Specific Assay Research Articles

Capsular Polysaccharide Production in Bacteria of the Mycoplasma Genus: A Huge Diversity of Pathways and Synthases for So-Called Minimal Bacteria.

Mycoplasmas are wall-less bacteria with many species spread across various animal hosts in which they can be pathogenic. Despite their reduced anabolic capacity, some mycoplasmas are known to secrete hetero- and homopolysaccharides, which play a role in host colonization through biofilm formation or immune evasion, for instance. This study explores how widespread the phenomenon of capsular homopolysaccharide secretion is within mycoplasmas, and investigates the diversity of both the molecules produced and the synthase-type glycosyltransferases responsible for their production. Fourteen strains representing 14 (sub)species from four types of hosts were tested invitro for their polysaccharide secretion using both specific (immunodetection) and nonspecific (sugar dosage) assays. We evidenced a new, atypical homopolymer of β-(1 → 6)-glucofuranose (named glucofuranan) in the human pathogen Mycoplasma (M.) fermentans, as well as a β-(1 → 6)-glucopyranose polymer for the turkey pathogen M. iowae and galactan (β-(1 → 6)-galactofuranose) and β-(1 → 2)-glucopyranose for M. bovigenitalium infecting ruminants. Sequence and phylogenetic analyses revealed a huge diversity of synthases from varied Mycoplasma species. The clustering of these membrane-embedded glycosyltransferases into three main groups was only partially correlated to the structure of the produced homopolysaccharides.

Assessment of the humoral immunity against diphtheria, tetanus, and hepatitis B among children with acute lymphocytic leukemia

Background: Approximately all systemic therapies for childhood affect the immune system. The behavior of the immune system in leukemia patients following chemotherapy is not yet clearly defined. The probability of vaccination failure and the need for revaccination remain challenging for these patients. Aim: To evaluate the humoral immunity against diphtheria, tetanus, and hepatitis B in children with acute lymphocytic leukemia (ALL) immediately and 6 months after chemotherapy. Materials and Methods: In the present prospective cohort study, 21 patients with ALL referred to Mofid Children’s Hospital were studied immediately and 6 months after chemotherapy. Serum samples were collected from patients, and the levels of immunoglobulins (IgG, IgM, IgE, and IgA) antibodies against diphtheria, tetanus, and hepatitis B were determined using specific enzymelinked immunosorbent assay kits. The obtained data were analyzed using Statistical Package for Social Sciences 21 software. Results: A total of 13 males and 8 females with an average age of 8.6 ± 2.5 years were included in the present study. Six months after chemotherapy, the mean level of IgG, IgM, IgE, and IgA displayed an increase of 563.1 units in IgG, 11 units in IgM, 11.3 units in IgE, and 5 units in IgA levels. Moreover, data revealed that 6 months after chemotherapy, the mean level of IgG antibodies displayed an increase of 7.09, 3.43, and 1.03 units against hepatitis B, diphtheria, and tetanus, respectively. A significant relationship was found between the antibody level against diphtheria and the age group of the patients (p = 0.003). Conclusion: Humoral immune status was boosted after 6 months of chemotherapy, though all patients had some extent of lasting immune dysfunction. We indicate that survivors of childhood cancer have ongoing humoral immunological defects and may remain at risk for infectious complications after completion of therapy. Relevance for Patients: The present study indicated that systemic therapies for pediatrics with leukemia affect the immune system. Pediatrics with leukemia may remain at risk for infectious complications after completion of therapy.

The expression of sICAM-1 influenced by Clonorchis sinensis co-infection in CHB patients.

Soluble Intercellular Adhesion Molecule-1 (sICAM-1) has emerged as an inflammatory biomarker of many essential functions. We investigated the level of sICAM-1 influenced by Clonorchis sinensis (C. sinensis) co-infection in chronic hepatitis B (CHB) patients to explore the degree of liver tissue inflammation and liver function damage after co-infection. The study included data from patients with C. sinensis mono-infection (n=27), hepatitis B virus (HBV) mono-infection (n=32), C. sinensis and HBV co-infection (n=24), post-hepatitis B liver cirrhosis (n=18), post-hepatitis B liver cirrhosis co-infected with C. sinensis (n=16), and healthy controls (n=39). The level of sICAM-1 was measured with specific enzyme-linked immunosorbent assay method. Compared to the healthy control group, all the experimental groups had significantly higher serum sICAM-1 levels. The levels of sICAM-1 in co-infected groups were significantly higher compared to the mono-infection groups and were positively correlated with the levels of glutamate aminotransferase (ALT) and aspartate aminotransferase (AST). Our research findings confirmed that co-infection could exacerbate liver tissue inflammation and liver function damage in patients, could raise the sICAM-1 level, and may lead to the chronicity of HBV infection. These results provide clues for pathological mechanism study and formulating treatment plans.

Wastewater-Based Epidemiology of Influenza Viruses: a systematic review

Abstract Background Influenza viruses (IVs) are responsible for annual epidemic outbreaks during the winter months. Beyond conventional surveillance systems, the circulation of these pathogens can be monitored using Wastewater-Based Epidemiology (WBE), an epidemiological tool that describes the health status of an entire community by analyzing the presence of pathogens within wastewater. Systematically reviewing existing experiences in this emerging field is crucial to highlight the best practices to establish a WBE surveillance system providing useful information for public health actions. Methods The study protocol for this systematic review has been registered on the PROSPERO website (registration ID: CRD42024532435). Three academic databases (PubMed, Web of Science and Scopus) were accessed to collect studies examining the presence of IVs in wastewater. Studies included were those reporting quantitative measures of viral concentration or detailing molecular/phylogenetic characterization of IVs in wastewater. Results Databases were searched on July 8th, 2024. After de-duplication and screening of the 400 remaining records, 42 were included in the review. IVs detection in wastewater was reported in all papers except from one, although with high variability in terms of positivity titers and concentration levels. More than half of the collected papers (52.4%) evaluated association between environmental viral concentration and clinical data, generally showing concordance between the two aspects. Moreover, 28.6% of papers described subtyping of IVs in wastewater through different methods (specific PCR assays being the most used). Conclusions WBE is a promising approach for surveilling viral circulation, and IVs represent a high value target for implementing this innovative system. Systematically reviewing previous experiences can be useful to obtain information for the establishment of a novel environmental surveillance system of Influenza Viruses Key messages • Nucleic acids belonging to Influenza Virus are detectable in wastewater, even though we report high levels of variability in sampling techniques, laboratory methods and data management systems. • Association between environmental data and number of clinical cases is reported to be generally strong in collected papers; subtyping of IVs present in wastewater is also feasible.

A highly sensitive and specific real-time quantitative polymerase chain reaction assay for Perkinsus marinus detection in oysters

Oyster aquaculture is one of the fastest-growing food production industries worldwide; however, it faces a significant challenge from the protist Perkinsus marinus, particularly in the USA. Although several quantitative molecular diagnostic methodologies are available for identifying diseases caused by P. marinus, the primer pairs used therein led to non-specific identification of other Perkinsus spp. Hence, a quantitative real-time PCR (Pm-qPCR) assay specific for P. marinus was developed using a TaqMan-based probe with the internal quencher in this study. A primer pair and probe specific to P. marinus were designed from a hypothetical protein of P. marinus collected from the whole-genome shotgun sequence database of the National Center for Biotechnology Information (NCBI). In silico analysis using homologous sequences of P. olseni and P. chesapeaki confirmed the high specificity of primers designed in this study. The Pm-qPCR assay was performed using seven different strains of P. marinus, P. olseni, and P. chesapeaki, revealing high specificity and sensitivity for detecting only P. marinus strains. In conclusion, it was demonstrated that Pm-qPCR can effectively and accurately diagnose P. marinus with high specificity and sensitivity. This assay is promising for monitoring oyster health and disease management in ecosystems and aquaculture.

Comparative evaluation of PCR and loop-mediated isothermal amplification (LAMP) assays for detecting Pasteurella multocida in poultry

ABSTRACT Aims To develop a colourimetric loop-mediated isothermal amplification (LAMP) assay for the detection of Pasteurella multocida in clinical poultry samples and compare the performance of this assay with PCR. A secondary aim was to evaluate a simple DNA extraction method that could enable LAMP-based testing in the field without the need for specialised laboratory equipment. Methods Primer sets for both LAMP and PCR were designed to amplify the KMT1 gene of P. multocida. DNA was extracted from 12 P. multocida isolates using a commercial extraction kit, and subjected to analysis using both LAMP and PCR. The analytical specificity of the LAMP assay was evaluated by testing it against a panel of 12 unrelated bacterial species, and the analytical sensitivity (limit of detection) was determined through testing of serial dilutions of the target DNA and compared to that of PCR. Subsequently, cloacal swabs (n = 40) from a commercial turkey flock were subjected to analysis using both LAMP and PCR assays, using a rapid DNA extraction method and a commercial extraction kit. Clinical sensitivity and specificity of the LAMP assay were calculated in comparison to PCR. Results A single DNA fragment of the expected size (∼ 200 base pairs), was amplified by PCR from 12 P. multocida isolates, which were also all positive by the LAMP assay. The identity of all PCR amplicons was confirmed by sequencing. Both PCR and LAMP showed similar analytical sensitivity, with a LOD of 20 pg of target DNA. As neither PCR nor LAMP assays produced positive results with 12 non-related bacterial species, the analytical specificity was assessed as 100%. However, LAMP demonstrated lower clinical specificity (94.74%) compared to PCR (100%) when 40 clinical samples were tested. None of the DNA samples extracted using the simplified DNA extraction method were amplified by either LAMP or PCR. Conclusion The LAMP assay developed in this study exhibits comparable performance to PCR in detecting P. multocida. Clinical relevance The use of a rapid and portable DNA extraction method, in conjunction with LAMP assays, could create opportunities for point-of-care testing for fowl cholera in field settings.

Development of a new tool to detect Melampsora medusae f.sp. tremuloidae causing rust disease on Populus tremuloides.

Melampsora medusae f. sp. tremuloidae is a quarantine organism for the EU. In North America, this fungus causes rust disease on Populus tremuloides. In Europe, Populus tremula, an aspen closely related to P. tremuloides, is widespread and plays an important ecological role. Introduction of M. medusae f. sp. tremuloidae into Europe could be a major risk if this forma specialis could evolve and become virulent on P. tremula. To date no PCR-based assay exists to specifically detect M. medusae f. sp. tremuloidae. In this study, a sensitive and specific real-time PCR assay has been developed based on the 28S rDNA. The assay proved to be reliable using many real-time PCR kits and platforms. It can be used to monitor the introduction and the spread of M. medusae f. sp. tremuloidae in the context of phytosanitary regulations.

Phytochemical Characterization and Assessment of the Wound Healing Properties of Three Eurasian Propolis

Objectives: The objective of this study is to evaluate the wound healing potential of Eurasian propolis by analyzing the phytochemical profile and the biological effects of three representative propolis samples. Methods: Specific colorimetric assays were used to estimate the total phenolic and flavonoid contents and the triterpenoids content. Some of the main components of Eurasian propolis (pinocembrin, pinobanksin, CAPE, chrysin and galangin) were analyzed using HPLC-DAD. Scavenging activity and total antioxidant capacity were assessed through DPPH and ORAC assays, respectively. Human keratinocyte, fibroblast, and monocytic cell lines were used for the biological in vitro analyses. The direct wound healing properties were tested through scratching assays and ELISA kits for the assessment of the production of growth factors (FGF-7, Latency Associated Peptide-LAP), while the indirect effects were evaluated through the estimation of the levels of MMP9, IL-1β, IL-8, and TNF-α using ELISA kits together with a cell-free test on the inhibition capacity on collagenases. Network Pharmacology analysis was employed to further explore possible mechanisms of the action of propolis on the healing process. Results: The analyses confirmed the high phenolic content of Eurasian propolis (142.50–211.30 mg GAE/g), dominated by flavonoids (95.50–196.80 mg Galangin Equivalents/g), and terpenes (431.50–650.00 mg β-sitosterol Equivalents/g), while also verifying the significant antioxidant (4.9–8.9 mM/g Trolox Equivalents) and antiradical (DPPH IC50 26.1–54.4 μg/mL) activities. The samples showed indirect wound healing properties by mitigating inflammation and remodeling (reduced IL-1β and MMP9) and potentially modulating the immune response (upregulated IL-8). In vitro studies confirmed these effects, demonstrating decreased MMP9 production and collagenase inhibition when cells were co-treated with propolis and a stressor. Propolis also suppressed IL-1β release in fibroblasts, although its impact on TNF-α was inconclusive. Notably, co-treatment upregulated IL-8 in monocytes, suggesting a potential immunomodulatory role. Conclusions: Eurasian propolis may not directly stimulate cell proliferation during wound healing. Its anti-inflammatory and immunomodulatory properties could indicate an indirect contribution in helping the process.

Optimisation of induction and purification protocols of recombinant 22.6kDa tegumental protein of Schistosoma spindale inprokaryotic vector

Schistosomosis is a prevalent zoonotic parasitic disease affecting both humans and animals on a global scale, with an estimated 165 million cattle and 200 million people impacted worldwide. Eventhough, serological methodologies designed for the identification of specific antibodies targeting parasitic antigens are esteemed for their high sensitivity, there are criticisms due to their incapacity to reliably indicate active infection, inability to correlate with the intensity of infection and lack of specificity. Enhancing the specificity of serological assays presents a significant challenge, primarily attributable to the identification and synthesis of specific antigens. In addressing these limitations, recombinant technology with specific immunogenic proteins as candidate antigens emerges as a viable alternative. In this study, induction of 22.6 kDa recombinant tegument protein of Schistosoma spindale was achieved using 0.6 mM concentration of IPTG at 37°C for four hours. Nickel chelating affinity chromatography was employed for protein purification, yielding maximum protein concentration at 75mM elution. Subsequently, the dialysis technique was employed to remove contaminants, while lyophilisation method was employed for protein concentration. The protein concentration post dialysis was measured at 0.220 mg/mL, while lyophilisation resulted in a concentration of 2mg/m Keywords: 22.6 kDa tegumental protein, Schistosoma spindale, dialysis, lyophilisation

An immunoassay for the quantification of phosphorylated α-synuclein at serine 129 in human cerebrospinal fluid.

The link between alpha Synuclein (α-Syn) phosphorylation and Parkinson's disease pathogenesis has not been fully elucidated, in part due to analytical methods with finite specificity and sensitivity, resulting in conflicting data on pathophysiological levels of the protein.One factor hindering the assessment of the role of pSer129 α-Syn is the lack of a fit for purpose assay. Antibodies were assessed for quantification of pSer129 α-Syn, resulting in a sensitive and specific assay suitable for use in Parkinson's disease and control CSF, with no significant difference found between the two populations. Total α-Syn was measured using a commercial kit, demonstrating a positive correlation between total and pSer129 α-Syn.This adds to available methods for pSer129 α-Syn in support of α-synucleinopathy research.

Clinical performance of a new lateral flow immunoassay for xylazine detection.

Xylazine is a potent sedative used in veterinary medicine. Recently, recreational drugs such as fentanyl havebeen found to contain xylazine, increasing the risk of respiratory depression and death. Despite a similar presentation to opioid overdose, patients who ingest xylazine donot respond to treatment with Narcan. Therefore, rapid detection of xylazine could improve patient management and prevent adverse outcomes. We evaluated the XYL500 one-step xylazine drug of abuse test for its ability to detect xylazine in 152 urine samples from patients on chronic opioid therapy for pain management or in treatment for substance use disorder. Results were compared to LC-MS/MS as the reference method. Precision, cross-reactivity, interference and stability studies were performed. Pooled patient samples were consistently negative or positive when tested five times on the same day and over three days of testing. The diagnostic sensitivity, specificity and accuracy of the XYL500 assay were 74, 98, and 82 % respectively, as compared with LC-MS/MS. XYL500 detected 77 of the 104 LC-MS/MS positive samples identified in our initial evaluation, including some that contained low levels of xylazine (n=8), <10 ng/mL. Minimal cross-reactivity with other opioid analgesics and commonly encountered drugs was seen with only one false positive result. Interferences by common urine contaminants were negligible. Specimens were stable up to 160 days refrigerated and up to 80 days at room temperature. XYL500 allows for rapid detection of xylazine, illustrating its utility in monitoring patients who ingested recreational drugs containing the additive, xylazine, and its potential to improve patient management.

Systematic review of the economic impact of novel Mycobacterium tuberculosis specific antigen-based skin tests for detection of TB infection compared with tuberculin skin test and interferon-gamma release assays.

The Purified Protein Derivative tuberculin skin tests (TST) and blood-based Mycobacterium tuberculosis (M.tb) specific interferon-gamma release assays (IGRA) are the currently used tests for identifying individuals with TB infection for preventive treatment. However, challenges around access and implementation have limited their use. Novel M.tb specific skin tests (TBST) such as Diaskintest, ESAT6-CFP10 (C-TST), C-Tb (also known as Cy-Tb), and DPPD may provide accurate and scalable options but evidence synthesis on their economic impact is lacking. We conducted two separate systematic reviews to compare the costs and cost-effectiveness of (1) the novel skin tests TBST (primary), and (2) TST and IGRA tests (secondary), to support WHO guideline development. We searched for articles presenting economic evaluations of the diagnostic tests using a health provider perspective and related to TB infection in humans. We considered papers written in English, Chinese or Russian. In the primary review, eight studies for novel TBST were found. One study in Brazil assessed cost-effectiveness of C-TST and Diaskintest and seven in Russia assessed the Diaskintest, while none evaluated C-Tb or DPPD. The review showed on average, Diaskintest kit costs (in 2021 USD) $1.60 (1.50 - 1.70), while full unit costs were estimated at $5.07. C-TST unit cost was $9.96. The second review found 32 articles on IGRA and/or the TST. These presented an average TST full unit cost of $37.88, and $87.81 for IGRA. Studies' quality for TBST was limited while high-quality studies were found for TST and IGRA tests. In conclusion, there is limited evidence regarding the costs and cost-effectiveness of novel TBST. Conversely, there is substantial evidence for TST and IGRA tests, but most studies were performed in high-income and low-TB burden settings and their cost-effectiveness varied between and within risk groups without clear economic consensus.

Anaplasma platys and Rickettsia massiliae in Rhipicephalus sanguineus sensu stricto ticks collected on dogs in the Patagonian region of Argentina.

The aim of this study was to examine the presence of tick-borne rickettsial bacteria in Rhipicephalus sanguineus sensu stricto ticks collected from dogs in the Patagonian region of Argentina. Fourteen stray dogs from Valcheta, Río Negro province, Argentina were examined for the presence of R. sanguineus s.s. ticks. Ninety ticks were collected and identified to species level. DNA was extracted and analysed by conventional PCR assays for the presence of tick-borne bacteria belonging to the genera Anaplasma, Ehrlichia and Rickettsia. Thirty-three tick pools were tested by different PCR assays of which 3 were positive for Anaplasmataceae bacteria. From the 3 Anaplasmataceae positive samples, 2 partial 16S rDNA sequences were generated and belonging to Anaplasma platys, the causative agent of canine cyclic thrombocytopenia. Two tick samples were positive in Rickettsia specific PCR assays and were identified by phylogenetic analysis as Rickettsia massiliae, a member of the spotted fever group rickettsiae. The results of this study demonstrate the molecular detection of 2 rickettsial bacteria in R. sanguineus s.s. in a region of Argentina where no data were available so far.

Development of a simple allele-specific PCR for the detection of pathogenic Vibrio cholerae O1 and O139 in seafood

This study developed a simple allele specific (AS) PCR assay for the detection of pathogenic Vibrio cholerae strains in seafood. A pair of primers, (1) an AS primer targeting the mutation at site G450 in the hapR2 allele and a (2) common forward primer, were designed to detect the pathogenic O1 and O139 strains. An artificial mismatch was also added to the AS primer to increase its specificity to the target allele. The PCR assays using different DNA polymerases of varying efficiencies were then optimized. The application of the AS primers in both conventional and real-time PCR assays was facilitated with enriched seafood samples spiked with known concentration of pathogenic V. cholerae. The designed AS primers showed high specificity and sensitivity to the hapR2 allele for both conventional and real-time PCR assays. Enrichment of seafood samples for 8 h is recommended to detect viable pathogenic V. cholerae presence in seafood. In this study, we developed an AS-PCR that is simple, rapid, and a low-cost alternative for routine detection of pathogenic V. cholerae in different seafood samples thus aiding in the surveillance and implementation of food safety measures at the seafood post-harvest level.

Koalas, friends and foes—The application of airborne eDNA for the biomonitoring of threatened species

Abstract Curbing global wildlife population declines will necessitate the protection of their habitat, and subsequent robust baseline information about wildlife that use and occupy it. Collating such information, however, remains a challenging and costly endeavour. Little did we know that traces of wildlife presence float in the air and can be detected through traces of DNA. Here, we deployed samplers to test the applicability of collecting airborne eDNA for the detection of a threatened species, the koala, and its co‐occurring terrestrial mammalian community. We develop a novel species specific qPCR assay to detect the presence of koalas and applied this in concert with a meta‐barcoding approach to detect the co‐occurring mammalian community. Through sampling of airborne particles, we successfully detected koala presence accurately to habitat patch level, alongside 16 unique taxonomic assignments, successfully assigning 11 of these to species level including detections belonging to the wallaby, antechinus, members of possum family and invasive species such as foxes, domestic dogs and hares. Synthesis and applications: We demonstrate the potential of airborne eDNA for the detection of threatened terrestrial wildlife and their surrounding ecological community under natural conditions. With achievable optimisations we detail how airborne eDNA may be applied for the management and monitoring of threated species for enhanced conservation efforts.

First molecular investigation to detect avian Mycoplasma species in clinical samples from laying-hen farms in Tunisia

BackgroundAvian mycoplasmas are known pathogens, which cause severe economic losses in poultry flocks. PCR is a rapid, sensitive, and less expensive diagnostic tool than culture for the identification of mycoplasmas in poultry farms. The objective of this study was to determine by PCR the presence of Mycoplasma spp., Mycoplasma gallisepticum (MG), Mycoplasma synoviae (MS), and Mycoplasma pullorum (MP) in laying hens located in the Sfax region, in the South of Tunisia.ResultsA total of 781 tracheal swabs were collected from 13 laying-hen farms without clinical signs at the date of sampling. MP was detected by a newly described specific PCR assay. The prevalence calculated from PCR results at the flock level was 100% for Mycoplasma spp., 0% for MG, 84.6% for MS and 61.5% for MP. The overall prevalence at the animal level was 38.7% for Mycoplasma spp., 0% for MG, 25% for MS and 6.4% for MP. The overall prevalence of 100% of avian mycoplasmas in laying-hen farms (38.7% prevalence at the animal level) shows an alarming situation.ConclusionsThese results underline the importance of monitoring the emergence and spread of Mycoplasma strains in farms in order to decrease economic losses due to mycoplasmoses.

Evaluation of the Seegene Allplex™ RV master assay for one-step simultaneous detection of eight respiratory viruses in nasopharyngeal specimens

BackgroundThe Seegene Allplex™ RV Master (RVM) assay is a one-step multiplex real-time reverse transcription polymerase chain reaction (RT-PCR) system for detecting eight viral respiratory pathogens from nasopharyngeal swab, aspirate, and bronchoalveolar lavage specimens. The eight RVM targets are: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Influenza A (Flu A), Influenza B (Flu B), Human respiratory syncytial virus (RSV), adenovirus (AdV), rhinovirus (HRV), parainfluenza virus (PIV), and metapneumovirus (MPV). The assay is based on Seegene’s multiple detection temperature (MuDT) technology and provides cycle threshold (Ct) values for each of its viral targets upon PCR completion. ObjectiveWe aimed to evaluate the diagnostic performance of the RVM assay by calculating sensitivity, specificity, accuracy, Positive Predictive Value (PPV), Negative Predictive Value (NPV), Positive Percent Agreement (PPA), Negative Percent Agreement (NPA), and Overall Percent Agreement (OPA) compared to definite diagnosis and analogous reference assays. Study designDiagnostic sensitivity, specificity, accuracy, PPV, and NPV were calculated by comparing the results of the RVM assay to that of definite diagnosis assays; while PPA, NPA, and OPA were calculated by comparing results of the RVM assay to that of analogous reference products. Definite diagnosis and reference methods comprised whole genome sequencing and PCR genotyping, the Allplex™ SARS-CoV-2/FluA/FluB/RSV and Respiratory Panels 1, 2, and 3 assays (Seegene), and the Xpert® Xpress SARS-CoV-2/FluA/FluB/RSV Plus assay (Cepheid). Reproducibility of the RVM assay using fully-automated and semi-automated nucleic acid (NA) extraction workflows and as performed by independent operators was also assessed. In total, 249 positive respiratory specimens and at least 50 negative specimens for each target tested were used for this evaluation study. ResultsSensitivity, specificity, accuracy, PPV, NPV, PPA, NPA, and OPA ranged from 95.7 % to 100 % for detecting all eight targets tested on the RVM assay. Reproducibility PPA, NPA, and OPA between automated and semi-automated NA extraction workflows were all >97.9 %, while the reproducibility PPA, NPA and OPA between independent operators were all 100 %. ConclusionThese results demonstrate a high level of sensitivity, specificity, accuracy and diagnostic predictive value of the RVM assay and high agreement with comparable reference assays in identifying all eight of its targets. Taken together, our study underscores the diagnostic utility of the RVM assay in detecting eight viral respiratory pathogens.

A preliminary study on detecting human DNA in aquatic environments: Potential of eDNA in forensics

Human environmental DNA (eDNA) application have not been fully applied or adequately considered in the fields of eDNA and forensics. Nonetheless, this technique holds great potential as a complementary tool for detecting human DNA in aquatic environments, particularly in cases involving crimes connect to such environments. However, the detectability or stability of eDNA can vary depending on several factors. Therefore, this preliminary study investigates the detection and degradation rates of human eDNA, as well as the recovery of nuclear short tandem repeat (STR) profiles and mitochondrial DNA (mtDNA) sequencing, using water samples from both saltwater and freshwater sources. To conduct the experiment, whole human blood was spiked into the water samples. Water samples were then filtered using a 5 µm pore size filter, and samples were collected at various time intervals up to 23 days. A human specific qPCR assay targeting HV1 region of human mtDNA was used to detect human eDNA. Results demonstrated that human eDNA remains detectable for up to 36 hours in freshwater samples and up 84 hours in saltwater samples. The limit of detection (LOD) of human eDNA, (205 copies/µl), was achieved after 60 hours in freshwater and 180 hours in saltwater samples. Partial STR profiles could be recovered up to 24 hours for freshwater and saltwater. Results from mtDNA sequencing indicate that full mtDNA profiles could be recovered from freshwater samples up to 48 hours and remained detectable up to 72 hours in saltwater. Overall, the findings of this study underscore the importance of considering and incorporating human eDNA analysis as a valuable tool in forensic practice. By harnessing the power of eDNA, law enforcement agencies can enhance their investigation capabilities, improve the accuracy of forensic reconstructions, and ultimately contribute to the resolution of cases involving aquatic environments. Further research and validation are needed to optimize and expand the utilization of eDNA techniques in forensic investigations.

On-Site Visualization Assay for Tumor-Associated miRNAs: Using Ru@TiO2 as a Peroxidase-like Nanozyme.

Accurate diagnosis of highly aggressive and deadly tumors is essential for effective treatment and improved patient outcomes, and microRNAs (miRNAs) have emerged as crucial biomarkers for their roles in tumor initiation, progression, and metastasis. Herein, we present an on-site visualization colorimetric assay for tumor-associated miRNAs using ruthenium nanoparticle decorated titanium dioxide nanoribbon (Ru@TiO2) as a peroxidase-like (POD) nanozyme. Remarkably, the Ru@TiO2 nanozyme can catalyze the oxidation of chromogenic substrates through its POD-like activity, which is effectively inhibited by pyrophosphate generated during the rolling circle amplification process, thereby enabling miRNA detection through a visible colorimetric readout. This approach provides a highly sensitive and specificity assay for miRNAs in diluted human serum with a detection limit of 100 pM. It shows great potential for clinical diagnostics and biological research, offering a promising tool for early cancer diagnosis and molecular diagnostics.

Construction of a chemiluminescent biosensor based on enzymatic extension and click chemistry for sensitive measurement of MGMT activity in human breast tissues

O6-methylguanine methyltransferase (MGMT) is responsible for dealkylation of naturally occurring O6-methylguanines, and it is closely related with DNA replication, transcription, and cancers. Herein, we develop a chemiluminescent biosensor based on enzymatic extension and click chemistry for sensitive measurement of MGMT activity. When MGMT is present, the MGMT-catalyzed demethylation reaction initiates the cleavage of biotinylated dumbbell probes by PvuII restrictive enzyme, releasing two DNA fragments with 3′-OH end. The resultant DNA fragments can trigger terminal transferase (TdT)- and click chemistry-assisted isothermal amplification to obtain abundant G-rich sequences. The G-rich sequences can be captured by magnetic beads to produce a high chemiluminescence signal. This biosensor can greatly amplify the chemiluminescence signal, facilitating label-free and template-free measurement of MGMT. Especially, the introduction of dumbbell probe and PvuII enzyme can efficiently eliminate the false positive and improve the assay specificity. This biosensor possesses high sensitivity with a detection limit of 1.4 × 10−9 ng/μL, and it may accurately quantify the intracellular MGMT. Importantly, this biosensor can be used to screen the MGMT inhibitors and distinguish the MGMT level in breast tumor tissues and normal tissues, with great potential in drug discovery and cancer diagnosis.