Detection Of Genetic Material Research Articles

Current Trends in Biophysical Modeling of eDNA Dynamics for the Detection of Marine Species

ABSTRACTMarine pest introductions continue to occur and increase at accelerated rates, threatening the marine environment and blue economy. Environmental DNA (eDNA) is a tool for determining the presence of both indigenous and nonindigenous species, via the detection of genetic material that is shed into the local environment. Although eDNA approaches have gained widespread adoption in the last decade, fundamental knowledge gaps remain around factors that can influence the probability of detection, and how to optimize eDNA sampling in aquatic environments. Here, we partition eDNA research into four major research themes: eDNA concentration (shedding and decay), transport (advection and mixing), sampling design strategies, and the modeling of these dynamics. We review current developments and challenges in each theme with a focus on field sampling strategies and the use of biophysical models for understanding the movement of modeling eDNA in complex aquatic environments. We then introduce three modeling case studies from a large embayment where we (1) quantify the spatial and temporal variability of eDNA dispersion, (2) use biophysical models to inform a field sampling strategy, and (3) demonstrate a backtracking modeling technique to identify upstream DNA sources to an existing sample (monitoring) site. We conclude by identifying specific recommendations to help improve future eDNA studies. This work highlights how biophysical models can be applied to improve early detection and informing response and management decisions.

Posicionamiento de la Sociedad Española de Infectología Pediátrica sobre el diagnóstico y tratamiento de la infección por Mycoplasma pneumoniae

Mycoplasma pneumoniae (MP) is a bacterium with particular characteristics that give rise to a broad clinical spectrum, and respiratory infection is the most frequent presentation. Infection by MP occurs in cyclical epidemics, and paediatricians in Spain have noticed an increase in cases since January 2024, establishing hospital registers to collect surveillance data (as it is not a notifiable disease in Spain). The diagnosis of infection by MP is made through serological testing and/or the detection of genetic material by means of polymerase chain reaction (PCR). Neither methods can differentiate between colonization and active infection, so a precise diagnosis is not possible and testing should only be requested in the case of high clinical suspicion. The role of antibiotherapy in infection by MP in its different clinical variants is not well defined. Most infections are self-limiting and mild, and there is insufficient evidence to support the use of antibiotherapy in these cases. Antibiotic treatment is justified in patients with risk factors for the development of severe disease (Down syndrome, anatomical or functional asplenia, immunosuppression), in hospitalized patients with respiratory infection and in patients with moderate or severe extrapulmonary forms. Taking into account aspects concerning the rational use of antimicrobials, the treatment of choice would be clarithromycin, with azithromycin as an alternative, reserving the use of doxycycline and levofloxacin for cases of antimicrobial resistance and/or infections of the central nervous system.

Proof of concept for multiplex detection of antibodies against Chlamydia species in chicken serum using a bead-based suspension array with peptides as antigens

The available differentiating tests for Chlamydia are based on detection of genetic material and only give information about the actual infection status, but reveal nothing of past infections. As the use of serological methods increases the window of detection, the goal of this study was to investigate if it is possible to develop a differentiating serological test for antibodies against Chlamydia species in chicken sera. Focus was on C. psittaci, C. gallinacea, and two closely related species, i.e. C. abortus and C. avium. To enable differentiating serology, a bead-based Luminex suspension array was constructed, using peptides as antigens, derived from known immunoreactive Chlamydia proteins. For the majority of these peptides, species-specific seroreactivity in mammalian sera has been reported in literature. The suspension array correctly identified antibodies against various Chlamydia species in sera from experimentally infected mice, and was also able to differentiate between antibodies against C. psittaci and C. gallinacea in sera from experimentally infected chickens. In field sera, signals were difficult to interpret as insufficient sera from experimentally infected chickens were available for evaluating the seroreactivity of all peptides. Nevertheless, results of the suspension array with field sera are supported by published data on the occurrence of C. gallinacea in Dutch layers, thereby demonstrating the proof of concept of multiplex serology for Chlamydial species in poultry.

Detection of Red-Eared Slider (Trachemys scripta elegans) using Environmental DNA with Cytochrome b Primer

Biomonitoring surveys for the presence of invasive species need effective and efficient methods. Molecular methods based on the detection of genetic material in environmental samples or environmental DNA (eDNA) provide better resolution for detection, especially in elusive species. The eDNA-based method is an approach that has been widely used for conservation and business management because it is a non-invasive method. Cytochrome b primer is a specific primer that can be used to amplify Red-Eared Slider eDNA from water samples, eDNA was amplified using qPCR. The results obtained after performing qPCR are in the form of Ct values that can be used to estimate the biomass of Red-Eared Slider.

Recent Advances in Single-Molecule Sensors Based on STM Break Junction Measurements

Single-molecule recognition and detection with the highest resolution measurement has been one of the ultimate goals in science and engineering. Break junction techniques, originally developed to measure single-molecule conductance, recently have also been proven to have the capacity for the label-free exploration of single-molecule physics and chemistry, which paves a new way for single-molecule detection with high temporal resolution. In this review, we outline the primary advances and potential of the STM break junction technique for qualitative identification and quantitative detection at a single-molecule level. The principles of operation of these single-molecule electrical sensing mainly in three regimes, ion, environmental pH and genetic material detection, are summarized. It clearly proves that the single-molecule electrical measurements with break junction techniques show a promising perspective for designing a simple, label-free and nondestructive electrical sensor with ultrahigh sensitivity and excellent selectivity.

Automatic thresholding method for high-throughput digital polymerase chain reaction fluorescence images with uneven illumination

The demand for nucleic acid detection is increasing worldwide as people are impacted by infectious diseases. Digital polymerase chain reaction (dPCR), which is a revolutionized nucleic acid absolute quantitative analysis technology, plays a significant role in the detection of genetic material. However, the traditional threshold-based methods fail to obtain high-accuracy segmentation results and real-time performance from dPCR fluorescence images with uneven illumination. Therefore, we proposed a robust two-stage automatic thresholding method to deal with dPCR fluorescence images. This method adopts the background subtraction method and the improved Otsu method to extract reaction chambers of dPCR images in two stages. Especially for the second stage, the peak-weaken method based on the improved Otsu method greatly improves the real-time segmentation of uneven illumination regions. Our method significantly improves the accuracy and processing speed of the dPCR image segmentation compared to several mainstream methods. This method could effectively segment the reaction chambers of dPCR images in complex environments. The average precision rate is 99.71%, the execution time is 0.139 s. Experimental results demonstrate that our proposed strategy has superior segmentation performance for high-throughput dPCR fluorescence images with uneven illumination.

Infecciones por gonococo

Neisseria gonorrhoeae is a strict pathogenic microorganism in human beings. Gonorrhea is an infection of the mucosa of the urogenital tract, rectum, or oropharynx. It can affect other areas such as the conjunctiva (especially in newborns) or spread in the form of gonococcemia. The diagnosis is based on identification of the germ following a culture or detection of genetic material in biological samples. Visualization of the intracellular germ is currently being replaced by these new techniques. Antibiotic therapy with ceftriaxone continues to be the treatment of choice.

On the Challenges for the Diagnosis of SARS-CoV-2 Based on a Review of Current Methodologies.

Diagnosis of COVID-19 has been challenging owing to the need for mass testing and for combining distinct types of detection to cover the different stages of the infection. In this review, we have surveyed the most used methodologies for diagnosis of COVID-19, which can be basically categorized into genetic-material detection and immunoassays. Detection of genetic material with real-time polymerase chain reaction (RT-PCR) and similar techniques has been achieved with high accuracy, but these methods are expensive and require time-consuming protocols which are not widely available, especially in less developed countries. Immunoassays for detecting a few antibodies, on the other hand, have been used for rapid, less expensive tests, but their accuracy in diagnosing infected individuals has been limited. We have therefore discussed the strengths and limitations of all of these methodologies, particularly in light of the required combination of tests owing to the long incubation periods. We identified the bottlenecks that prevented mass testing in many countries, and proposed strategies for further action, which are mostly associated with materials science and chemistry. Of special relevance are the methodologies which can be integrated into point-of-care (POC) devices and the use of artificial intelligence that do not require products from a well-developed biotech industry.

Noninvasive Biological Samples to Detect and Diagnose Infections due to Trypanosomatidae Parasites: A Systematic Review and Meta-Analysis.

Unicellular eukaryotes of the Trypanosomatidae family include human and animal pathogens that belong to the Trypanosoma and Leishmania genera. Diagnosis of the diseases they cause requires the sampling of body fluids (e.g., blood, lymph, peritoneal fluid, cerebrospinal fluid) or organ biopsies (e.g., bone marrow, spleen), which are mostly obtained through invasive methods. Body fluids or appendages can be alternatives to these invasive biopsies but appropriateness remains poorly studied. To further address this question, we perform a systematic review on clues evidencing the presence of parasites, genetic material, antibodies, and antigens in body secretions, appendages, or the organs or proximal tissues that produce these materials. Paper selection was based on searches in PubMed, Web of Science, WorldWideScience, SciELO, Embase, and Google. The information of each selected article (n = 333) was classified into different sections and data were extracted from 77 papers. The presence of Trypanosomatidae parasites has been tracked in most of organs or proximal tissues that produce body secretions or appendages, in naturally or experimentally infected hosts. The meta-analysis highlights the paucity of studies on human African trypanosomiasis and an absence on animal trypanosomiasis. Among the collected data high heterogeneity in terms of the I2 statistic (100%) is recorded. A high positivity is recorded for antibody and genetic material detection in urine of patients and dogs suffering leishmaniasis, and of antigens for leishmaniasis and Chagas disease. Data on conjunctival swabs can be analyzed with molecular methods solely for dogs suffering canine visceral leishmaniasis. Saliva and hair/bristles showed a pretty good positivity that support their potential to be used for leishmaniasis diagnosis. In conclusion, our study pinpoints significant gaps that need to be filled in order to properly address the interest of body secretion and hair or bristles for the diagnosis of infections caused by Leishmania and by other Trypanosomatidae parasites.

New insights into the detection and molecular characterization of Cryptosporidium with emphasis in Brazilian studies: a review.

ABSTRACTCryptosporidium spp. is a pathogenic protozoan present in the gastrointestinal tract of several hosts. This protozoan was originally classified as within the Coccidia Class and has recently been reclassified to gregarine based on studies that observed the evolutionary phases from the process of excision and sequencing of the 18S rRNA gene. Molecular biology techniques have become diagnostic tools and have also been used to understand the epidemiology of Cryptosporidium spp., since several species of this genus are very similar morphologically and morphometrically. Molecular techniques have been used in the identification of parasites, at the species and subtypes levels and to study disease transmission. The laboratory diagnosis of human cryptosporidiosis can be made by parasite detection methods, such as optical microscopy, antigens or genetic material detection, as well as serum antibodies raised to Cryptosporidium spp. Molecular methods were developed and allowed, not only an extensive revision of the taxonomy, but also an improvement in the laboratory diagnosis. In Brazil, there are few reports of Cryptosporidium spp. outbreaks in humans and all of them took place in nurseries. A few epidemiological studies developed in Brazil have used molecular methods for the detection of Cryptosporidium spp., as well as genotyping studies of their species and subtypes. The use of real-time PCR, together with microscopy and immunochromatography techniques, would result in a more precise diagnosis of cryptosporidiosis. The analysis of genotypes, subtypes and clonality of Cryptosporidium could be useful to understand and define the prognosis and severity of infections.

Detección de Chlamydia abortus en pérdidas reproductivas de bovinos en la provincia de La Pampa, Argentina

Reproductive losses linked to an infectious etiology in bovine cattle are a major economic concern worldwide. In Argentina, more than 50% of abortion cases have unknown causes. Species belonging to Chlamydiaceae family are frequent etiologic agents of abortion around the world; however, there is yet no information on their prevalence in Argentina. The objective of this work was to identify Chlamydia spp., and particularly C. abortus in reproductive losses from bovine cattle in La Pampa, Argentina. Real time PCR targeting Chlamydiaceae-specific DNA fragments was performed on 251 samples obtained from bovine abortions and stillborns, and ArrayTube was used for species identification on positive samples. Chlamydiaceae DNA was detected in 12 samples of aborted fetuses (4.78%), 83.33% (10/12) accounting for abortions and 16.66% (2/12) for stillborns. C. abortus was detected by ArrayTube in 5 cases (1.99% of all samples, and 41.67% of Chlamydiaceae positive samples). This study shows the first detection of Chlamydiaceae and C. abortus DNA on reproductive losses of bovine cattle in Argentina, and the described prevalence value (4.78%) should be taken as baseline value due to the type of samples analyzed. Detection of genetic material from Chlamydiaceae not matching any of the studied species could be due to intraspecies variants or local species not yet described. Further research on Chlamydia infections in bovine cattle in Argentina is imperative to describe their range, to analyze their economic and zoonotic implications and to make recommendations about prevention and control measures.

Wafer scale fabrication of graphene microelectrode arrays for the detection of DNA hybridization

Since the discovery of graphene, and due to its unique properties, we have witnessed a growing interest in the use of graphene-based devices for applications in the most diverse areas, namely in biosensing, particularly in the detection of genetic material. DNA can bind directly to graphene without the need of a linker and that makes this platform highly interesting for biosensor development.Here, electrochemical chips consisting of 6 independent gold microelectrode arrays as working electrode, and platinum reference and counter electrodes were fabricated at the wafer scale and, after graphene transfer and patterning, were used in the detection of DNA hybridization. Combining the sensitivity of electrochemical impedance spectroscopy and the selectivity of DNA beacons, we were able to detect DNA hybridization in a linear range between 5pM and 5nM, which is in the relevant clinical range for many diseases, with sensitivity to single nucleotide polymorphism.

Controls on eDNA movement in streams: Transport, Retention, and Resuspension

Advances in detection of genetic material from species in aquatic ecosystems, including environmental DNA (eDNA), have improved species monitoring and management. eDNA from target species can readily move in streams and rivers and the goal is to measure it, and with that infer where and how abundant species are, adding great value to delimiting species invasions, monitoring and protecting rare species, and estimating biodiversity. To date, we lack an integrated framework that identifies environmental factors that control eDNA movement in realistic, complex, and heterogeneous flowing waters. To this end, using an empirical approach and a simple conceptual model, we propose a framework of how eDNA is transported, retained, and resuspended in stream systems. Such an understanding of eDNA dispersal in streams will be essential for designing optimized sampling protocols and subsequently estimating biomass or organismal abundance. We also discuss guiding principles for more effective use of eDNA methods, highlighting the necessity of understanding these parameters for use in future predictive modeling of eDNA transport.

Detection of Leishmania infantum in Peripheral Blood Smear and Lymph Node of a Domestic Feline

Background: Cats can become infected and develop feline visceral leishmaniasis (LVF) and manifest variable clinical signs. The diagnosis of the disease in this species has been made more frequently by means of serological and molecular techniques. Different from what happens with dogs, the use of parasitological analysis of lymph node, in clinical practice, for agent detection in felines are infrequent. Therefore, the aim of this study was to disclose the diagnosis of visceral leishmaniasis in a domestic cat from the blood smear analysis and aspiration cytology of lymph node.Case: An adult mixed breed queen, from the city of Campo Grande, Mato Grosso do Sul state, Brazil was referred for clinical care due to the presence of a nodule, with approximately 4 cm, near the inguinal breast. Other parameters, on physical examination, are within the normal range for the specie. For the investigation, additional tests were requested. Eosinophilia was found in a complete blood count (CBC). The association between macroscopic characteristics and cyto­logical findings allowed the diagnosis of inguinal lymph node hyperplastic. Furthermore, amastigotes of Leishmania sp. were visualized both in the peripheral blood sample and in the analyzed material mass. The diagnosis of visceral leishmani­asis and identification of specie of Leishmania was performed by PCR technique (Polymerase Chain Reaction), from the detection of genetic material from Leishmania infantum. After diagnosis of the disease, the owner opted for the conduct of animal euthanasia in the Zoonosis Control Center (CCZ), making impossible the realization of post-mortem examination.Discussion: Hematologic changes described for cats with feline visceral leishmaniasis may be absent or multiple and vari­able, such as for example, anemia, agglutination of erythrocytes, leukocytosis, leukopenia and thrombocytopenia, however, isolated eosinophilia, identified in this report, has not been observed by other authors. The viewing of the amastigote forms in blood smears is considered unusual. A study in which there were examined 1438 samples of dogs, the agent detection did not exceed 0.28%. In cats, there is only one report, of a pancytopenic feline, describing this finding and corroborates for the rarity of the observation of amastigote forms in peripheral blood feline of this report. The use of serologic and mo­lecular methods for the diagnosis of disease in felids has been most frequently used, however, high titers of anti-Leishmania antibodies associated with protective humoral immune response and degenerated genetic material of the amastigotes can be detected by PCR techniques, not identifying active infection. The parasitological diagnosis by means of cytological evaluation of lymph nodes is fast, low cost, presents high specificity and is frequently used in research of canine visceral leishmaniasis, however, the use of this technique for the diagnosis of the disease in cats is still restricted to academic environment. The identification of amastigotes in peripheral blood and lymph nodes, detected in this study, indicates the possibility of using the blood test and, especially, the cytological examination of lymph node for parasitological research of feline visceral leishmaniasis and highlights the need of studies to determine the sensitivity of these tests in this species.Keywords: cat, diagnosis, leishmaniasis, complete blood count, cytology.

Exploring the recovery and detection of messenger RNA and DNA from enhanced fingermarks in blood.

Often in the examination of bloodstained fingermarks discussion occurs around whether to prioritise the fingerprint evidence or focus on the biological evidence. Collecting a sample for genetic profiling may result in the loss of ridge detail that could have been used for fingerprint comparison. Fingermark enhancement and recovery methods along with sample collection methods could also compromise downstream genetic analysis. Previous forensic casework has highlighted circumstances where, after enhancement had been performed, it would have been extremely valuable to both identify the body fluid and generate a DNA profile from the same sample. We enhanced depletion series of fingermarks made in blood, using single treatments consisting of aqueous amido black, methanol-based amido black, acid yellow and leucocrystal violet, and exposure to long wave UV light. We then extracted the DNA and RNA for profiling, to assess the recovery and detection of genetic material from the enhanced fingermarks. We have shown that genetic profiling of bloodstained fingermarks can be successful after chemical enhancement; however it may still be necessary to prioritise evidence types in certain circumstances. From our results it appears that even with visible bloodstained fingermarks, leucocrystal violet can reduce the effectiveness of subsequent messenger RNA profiling. Aqueous amido black and acid yellow also have adverse effects on messenger RNA profiling of depleted fingermarks with low levels of cellular material. These results help with forensic decision-making by expanding knowledge of the extent of the detrimental effects of blood-enhancement reagents on both DNA profiling and body fluid identification using messenger RNA profiling.

Detection of Influenza and Other Respiratory Viruses Carried Out in the Influenza Project - Monitoring Vaccine Effectiveness (I-MOVE).

The project Influenza Vaccine Effectiveness-Monitoring (I-MOVE) is part of the European research carried out by the ECDC (European Center for Disease Prevention and Control), aimed at monitoring the effectiveness of vaccination in Europe during the growing incidence of flu and influenza-like illnesses in the coming epidemic seasons. Laboratory studies using molecular RT-PCR biology methods for detection of genetic material of influenza virus and other respiratory viruses were performed by Voivodeship Sanitary-Epidemiological Stations in Poland. The validation of the results of swabs taken from the nose and throat were carried out in the Department of Influenza Research, National Influenza Center in Warsaw. The study involved 210 samples from patients across Poland. Positive results were recorded for 72.4 % of the samples; influenza virus type A was detected in 43 and type B in 38 cases, whereas in 71 cases other respiratory viruses were detected, which included Human parainfluenza virus type 1–4; Human respiratory syncytial virus type A and B; Human coronavirus 229E/NL63, OC43; Human rhinovirus type A, B, and C; Human enterovirus; and Human adenovirus. The results show that although influenza viruses predominated in the 2010/2011 season in Poland, other flu-like viruses also abounded.

Coxiella burnetiiInfection in Roe Deer during Q Fever Epidemic, the Netherlands

<i>Coxiella burnetii</i>Infection in Roe Deer during Q Fever Epidemic, the Netherlands