Two-step Assay Research Articles

Development and validation of a two-step assay for differentiation of Penis et testis cervi from cervi nippon Temmink and cervi elaphus Linnaeus based on specific-species polymerase chain reaction-restriction fragment length polymorphism patterns

Penis et testis cerviis one of the precious traditional Chinese medicines (TCMs) in China, which, whenauthentic, is sourcedfrom species ofCervi nippon Temmink(C. Nippon)andCervi elaphus Linnaeus(C.elaphus L).Authentication of species ofPenis et testis cerviremainstechnically challenging. This study attempted to modify the species-specific polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (PCR-RFLP) method using restriction enzymeHpy188III for the identification ofPenis et testis cervispecies.First, thecytochrome b (Cytb)and cytochrome c oxidase subunit I (COI) genes in mitochondrial DNAfrom two authentic species and their counterfeitsor adulterantswere downloaded and analyzed with bioinformatics tools to establish the phylogenetic tree, then a series of primers were designed, and the restriction enzymesites were confirmed. Genomic DNA was extracted fromall samplesby the improved SDS method. Species-specific PCR reactions were optimizedto differentiate authentic Sika deer and ReddeerPenis et testis cervifrom their counterfeits or adulterants. PCR-RFLP using single restriction enzyme Hpy188III was performed inCytb regionsto identify the species of Sika deer and Reddeer. Simultaneously, recombination plasmid carrying thecytb fragmentcould be cloned, sequenced, and blasted.The assay specificity and sensitivity were evaluated, and30 batches of commercial samples werevalidated. Thephylogenetic treeshowed that the physico-chemical properties of Sika deer and Reddeer variants were similar.Sufficient DNA extracts were extracted from all samples used inthe improved SDS method.Establishment and optimization of a species-specific PCR reactionsand PCR-RFLPsystem were undertaken to identify deer species inPenis et testis cervisamples. Aspecific PCR-RFLP pattern with three distinct products of sizes 155, 113, and 204 bp was exhibited in Sika deerspecies,and two distinct products of 204 and 268 bp in size were evidentin Reddeerspecies,which were significantly different from the pattern of thecounterfeitsor adulterants.The distinctive PCR-RFLP fingerprint pattern using the restriction enzyme Hpy188III is a rapid and reliable assay for identification of thePenis et testis cervispecies.

One-tube, two-step isothermal amplification of histatin 3 mRNA for saliva screening

Saliva samples are frequently collected at crime scenes. Salivary mRNA profiling, such as that of histatin 3 (HTN3), is a highly specific approach that overcomes the limitation of traditional amylase tests. However, typical mRNA detection methods based on reverse transcription PCR (RT-PCR) are time-consuming and labor-intensive. Here, we report a one-tube, two-step isothermal amplification assay for HTN3 mRNA, which enables rapid, simple, and sensitive screening of saliva. The first step is an RT-recombinase polymerase amplification (RT-RPA) assay at 42 °C for 20 min; the second step is a loop-mediated isothermal amplification (LAMP) assay at 65 °C for 30 min. The reactions can be performed in a closed tube, and the products are detected using real-time fluorescence analysis. The assay sensitivity was 0.5 µL of saliva samples. It also detected HTN3 mRNA in mixed and mock samples, demonstrating its applicability to actual forensic samples. These findings suggest that our strategy is promising for screening of saliva from forensic samples.

Melt-Encoded-Tags for Expanded Optical Readout in Digital PCR (METEOR-dPCR) Enables Highly Multiplexed Quantitative Gene Panel Profiling.

Digital PCR (dPCR) is an important tool for precise nucleic acid quantification in clinical setting, but the limited multiplexing capability restricts its applications for quantitative gene panel profiling. Here, this work describes melt-encoded-tags for expanded optical readout in digital PCR (METEOR-dPCR), a simple two-step assay that enables simultaneous quantification of a large panel of arbitrary genes in a dPCR platform. Target genes are quantitatively converted into DNA tags with unique melting temperatures through a ligation approach. These tags are then counted and distinguished by their melt-curve profiles on a dPCR platform. A multiplexing capacity of M^N, where M is the number of resolvable melting temperature and N is the number of fluorescence channel, can be achieved. This work validates METEOR-dPCR with simultaneous DNA copy number profiling of 60 targets using dPCR in cancer cells, and demonstrates its sensitivity for estimating tumor fraction in mixed tumor and normal DNA samples. The rapid, quantitative, and highly multiplexed METEOR-dPCR assay will have wide appeal for many clinical applications.

Exploring the use of modified in vitro digestion assays for the evaluation of ritonavir loaded solid lipid-based formulations

Solid lipid-based formulations (sLBFs) have the potential to increase the oral bioavailability of drugs with poor solubility in water, while counteracting some of the disadvantages of liquid LBFs. The most common experimental set-up to study the performance of LBFs in vitro is the lipolysis assay, during which the LBFs are digested by lipases in an environment mimicking the human small intestine. However, this assay has failed in many cases to correctly predict the performance of LBFs in vivo, highlighting the need for new and improved in vitro assays to evaluate LBFs at the preclinical stage. In this study, the suitability of three different in vitro digestion assays for the evaluation of sLBFs was assessed; the classic one-step intestinal digestion assay, a two-step gastrointestinal digestion assay and a bicompartmental assay permitting the simultaneous monitoring of digestion and permeation of the active pharmaceutical ingredient (API) across an artificial membrane (Lecithin in Dodecane – LiDo). Three sLBFs (M1-M3) with varied composition and ritonavir as model drug were prepared and examined. When comparing the ability of these formulations to keep the drug solubilized in the aqueous phase, all three assays show that M1 performs better, while M3 presents poor performance. However, the classic in vitro intestinal digestion assay fails to provide a clear ranking of the three formulations, something that is more evident when using the two modified and more physiologically relevant assays. Also, the two modified assays provide additional information about the performance of the formulations including the performance in the gastric environment and intestinal flux of the drug. These modified in vitro digestion assays are valuable tools for the development and evaluation of sLBFs to make better informed decisions of which formulations to pursue for in vivo studies.

Role of probe design and bioassay configuration in surface enhanced Raman scattering based biosensors for miRNA detection

The accurate design of labelled oligo probes for the detection of miRNA biomarkers by Surface Enhanced Raman Scattering (SERS) may improve the exploitation of the plasmonic enhancement. This work, thus, critically investigates the role of probe labelling configuration on the performance of SERS-based bioassays for miRNA quantitation. To this aim, highly efficient SERS substrates based on Ag-decorated porous silicon/PDMS membranes are functionalized according to bioassays relying on a one-step or two-step hybridization of the target miRNA with DNA probes. Then, the detection configuration is varied to evaluate the impact of different Raman reporters and their labelling position along the oligo sequence on bioassay sensitivity. At high miRNA concentration (100–10 nM), a significantly increased SERS intensity is detected when the reporters are located closer to the plasmonic surface compared to farther probe labelling positions. Counterintuitively, a levelling-off of the SERS intensity from the different configurations is recorded at low miRNA concentration. Such effect is attributed to the increased relative contribution of Raman hot-spots to the whole SERS signal, in line with the electric near field distribution simulated for a simplified model of the Ag nanostructures. However, the beneficial effect of reducing the reporter-to-surface distance is partially retained for a two-step hybridization assay thanks to the less sterically hindered environment in which the second hybridization occurs. The study thus demonstrates an improvement of the detection limit of the two-step assay by tuning the probe labelling position, but sheds at the same time light on the multiple factors affecting the sensitivity of SERS-based bioassays.

Relationship between anthropometric and body composition parameters and anti-SARS-CoV-2 specific IgG titers in females vaccinated against COVID-19 according to the heterologous vaccination course: A cohort study.

The aim of this cohort study was to evaluate the relationship between anthropometric and body composition parameters and anti-SARS-CoV-2 IgG titers in a group of females who were vaccinated against COVID-19 with two doses of ChAdOx1 vaccine and then boosted with the BNT162b2 vaccine. The study group consisted of 63 women. Basic demographic and clinical data were collected. To assess the anti-SARS-CoV-2 immunoglobulin G titers following the vaccination, five blood draws were performed: 1) before the first dose, 2) before the second dose, 3) 14-21 days after the primary vaccination, 4) before the booster, and 5) 21 days after the booster. Blood samples were analyzed using a two-step enzymatic chemiluminescent assay. Body mass index and body composition were evaluated using bioelectrical impedance analysis. To select the most distinguishing parameters and correlations between anthropometric and body composition parameters and anti-SARS-CoV-2 IgG titers, factor analysis using the Principal Component Analysis was conducted. Sixty-three females (mean age: 46.52 years) who met the inclusion criteria were enrolled. 40 of them (63.50%) participated in the post-booster follow-up. After receiving two doses of the ChAdOx1 vaccine, the study group's anti-SARS-CoV-2 IgG titers were 67.19 ± 77.44 AU/mL (mean ± SD), whereas after receiving a heterologous mRNA booster, the level of anti-SARS-CoV-2 IgG titers was about three-times higher and amounted to 212.64 ± 146.40 AU/mL (mean ± SD). Our data shows that seropositivity, obesity, non-fat-related, and fat-related body composition parameters all had a significant effect on the level of IgG titer after a two-dose vaccination of ChAdOx1. However, only non-fat-related and fat-related body composition parameters had a significant effect on the IgG titer after booster vaccination. COVID-19 infection before the first dose of vaccination is not related to IgG titer after booster administration. Body composition has a significant effect on the production of anti-SARS-CoV-2 IgG after booster vaccination in females.

Validation of the AnticFast® Beta-Lactams Rapid Test Kit for Detection of Beta-Lactams (Penicillins and Cephalosporins) in Raw Cow's Milk: AOAC Performance Tested MethodSM 032303.

AnticFast® Beta-Lactams Rapid Test Kit is a qualitative two-step (2 min + 5 min) rapid lateral flow assay to detect β-lactam (penicillins and cephalosporins) antibiotic residues in raw commingled cow's milk. The method performance was evaluated according to Commission Decision 2002/657/EC, Commission Implementing Regulation 2021/808, and Community Reference Laboratories Residues Guidelines for the Validation of Screening Methods for Residues of Veterinary Medicines. The AnticFast Beta-Lactams Rapid Test Kit was evaluated for detection capability, selectivity, false-positive results, repeatability, robustness, suitability for various milk types and milk compositions, milks from various species, and test kit consistency and stability. Samples included milks spiked at concentrations bracketing the EU maximum residue limits (MRLs) for β-lactams as well as bulk farm and tanker milks. The AnticFast Beta-Lactams Rapid Test Kit is specific for the detection of β-lactams in milk and does not detect compounds from other antibiotic families. Interference was seen with clavulanic acid, a β-lactamase inhibitor, which was expected. The test can detect all residues of β-lactams (penicillins and cephalosporins) present on the EU-MRL list for milk at their respective MRL except for desfuroylceftiofur and cephalexin, which were above the MRL. No false positives were detected in the 602 (300 blank farm and 302 tanker load) samples tested. Robustness testing indicated that the detection in heat-treated milk types may be slightly hampered. For substances with a detection capability well below the MRL, this interference does not cause problems since detection at MRL remains guaranteed, but care should be taken for substances with a CCβ at or near their MRL. Diminished sample flow was seen with reconstituted milk powder and blank ewes' milk, so sample flow should always be verified for these milk types. Results of this validation show that the AnticFast Beta-Lactams Rapid Test Kit is a reliable test for rapid screening of raw cows' milk for residues of β-lactam antibiotics. AnticFast Beta-Lactams Rapid Test Kit is an easy, realiable, robust and highly specific test for screening of raw cows' milk for residues of penicillins and cephalosporins.

The relation between vitamin D insufficiency and thyroid hormones in Iraqi women living in Al-Khalidiya, Anbar Province

Background: Vitamin D insufficiency is a worldwide issue in all age groups. It wasdiscovered that vitamin D insufficiency is linked to autoimmune disorders such as Irritablebowel illness, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, andrheumatoid arthritis. Vitamin D is also connected to the development of thyroid autoimmunedisease and plays an important role in endocrinopathies including type 1 and type 2 diabetes,adrenal diseases, and polycystic ovarian syndrome.Materials and methods: 100 samples were collected for women from Al-Khalidiya city inAnbar province (50 samples likely to have thyroid problems, 50 healthy samples). The levelsof vitamin D and thyroid hormones were measured using A Minividus system (whichcombines a two-step enzyme sands assay with an ELFA, or End Fluorescent DetectionMethod).Results: The current findings revealed that there are significant variations in vitamin D andT4 levels between patients and healthy controls ( P-value 0.05). There is also a positiveassociation between vitamin D levels and T4 (r = 0.378, P-value = 0.007), as well as anegative correlation between vitamin D levels and TSH (r = -0.373, P-value = 0.008).Conclusion: According to our result, Individuals with hypothyroidism in Alkhaldia have avitamin D3 deficiency. In addition, blood vitamin D levels show a positive correlation with T4and a negative correlation with TSH levels. To avoid developing osteoporosis ifhypothyroidism persists, all hypothyroid Iraqi patients should be checked for Vitamin Ddeficiency.

Development of a Pan-Filoviridae SYBR Green qPCR Assay for Biosurveillance Studies in Bats.

Recent studies have indicated that bats are hosts to diverse filoviruses. Currently, no pan-filovirus molecular assays are available that have been evaluated for the detection of all mammalian filoviruses. In this study, a two-step pan-filovirus SYBR Green real-time PCR assay targeting the nucleoprotein gene was developed for filovirus surveillance in bats. Synthetic constructs were designed as representatives of nine filovirus species and used to evaluate the assay. This assay detected all synthetic constructs included with an analytical sensitivity of 3-31.7 copies/reaction and was evaluated against the field collected samples. The assay's performance was similar to a previously published probe based assay for detecting Ebola- and Marburgvirus. The developed pan-filovirus SYBR Green assay will allow for more affordable and sensitive detection of mammalian filoviruses in bat samples.

Aminosalicylates target GPR35, partly contributing to the prevention of DSS-induced colitis

GPR35, a class A G-protein-coupled receptor, is considered an orphan receptor; the endogenous ligand and precise physiological function of GPR35 remain obscure. GPR35 is expressed relatively highly in the gastrointestinal tract and immune cells. It plays a role in colorectal diseases like inflammatory bowel diseases (IBDs) and colon cancer. More recently, the development of GPR35 targeting anti-IBD drugs is in solid request. Nevertheless, the development process is in stagnation due to the lack of a highly potent GPR35 agonist that is also active comparably in both human and mouse orthologs. Therefore, we proposed to find compounds for GPR35 agonist development, especially for the human ortholog of GPR35. As an efficient way to pick up a safe and effective GPR35 targeting anti-IBD drug, we screened Food and Drug Administration (FDA)-approved 1850 drugs using a two-step DMR assay. Interestingly, we found aminosalicylates, first-line medicine for IBDs whose precise target remains unknown, exhibited activity on both human and mouse GPR35. Among these, pro-drug olsalazine showed the most potency on GPR35 agonism, inducing ERK phosphorylation and β-arrestin2 translocation. In dextran sodium sulfate (DSS)-induced colitis, the protective effect on disease progression and inhibitory effect on TNFα mRNA expression, NF-κB and JAK-STAT3 pathway of olsalazine are compromised in GPR35 knock-out mice. The present study identified a target for first-line medicine aminosalicylates, highlighted that uncleaved pro-drug olsalazine is effective, and provided a new concept for the design of aminosalicylic GPR35 targeting anti-IBD drug.

Musk mallow (Abelmoschus moschatus): a new host of Cucurbit aphid‐borne yellows virus

Musk mallow (Abelmoschus moschatus, family Malvaceae) is a medicinal plant cultivated across Asia, Africa and South America. It is found as a weed all over the Deccan region of India (Pawar & Vyawahare, 2017). The seeds of the plant are used for medicinal purposes and as a source of fragrant compounds (Gul et al., 2011). Musk mallow plants exhibiting yellowing of older leaves, midvein clearing, leaf reduction, mottling and slight downward curling (Fig. 1) were observed in October 2022 at the experimental research farm of the Indian Agricultural Research Institute in Pune, Maharashtra, India. The plants occurred as weeds and the disease incidence was 4% (c. 16 plants) in an eight-acre area. Ten diseased and four asymptomatic plants were sampled to ascertain the viruses associated with the symptoms. ELISA was done using antisera against Cucurbit aphid-borne yellows virus (CABYV) (DSMZ, Germany), Cucurbit yellow stunting disorder virus (CYSDV) (Arsh Biotech, India), Cucumber mosaic virus (CMV), Papaya ringspot virus (PRSV) and Zucchini yellow mosaic virus (ZYMV) (Agdia, USA). CABYV was detected in all diseased samples but CMV, CYSDV, PRSV and ZYMV were not detected. None of the five viruses were detected by ELISA in the asymptomatic samples. Total RNA was extracted from both diseased and asymptomatic samples using an RNeasy Plant Mini Kit (Qiagen, USA). Two-step RT-PCR assay was done using the extracted RNA and CABYV-specific primers which amplify c. 484 bp of the partial coat protein region (Boubourakas et al., 2006). RT-PCR was also performed using primers specific for CMV, CYSDV, PRSV, ZYMV, Beet pseudo-yellows virus and Cucumber vein yellowing virus. Amplicons of the expected size were obtained in all diseased plants with CABYV primers but not with primers for any of the other viruses. No amplification was obtained in the asymptomatic samples for any of the viruses. The amplicons of five samples were Sanger sequenced bidirectionally and found to be identical (GenBank Accession No. OP455156). A BLASTn search of the sequence revealed that it shared 100% identity with an Indian CABYV sequence from Praecitrullus fistulosus (OP455157). A phylogenetic tree was constructed using the neighbour-joining method (MEGA X program) with CABYV sequences from different hosts and geographies, and the sequence clustered with other Indian CABYV sequences (Fig. 2). CABYV primarily infects cucurbits but also infects other crops and weeds including three Malvaceae species, Abutilon theophrasti, Gossypium hirsutum and Malva parviflora (Lecoq et al., 2022). To our knowledge, this is the first report of CABYV infecting Abelmoschus moschatus in the world. The present finding is of epidemiological importance as it indicates the further broadening of the host range of CABYV and the possibility of infecting other economically important Malvaceae members, such as G. hirsutum which is grown for cotton in India. Furthermore, musk mallow plants could serve as an alternative / reservoir host for CABYV aiding its transmission to other commercial crops. The authors thank the Director, ICAR-IARI, New Delhi for providing lab facilities.

Elucidating the Role of CRISPR/Cas in Single-Step Isothermal Nucleic Acid Amplification Testing Assays.

Developing assays that combine CRISPR/Cas and isothermal nucleic acid amplification has become a burgeoning research area due to the novelty and simplicity of CRISPR/Cas and the potential for point-of-care uses. Most current research explores various two-step assays by appending different CRISPR/Cas effectors to the end of different isothermal nucleic acid amplification methods. However, efforts in integrating both components into more ideal single-step assays are scarce, and poor-performing single-step assays have been reported. Moreover, lack of investigations into CRISPR/Cas in single-step assays results in incomplete understanding. To fill this knowledge gap, we conducted a systematic investigation by developing and comparing assays that share the identical recombinase polymerase amplification (RPA) but differ in CRISPR/Cas12a. We found that the addition of CRISPR/Cas12a indeed unlocks signal amplification but, at the same time, impedes RPA and that CRISPR/Cas12a concentration is a key parameter for attenuating RPA impediment and ensuring assay performance. Accordingly, we found that our protospacer adjacent motif (PAM)-free CRISPR/Cas12a-assisted RPA assay, which only moderately impeded RPA at its optimal CRISPR/Cas12a concentration, outperformed its counterparts in assay design, signal, sensitivity, and speed. We also discovered that a new commercial Cas12a effector could also drive our PAM-free CRISPR/Cas12a-assisted RPA assay and reduce its cost, though simultaneously lowering its signal. Our study and the new insights can be broadly applied to steer and facilitate further advances in CRISPR/Cas-based assays.

A semi high-throughput whole blood-based flow cytometry assay to detect and monitor Bordetella pertussis-specific Th1, Th2 and Th17 responses.

The characterization of B. pertussis (Bp) antigen-specific CD4+ T cell cytokine responses should be included in the evaluation of immunogenicity of pertussis vaccines but is often hindered by the lack of standardized robust assays. To overcome this limitation, we developed a two-step assay comprising a short-term stimulation of fresh whole blood with Bp antigens and cryopreservation of the stimulated cells, followed later on by batch-wise intracellular cytokine analysis by flow cytometry. Blood samples collected from recently acellular (aP) vaccine boosted subjects with a whole-cell- or aP-primed background was incubated for 24 hrs with Pertussis toxin, Filamentous hemagglutinin or a Bp lysate (400µl per stimulation). Antigen-specific IFN-γ-, IL-4/IL-5/IL-13-, IL-17A/IL-17F- and/or IL-22-producing CD4+ T cells were quantified by flow cytometry to reveal Th1, Th2, and Th17-type responses, respectively. The frequencies of IFN-γ-producing CD8+ T cells were also analyzed. We demonstrate high reproducibility of the Bp-specific whole blood intracellular staining assay. The results obtained after cryopreservation of the stimulated and fixed cells were very well correlated to those obtained without cryopreservation, an approach used in our previously published assay. Optimization resulted in high sensitivity thanks to very low non-specific backgrounds, with reliable detection of Bp antigen-specific Th1, Th2 and Th17-type CD4+ T cells, in the lowest range frequency of 0.01-0.03%. Bp antigen-specific IFN-γ+ CD8+ T lymphocytes were also detected. This test is easy to perform, analyse and interpret with the establishment of strict criteria defining Bp antigen responses. Thus, this assay appears as a promising test for evaluation of Bp antigen-specific CD4+ T cells induced by current and next generation pertussis vaccines.

CRISPR-Cas12-based field-deployable system for rapid detection of synthetic DNA sequence of the monkeypox virus genome.

The global outbreak of the monkeypox virus (MPXV) highlights the need for rapid and cost-effective MPXV detection tools to effectively monitor and control the monkeypox disease. Herein, we demonstrated a portable CRISPR-Cas-based system for naked-eye detection of MPXV. The system harnesses the high selectivity of CRISPR-Cas12 and the isothermal nucleic acid amplification potential of recombinase polymerase amplification. It can detect both the current circulating MPXV clade and the original clades. We reached alimit of detection (LoD) of 22.4 aM (13.5 copies/µl) using a microtiter plate reader, while the visual LoD of the system is 75 aM (45 copies/µl) in a two-step assay, which is further reduced to 25 aM (15 copies/µl) in a one-pot system. We compared our results with quantitative polymerase chain reaction andobtained satisfactory consistency. For clinical application, we demonstrated a sensitive and precise visual detection method with attomolar sensitivity and a sample-to-answer time of 35 min.

Rapid detection methods and modelling simulations provide new insights into cyanobacteria detection and bloom management in a tropical reservoir

The increasing occurrence of cyanobacteria blooms is of global concern, and is often associated with environmental and socio-economic problems, such as degenerated ecosystems and aquaculture impairment. The diazotrophic cyanobacterium Raphidiopsis raciborskii (R. raciborskii) grows rapidly in the tropics, and produces the toxin, cylindrospermopsin (CYN), which has harmful effects on aquatic organisms. Thus, to protect water quality and ecosystem, it is essential to have rapid and reliable methods for cyanobacteria and R. raciborskii detection and prediction so that early warning can be provided for management. Molecular assays, such as PCR, real-time quantitative PCR (qPCR), two-step PCR assays are accurate and widely used, but still require several hours from sample preparation to data analysis. In this study, insulated isothermal PCR (iiPCR) assays in conjunction with fast DNA extraction method, were developed and verified as a rapid detection assay in detecting cyanobacteria and R. raciborskii within 50 min, and also with high detection accuracy (98.8%) and the overall high agreement level (98.8%, k = 97.5%)) comparing to conventional qPCR assay. However, the limitation of the iiPCR assay is that it only generates qualitative results. Therefore, the quantified iiPCR assay, named as A-iiPCR, by coupling iiPCR device with fluorescence signal catching and interpretation instrument (Andor spectrometer with Solis spectroscopy software) was developed and verified with in situ environmental samples. The fluorescence intensity decreased accordingly with the drop of DNA concentration until reaching 1.32 ng/μL. Also, Delft 3D modelling was established to simulate R. raciborskii change in predicting spatial and temporal variabilities for reservoir management, as the simulated R. raciborskii concentration was the highest at sampling site 1, as well as temporally highest in April and October, posing as the most high-risk location and time periods for R. raciborskii bloom-forming requiring corresponding governance measures.

C9orf72 repeat length might influence clinical sub-phenotypes in dementia patients

BackgroundC9orf72 repeat expansions have been observed in a wide variety of neurodegenerative disorders. The cut-off between normal and pathogenic alleles is not well established as repeat sizing methods are often semi-quantitative. However, intermediate alleles might influence disease prevalence and phenotype, as seen for other repeat expansion disorders. We aimed to further delineate the prevalence of small, intermediate and expanded C9orf72 alleles and elucidate their potential influence on the disease phenotype. MethodsDNA derived from patients (n = 1804) and healthy individuals (n = 643) was obtained from multiple collectives in Austria. Genotyping was performed using a two-step PCR assay followed by Southern blotting. Results3.4% of clinically diagnosed frontotemporal dementia (FTD; n = 5/147) cases and 0.8% of clinically diagnosed Alzheimer's disease (AD; n = 5/602) cases were carriers of a pathological C9orf72 repeat expansion. A significantly earlier disease onset was detected in expansion carriers compared to non-carriers in the FTD and AD cohorts (median 50 years, range 39–64 vs. median 64 years, range 36–92, p = 0.018 and median 63 years, range 54–71 vs. median 74 years, range 45–92, p = 0.006, respectively). C9orf72 intermediate alleles were significantly associated with cerebellar symptoms (p = 0.0004) and sensory deficits in the dementia cohort (p = 0.01). ConclusionsC9orf72 repeat expansion carriers showed earlier disease onset compared to non-carriers with clinical diagnosis of FTD and AD. Furthermore, C9orf72 intermediate repeats might modify the phenotypic expression in dementia.

A Simplified Sanger Sequencing Method for Detection of Relevant SARS-CoV-2 Variants.

Molecular surveillance of the new coronavirus through new genomic sequencing technologies revealed the circulation of important variants of SARS-CoV-2. Sanger sequencing has been useful in identifying important variants of SARS-CoV-2 without the need for whole-genome sequencing. A sequencing protocol was constructed to cover a region of 1000 base pairs, from a 1120 bp product generated after a two-step RT-PCR assay in samples positive for SARS-CoV-2. Consensus sequence construction and mutation identification were performed. Of all 103 samples sequenced, 69 contained relevant variants represented by 20 BA.1, 13 delta, 22 gamma, and 14 zeta, identified between June 2020 and February 2022. All sequences found were aligned with representative sequences of the variants. Using the Sanger sequencing methodology, we were able to develop a more accessible protocol to assist viral surveillance with a more accessible platform.

Direct Capture and Early Detection of Lyme Disease Spirochete in Skin with a Microneedle Patch.

Borrelia burgdorferi sensu lato family of spirochetes causes Lyme disease (LD) in animals and humans. As geographic territory of ticks expands across the globe, surveillance measures are needed to measure transmission rates and provide early risk testing of suspected bites. The current standard testing of LD uses an indirect two-step serological assay that detects host immune reactivity. Early detection remains a challenge because the host antibody response develops several weeks after infection. A microneedle (MN) device was developed to sample interstitial fluid (ISF) and capture spirochetes directly from skin. After sampling, the MN patch is easily dissolved in water or TE buffer, and the presence of spirochete DNA is detected by PCR. Performance was tested by spiking porcine ear skin with inactivated Borrelia burgdorferi, which had an approximate recovery of 80% of spirochetes. With further development, this simple direct PCR method could be a transformative approach for early detection of the causative agent of Lyme disease and enable rapid treatment to patients when infection is early, and numbers of systemic spirochetes are low.

(Keynote, Digital Presentation) Why Au Nanoparticles Could Behave As a Glucose Oxidase Mimic

The Au nanoparticle has been found to be the excellent glucose oxidase mimic, while the catalytic process has rarely been studied. Here, we revealed that the process of glucose oxidation catalyzed by Au nanoparticles is the same as that of natural glucose oxidase, namely, a two-step reaction including the dehydrogenation of glucose and the subsequent reduction of O2 to H2O2 by two electrons. Pt, Pd, Ru, Rh, and Ir nanoparticles can also catalyze the dehydrogenation of glucose, except that O2 is preferably reduced to H2O. Using the electron transfer property of noble metal nanoparticles, we overcame the limitation that H2O2 must be produced in the traditional two-step glucose assay and realized the rapid colorimetric detection of glucose. Noble metal nanoparticles have also been found to mimic various enzymatic electron transfer reactions including cytochrome c, coenzymes as well as nitrobenzene reductions.

Establishment and Validation of a Two-Step LAMP Assay for Detection of Bacillus cereus-Group Isolates in Food and Their Possibility of Non-haemolytic Enterotoxin Production.

The closely related members of the Bacillus cereus-group can mainly only be differentiated by whole genome sequencing. Among them, there are potentially toxin-producing bacteria. When consumed with food, these can cause vomiting or diarrhea and abdominal cramps. To date, although no EU-wide threshold exists, a bacterial count of 105 CFU/g can be regarded as critical. Specific and rapid detection of the bacteria is difficult due to their close relationship, and no loop-mediated isothermal amplification (LAMP) assay has been developed so far to detect potentially toxin-producing members of the B. cereus-group. Aim of this study was to develop a LAMP method to detect critical cell counts specifically and rapidly of potentially non-haemolytic enterotoxin (NHE)-producing cells of this group. A two-step LAMP assay was developed. First, the target sequence groEL was used to determine the representatives of the B. cereus-group. Second, since bacteria in which nheB is present are basically capable of producing enterotoxins, this gene was chosen for detection. The specificity of the developed assay was 100% for B. cereus-group isolates and 93.7% for the detection of nheB. The analytical sensitivity was 0.1 pg DNA/μl. Using simplified DNA extraction by boiling, cell-based sensitivity was determined. Targeting groEL and nheB, 11.35–27.05 CFU/reaction and 11.35–270.5 CFU/reaction were detectable, respectively. Artificially contaminated samples were investigated to prove the application in foods. Direct detection of the critical value of B. cereus-group cells was possible in 83.3% of samples and detecting the toxin-gene 50% thereof. After a 6-h incubation period, the detection rate increased to 100 and 91.7%, respectively. Additionally, 100 natively contaminated food samples were tested, also quantitatively and culturally. Samples with relevant contamination levels were reliably detected using groEL-LAMP. After a 6-h incubation period, isolates bearing the toxin gene nheB could also be reliably detected. In addition, colony material was boiled and used as a LAMP template for simple detection. Specificity for the B. cereus-group was 100 and 93.22% detecting nheB. The study demonstrated that screening of food samples with the groEL/nheB-LAMP assay can be performed within 1 day, making it possible to detect critical levels of potentially NHE-toxin-producing cells of the B. cereus-group.