristics, such as its commercial availability at a low cost with very good tolerance and versatility, due to its multilayer characteristics; that is, the possibility of using several metals and substrate layers. The alternative uses of copper, gold, Flame Retardant 4 (FR4) and silver layers, together with the use of vias, solder masks and a rigid and flexible substrate, are noted. Among other uses, these characteristics have been using to develop many sensors, biosensors and actuators, and PCB-based lab-on chips; for example, deoxyribonucleic acid (DNA) amplification devices for Polymerase Chain Reaction (PCR). In addition, several applications of these devices are going to be noted in this paper, and two tables summarizing the layers’ functions are included in the discussion: the first one for metallic layers, and the second one for the vias, solder mask, flexible and rigid substrate functions.

Infectious mimics of rheumatoid arthritis.

Rheumatoid arthritis (RA) can have various infectious mimics. As immunosuppressive agents used in treatment can aggravate the underlying infections, correct diagnosis of RA and ruling out infections is important. Numerous viral infections (Parvovirus B19, Hepatitis B, Hepatitis C, Chikungunya and other alphaviruses, human immunodeficiency virus (HIV) and various other viruses), mycobacterial infections (Poncet's disease, tubercular septic arthritis, and leprosy), bacterial arthritis, brucellosis and Lyme disease are among common infections that mimic RA. Widespread travel and tourism, especially to exotic areas, high risk sexual behavior and widespread use of immunosuppressive and chemotherapeutic agents has led to numerous outbreaks of infections in areas where these infections were never reported before. Hence, rheumatologists all over the world should be familiar with musculoskeletal manifestations of infections. History of travel, comorbid fever, skin rash, genital ulcers, urethral discharge, the consumption of unpasteurized milk, lymphadenopathy, tenosynovitis, low platelet count, and positive Mantoux test can offer potential diagnostic clues. Serological testing, cultures, specific radiological signs and deoxyribonucleic Acid (DNA) amplification techniques often aid in diagnosis. Treatment mainly consists of antimicrobial agents, analgesics, and nonsteroidal anti-inflammatory drugs (NSAIDs). However, immunosuppressive agents including steroids and disease modifying anti-rheumatic drugs (DMARDs) are needed occasionally in different refractory and prolonged illnesses. Most of the times, episodes of arthritis are self-limiting and respond to treatment of underlying cause. However, few infections like Chikungunya and Lyme's disease can lead to chronic arthritis as well.

GeneXpert Based Confirmed Cases among Suspected Cases of Tuberculosis in a Tertiary Care Centre: A Descriptive Cross-sectional Study.

Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis mostly affecting the lungs. Due to the low sensitivity of conventional microscopy and time-consuming culture method, Nucleic acid Amplification Assay Technique is preferred because of its rapidity and sensitivity. This test also helps in finding drug resistance to Rifampicin and also curtails the transmission of disease. The study is aimed to find the prevalence of GeneXpert confirmed cases among suspected cases of tuberculosis in a tertiary care centre. A descriptive cross-sectional study in 104 patients was conducted in a tertiary care centre from 30th Dec 2021 to 3rd Feb 2022. Ethical clearance was taken from the Institutional Review Committee (Reference number: 464/078/079). Sputum samples were collected from patients and were processed for GeneXpert under biological safety standards. GeneXpert Mycobacterium tuberculosis/rifampicin assay, sample processing, deoxyribonucleic acid extraction, and deoxyribonucleic acid amplification occurred in a fully automated cartridge-based real-time Polymerase chain reaction. A convenience sampling method was done. Collected data were coded as per variables and entered in Statistical Package for the Social Sciences version 25. Point estimate at 95% Confidence Interval was calculated along with frequency and percentage for binary data. In all 104 patients, GeneXpert detected 10 (9.62%) (3.94-15.26 at 95% Confidence Interval) positive tuberculosis cases. Out of total positive cases, there were 6 (60%) males and 4 (40%) females and there was 1 (10%) rifampicin-resistant case. The prevalence of pulmonary tuberculosis among presumptive cases in our study was found to be similar to reported literature. multidrug-resistant; nucleic acid amplification test; pulmonary tuberculosis.

Review on Molecular Diagnosis of Cestode and Metacestode in Cattle

Cestode infestations in animals are the most important parasite of livestock and humans because most of these parasites are zoonotic causing cysticercosis and hydatidosis in man and it causes economic and production losses in livestock. Diagnosis of Taenia Spp by microscopic observation lack sensitivity and specificity and detection by enzyme-linked immunosorbent assay (ELISA) technique form cross-reaction. The molecular diagnostic can be best to detect in adult and larval stage in definitive and intermediate host based on the amplification of deoxyribonucleic acid (DNA) of target gene with the primer using a different technique of polymerase chain reaction (PCR) such as multiplex PCR. Conventional PCR, real-time PCR, nested PCR, and PCR-restriction fragment length polymorphism (RFLP) are highly sensitive for the diagnosis of cestode and metacestode. Those diagnoses are used for differentiation of Taenia species and differentiation of Taenia and Echinococcus species. As compared to other diagnostic techniques most molecular methods have higher sensitivity and specificity but due to the relatively higher cost, few are commercially available. Most of the molecular diagnostic tests developed to date are generally applicable for laboratory research purposes. The developments in the genomic and proteomic analysis should be used for further understanding of parasite-animal host interaction to find additional targets for diagnosis.

Thermal study of polymerase chain reaction with capillary tubes

Polymerase Chain Reaction (PCR) amplifies specific deoxyribonucleic acid (DNA) targets whereby the reaction vessels are placed in high thermal inertia media and subjected to multiple cycles of discrete temperatures to effect denaturation, annealing and extension events. Precise temperature control and efficient heat transfer are crucial factors in achieving amplification reproducibility and reliability as well as in reducing the time required for PCR. Understanding the temperature distribution and transfer characteristics during PCR will facilitate system optimization to enhance the efficiency of DNA amplification. We describe a numerical thermal study investigating temperature profiles achieved using the classical method of manual transfer of PCR reaction vessels through a series of temperature-controlled media compared with that of a more recently reported method that displaces reaction vessels spatially by tilting through different temperature zones. It was found that while the classical method allows the reagent to achieve uniform temperature distribution, natural convection effects would necessitate translation speeds in excess of 150 mm/min in order to limit the reagent temperature drop to within 10 °C. With the tilt method, uniform temperature distributions as well as temperature settling times within 5 s could be attained. The simulation results demonstrated here indicate that the tilt method can be developed further to augment extreme PCR requiring less than 2s for each cycle.

Molecular identification and subtyping of Blastocystis sp. in hospital patients in Central China

Blastocystis is a common enteric protist that inhabits the gastrointestinal tract of approximately 1 billion people worldwide. In this study, a total of 1,070 patients from two hospitals in Zhengzhou, Central China were enrolled to know molecular characteristics of Blastocystis sp. The microorganism was identified and subtyped with a PCR amplification and sequencing of the small subunit ribosomal deoxyribonucleic acid (SSU-rDNA). The overall minimum prevalence of Blastocystis sp. in participants was 3.1% (33/1070). Although there were no significant differences on Blastocystis sp. infections among study sites, age groups, and gender, the higher infection was observed in the patients with gastrointestinal diseases (8.8%, 15/170). Sequence analysis of the 33 isolates revealed three known subtypes, such as ST1 (n = 7), ST3 (n = 23), and ST7 (n = 3). Among them, ST3 was the dominant subtype being detected in 23 isolates (69.7%), followed by ST1 (21.2%, 7/33) and ST7 (9.1%, 3/33). The phylogenetic analysis demonstrated that three subtypes (ST1, ST3 and ST7) were clustered with their reference sequences with good bootstrap support. The subtype determination of Blastocystis sp. isolates by the phylogenetic analysis was well supported by online platform. The present study provides the first molecular report of Blastocystis sp. infections in hospital patients in Central China.

Discrimination of Vitis vinifera varieties using DNA fingerprinting and NMR coupled with chemometrics and their impact on the efficacy of fluoxetine and indomethacin in vivo

The grapes, Vitis vinifera, belong to family Vitaceae. Some fruit juices substantially decrease the oral absorption of certain drugs undergoing intestinal uptake transport. Green seedless “Thompson” type (GT) and reddish black seeded “Magic black” type (BM) of V. vinifera L. were discriminated using deoxyribonucleic acid (DNA) fingerprinting and nuclear magnetic resonance (NMR) coupled with chemometrics. Their effects on the bioavailability of fluoxetine and indomethacin were assessed in vivo. Practical applications Random amplification of polymorphic DNA (RAPD) analysis of GT and BM revealed that primers OPA-02 and OPB-10 indicate a taxonomical relationship between them as they produced high similarity coefficients estimated by 80 and 66.67%, respectively. OPC-03, OPB-13, and OPC-07 primers help in their discrimination based on their minimal levels of similarity coefficients estimated by zero percentages accompanied by the high levels of polymorphism. Principal component analysis score plot obtained from processing of proton nuclear magnetic resonance (1H NMR) showed very good discrimination between GT and BM in PC1 that accounts for most of the variance. Oral administration of 100 mg/kg of GT and BM separately produce no significant change in immobility period of rats dropped individually into a glass cylinder. Concomitant administration of either 100 mg/kg of GT or BM with 20 mg/kg of fluoxetine significantly decreased the efficacy of antidepressant reference drug (fluoxetine) by decreasing time before immobility showing a percentage of depression inhibition estimated by 17.2 and 29.1%, respectively, that obviously decreased when compared to fluoxetine alone. Regarding anti-inflammatory activity, both GT and BM exhibited a notable activity by inhibiting the increase in paw edema and inflammation induced by carrageen in rats where BM is more potent approaching indomethacin. Administration of GT or BM with indomethacin reduces its efficacy revealing 27.5 and 26.1% in edema percentage after 1 hr, respectively, that decreased minimally to be 19.1% and 15.6% after 4 hr, respectively, instead of 7% in case of indomethacin alone. Thus, it can be concluded that DNA and NMR fingerprinting could successfully differentiate between Vitis varieties. Grape juice decreased the efficacy of fluoxetine and indomethacin. Further studies should be conducted to declare the effect of Vitis varieties on the bioavailability of additional drugs that treat chronic diseases as diabetes and hypertension.

Noninvasive preimplantation genetic testing for aneuploidy exhibits high rates of deoxyribonucleic acid amplification failure and poor correlation with results obtained using trophectoderm biopsy

To validate a commercially available noninvasive preimplantation genetic testing for aneuploidy (niPGT-A) assay by investigating the following: prevalence of deoxyribonucleic acid (DNA) amplification failure with niPGT-A; factors affecting amplification failure with niPGT-A; and frequency of discordant results between niPGT-A and traditional preimplantation genetic testing for aneuploidy. Prospective cohort study SETTING: Academic-affiliated private practice PATIENT(S): One hundred sixty-six blastocysts and their surrounding culture media from couples undergoing invitro fertilization between July 2019 and May 2020 were analyzed. Blastocyst-stage spent culture media samples underwent niPGT-A using a commercially available kit that used whole-genome amplification with a modified multiple annealing and looping-based amplification cycle protocol followed by next-generation sequencing. Preimplantation genetic testing for aneuploidy of trophectoderm (TE) biopsies was performed using targeted next-generation sequencing. The primary outcome was failure to achieve an interpretable result with niPGT-A. Factors affecting DNA amplification were also assessed. Discrepancies between niPGT-A and TE biopsy results were analyzed, and clinical outcomes were evaluated. Deoxyribonucleic acid amplification failures with niPGT-A were observed in 37.3% (62/166) of the samples. With TE biopsy, no embryos exhibited DNA amplification failure. Embryos with a shorter duration of exposure to the culture media and no evidence of whole-chromosome aneuploidy on the TE biopsy displayed high rates of DNA amplification failure with niPGT-A. Of 104 embryos with both niPGT-A and TE biopsy results available, whole-chromosome discordance was noted in 42 cases (40.4%). Three embryos classified as aneuploid based on the niPGT-A result progressed to successful delivery. The rates of DNA amplification failure were high among the niPGT-A samples, virtually precluding the clinical applicability of niPGT-A in its current form.

Secondary metabolites profiling of Vernonia amygdalina Del. in response to copper-induced abiotic stress

Background: Vernonia amygdalina is a green leafy vegetable that grows in tropical Africa and popularly cultivated in the southern part of Nigeria for its economic, nutritional and ethnomedicine value.Aims: This study analysed the influence of copper-induced abiotic stress on the deoxyribonucleic acid and secondary metabolites of V. amygdalina.Setting: Analyses of plant material were carried out in the Department of Botany, Lagos State University.Methods: Deoxyribonucleic acid (DNA) extraction and Inter Simple Sequence Repeat (ISSR) amplification were performed. Copper (Cu) concentration in leaves and stem of V. amygdalina was determined using inductively coupled plasma-optical emission spectroscopy (ICP-OES), secondary metabolites were determined using gas chromatography–mass spectrometry (GC-MS) and the effect on DNA amplification and profiling was analysed using gel electrophoresis.Results: After 41 days of cultivation, the stem of V. amygdalina significantly bioaccumulated 37 mg/L, 50 mg/L and 215 mg/L of Cu after spiking with 400 mg/L, 1000 mg/L and 2000 mg/L, respectively, whilst the leaves accumulated 52 mg/L, 100 mg/L and 350 mg/L of Cu, respectively. Copper-impregnated soil influenced phytochemicals of the plants by causing inconsistent increase and decrease in specific compounds such as levomenthol, methyl stearate and glycerine. Deoxyribonucleic acid bands of the stem and leaf of V. amygdalina from control site showed no band shift, whilst band shift occurred in the stem and leaf of the Cu-spiked V. amygdalina.Conclusion: This study revealed the leaves of V. amygdalina having higher Cu accumulation capacity than the stems. Also, Cu alters the quality and quantity of phytochemicals in plant parts.

Trend Analysis of the Profiles of 12 Sexually Transmitted Disease Pathogens in the Republic of Korea in 2019.

Background: Sexually transmitted diseases (STDs) are common infectious diseases in humans transmitted through unprotected sexual activities. In South Korea, despite the high annual incidence of STDs, detailed examinations of pathogen-specific factors and causes for delays in diagnosis and treatment are still lacking. Furthermore, STD prevalence patterns and important pathogen-specific factors remain unclear. Herein, we retrospectively analyzed the epidemiology of STDs in South Korea in 2019 by analyzing the association of pathogen-specific infection patterns with factors such as sex, age, region, and month. Methods: We obtained the STD test results of 172,973 individuals from the Seoul Clinic Laboratory in 2019, most of whom had multiple infections; hence, 275,296 STD-positive cases were included in this analysis. Through deoxyribonucleic acid (DNA) amplification, they were categorized by pathogen type. Subsequently, they were further classified by month, region, and age while concurrently being stratified according to sex. Results: Among the 12 pathogens detected in this study, Gardnerella vaginalis had the highest prevalence, with 92,490 cases in both sex groups; moreover, many of them were concurrently infected by two or more pathogens. The prevalence of STDs did not differ according to month or region. Conversely, the pathogen-specific prevalence rates significantly differed according to age. Older adults had higher prevalence rates of Chlamydia trachomatis, Trichomonas vaginalis, Candida albicans, and herpes simplex virus type 1 infections than younger adults. Conclusion: These pathogen-specific prevalence patterns provide information that helps to understand population vulnerability according to region and age and helps develop STD prevention and treatment strategies in South Korea.

Miniaturized Thermal Monitoring Module With CO₂ Laser Ablated Microfluidic Device for Electrochemically Validated DNA Amplification

Polymerase chain reaction (PCR) is the gold standard method to amplify deoxyribonucleic acid (DNA) fragments. Several methods have been used to miniaturize and integrate PCR to execute in a microfluidic platform. A continuous-flow-based microfluidic PCR device permits the required rapid thermal cycling and multiplexing, which are hard to realize in a benchtop PCR or microchamber PCR. This work aims to develop a microfluidic PCR platform using a custom-made Internet of Things (IoT)-enabled portable thermal monitoring module. The microfluidic device was fabricated on polymethyl methacrylate (PMMA) using a CO <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> laser. Apart from 30 serpentine cycles, the PCR microchannel also had initial denaturation and final elongation regions to enhance the overall amplification. As a proof-of-application, rat GAPDH gene, with a fragment size of 594 base pair, has been amplified on the miniaturized platform. Furthermore, the agarose gel electrophoresis technique was used to separate the DNA molecules validated using a gel-doc system. Subsequently, the acquired results were quantitatively compared with the conventional thermocycler using ImageJ software. Furthermore, this was also validated with electrochemical DNA detection using differential pulse voltammetry (DPV). The results from the smart low-power device showed promising performance for the nucleic acid amplification process while reducing the overall amplification time and achieving benchmarked efficiency.