Method For Detection Of DNA Research Articles

CRISPR-Cas12a based HSV DNA detection method using quantum dot-labeled immunochromatographic strips

CRISPR-Cas12a based HSV DNA detection method using quantum dot-labeled immunochromatographic strips

A comparative study of extraction free detection of HBV DNA using sodium dodecyl sulfate, N-lauroylsarcosine sodium salt, and sodium dodecyl benzene sulfonate

This study aimed to develop an extraction-free method for quantitative and qualitative detection of HBV DNA compared to traditional nucleic acid extraction. Paired serum and dried blood spot (DBS) samples from 67 HBsAg-positive and 67 HBsAg-negative individuals were included. Two samples with known HBV DNA titers (~ 109 copies/mL) were examined by extraction-free detection using three surfactants (0.2 to 1% of Sodium dodecyl sulfate:SDS, N-Lauroylsarcosine sodium salt:NL, Sodium dodecyl benzene sulfonate:SDBS), two stabilizing agents (0.1 or 0.01% 2-Mercaptoethanol:2ME and 3.5 or 7% Bovine Serum Albumin:BSA) and two Taq polymerases (Fast Advanced and Prime Direct Probe). HBV DNA in all 67 HBsAg-positive and 67 HBsAg-negative serum and DBS samples was directly quantified by Rt-PCR using 0.4% SDS or 0.4% NL with Fast Advance or Prime Direct Probe Taq. Extraction-free amplification was also performed. Detection limits were varied by different surfactants and Taq. SDS combined with Fast Advanced Taq showed lower detection limits, while SDS with Prime Direct Probe Taq outperformed NL or SDBS-based detection. Adding 2ME to SDS improved detection limit with Prime Direct Probe Taq but not significantly compared to SDS alone. BSA did not significantly enhance detection limits but provided insights into sample stability. The senitivity and specificity of 0.4% SDS and NL in combination with either Fast advanced or Prime Direct Probe Taq polymerase in serum samples were > 90% and 100% resepctively, while it was > 80% and 100% respectively in DBS samples. Extraction-free HBV DNA amplification provided 100% identity with original genomes. Our study suggests that SDS, NL or SDBS-based extraction-free HBV DNA detection strategies using Prime Direct Probe Taq have potential to simplify and accelerate HBV DNA detection with high sensitivity and specificity in both serum and DBS samples, with implications for resource-limited settings and clinical applications. Utilizing surfactants with 2ME is optional, and further research and validation are necessary to broaden its application in real-world diagnostics.

Comparison of different diagnostic protocols for the detection of Toxocara spp. in faecal samples of cats and dogs

BackgroundToxocara canis and Toxocara cati are parasitic nematodes that occur worldwide. As embryonated Toxocara spp. eggs in the environment pose a zoonotic risk, especially for children, optimal diagnostic approaches are necessary for effective disease response and management, including surveillance. However, little is known about the performance of different diagnostic protocols for detecting Toxocara spp. in the faeces of cats and dogs, hampering movement towards an optimal diagnostic process. This study aimed to compare detection methods, including a newly developed sequential sieving protocol (SF-SSV) and a high-throughput multiplex qPCR-based method to facilitate epidemiological studies.MethodsSpecies-specific Toxocara spp. egg suspensions and canine and feline faecal samples from the field were used to estimate analytical and diagnostic sensitivity of the protocols. The performance of two automated DNA extraction protocols using enzymatic and mechanical lysis were compared by multiplex qPCR, targeting both T. canis and T. cati-specific genomic sequences. All samples were examined by microscopy-based techniques, the sedimentation flotation technique (SF) and a newly developed SF-SSV for the detection, enrichment and purification of parasite eggs. The costs and processing times necessary for all protocols were estimated and compared for both single samples and sets of 100 samples.ResultsTo detect Toxocara spp. eggs, SF-SSV showed the highest analytical sensitivity and a significantly higher diagnostic sensitivity than the DNA detection methods. Mechanical lysis performed better than enzymatic lysis for automated DNA extraction. In automated DNA extraction, 96-well plates performed better than 24-well plates. DNA detection and microscopy-based parasitological methods showed substantial agreement between the results generated by each method. Microscopy-based techniques required the lowest costs and least hands-on time for a single sample. However, when costs and labour were estimated for a set of 100 samples, the DNA detection protocol using 96-well plates for extraction revealed costs similar to SF-SSV and the fastest processing times.ConclusionsSF-SSV was superior in terms of analytical and diagnostic sensitivity for the detection of Toxocara spp. eggs. For larger sets of samples, multiplex qPCR-based DNA detection represents an alternative to microscopy-based methods, based on the possibility of faster sample processing at similar costs to SF-SSV, and the ability to provide species-specific diagnoses.Graphical

Comparison of multi-parallel quantitative real-time PCRs targeting different DNA regions and detecting soil-transmitted helminths in stool

BackgroundSoil-transmitted helminths infect an estimated 18% of the world’s population, causing a significant health burden. Microscopy has been the primary tool for diagnosing eggs from fecal samples, but its sensitivity drops in low-prevalence settings. Quantitative real-time polymerase chain reaction (qPCR) is slowly increasing in research and clinical settings. However, there is still no consensus on preferred qPCR targets.MethodsWe aimed to compare soil-transmitted helminth (STH) DNA detection methods by testing naïve stool samples spiked with known quantities of STH eggs and larvae. DNA extracts from spiked samples were tested using independent quantitative realtime PCR (qPCR) assays targeting ribosomal or putative non-protein coding satellite sequences.ResultsFor Trichuris trichiura, there was a strong correlation between egg/larvae counts and qPCR results using either qPCR method (0.86 and 0.87, respectively). Strong correlations also existed for A. lumbricoides (0.60 and 0.63, respectively), but weaker correlations were found for Ancylostoma duodenale (0.41 for both assays) and Strongyloides stercoralis (0.48 and 0.65, respectively). No correlation for Necator americanus was observed when testing with either qPCR assay. Both assays had fair-to-moderate agreement across targets when using field-collected stool samples (0.28–0.45, for all STHs), except for S. stercoralis (0.12) with slight agreement.ConclusionsThere is a strong correlation between qPCR results and egg/larvae counts. Our study confirms that qPCR is an effective diagnostic tool, even with low-intensity infections, regardless of the DNA-based diagnostic marker used. However, the moderate agreement between the two different qPCR assays when testing field samples highlights the need to understand the role of these targets in the genome so that the parasite burden can be quantified more accurately and consistently by qPCR.Graphical abstract

Rapid detection of murine-derived ingredients in meat products using real-time enzymatic recombinase amplification.

Accurate identification of meat species is critical to prevent economic fraud and safeguard public health. The use of inappropriate meat sources, such as murine, poses significant health risks because of potential contamination with pathogens and allergens, leading to foodborne illnesses. The present study aimed to develop a novel real-time enzymatic recombinase amplification (ERA) method for the rapid and specific detection of murine DNA in meat products. A novel ERA primer and probe set was designed, targeting a murine-specific single-copy nuclear gene identified through bioinformatics analysis. The assay demonstrates high specificity, showing no amplification in commonly consumed meats, other animals or major crops. Additionally, it exhibits remarkable sensitivity, detecting as few as five copies of murine genomic DNA. For practical application, the ERA method could effectively identify mouse DNA in laboratory-prepared samples at concentrations as low as 0.5% and also quantify samples with mouse DNA content as low as 5%. It also accurately detects the presence of murine-derived ingredients in commercially available meat products. The detection process is straightforward, utilizing a simple isothermal device for incubation, blue light excitation and a smartphone camera for result interpretation. This rapid analysis can be completed within 20 min. The newly developed real-time ERA method provides a valuable tool for standardizing meat trade practices, promoting food safety and enhancing consumer confidence in the authenticity of meat products. © 2024 Society of Chemical Industry.

A micro-disc-based multiplex method for monitoring emerging SARS-CoV-2 variants using the molecular diagnostic tool Intelli-OVI

BackgroundHighly transmissible viruses including SARS-CoV-2 frequently accumulate novel mutations that are detected via high-throughput sequencing. However, there is a need to develop an alternative rapid and non-expensive approach. Here we developed a novel multiplex DNA detection method Intelli-OVI for analysing existing and novel mutations of SARS-CoV-2.MethodsWe have developed Intelli-OVI that includes the micro-disc-based method IntelliPlex and computational algorithms of objective variant identification (OVI). More than 250 SARS-CoV-2 positive samples including wastewater ones were analysed to verify the efficiency of the method.ResultsIntelliPlex uses micro-discs printed with a unique pictorial pattern as a labelling conjugate for DNA probes, and OVI allows simultaneous identification of several variants using multidimensional data obtained by the IntelliPlex method. Importantly, de novo mutations can be identified by decreased signals, which indicates that there is an emergence of de novo variant virus as well as prompts the need to design additional primers and probes. We have upgraded probe panel according to the emergence of new variants and demonstrated that Intelli-OVI efficiently identified more than 20 different SARS-CoV-2 variants by using 35 different probes simultaneously.ConclusionsIntelli-OVI can be upgraded to keep up with rapidly evolving viruses as we showed in this study using SARS-CoV-2 as an example and may be suitable for other viruses but would need to be validated.

Ultrafast DNA detection based on turn-back loop primer-accelerated LAMP (TLAMP)

Rapid DNA detection is a long-pursuing goal in molecular detection, especially in combating infectious diseases. Loop-mediated isothermal amplification (LAMP) is a robust and prevailing DNA detection method in pathogen detection, which has been drawing broad interest in improving its performance. Herein, we reported a new strategy and developed a new LAMP variant named TLAMP with a superior amplification rate. In this strategy, the turn-back loop primers (TLPs) were devised by ingeniously extending the 5’ end of the original loop primer, which conferred the new role of being the inner primer for TLPs while retaining its original function as the loop primer. In theory, based on the bifunctional TLPs, a total of eight basic dumbbell-like structures and four cyclic amplification pathways were produced to significantly enhance the amplification efficiency of TLAMP. With the enhancing effect of TLPs, TLAMP exhibited a significantly reduced amplification-to-result time compared to the conventional six-primer LAMP (typically 1 h), enabling rapid DNA detection within 20 min. Furthermore, TLAMP proved to be about 10 min faster than the fast LAMP variants reported so far, while still presenting comparable sensitivity and higher repeatability. Finally, TLAMP successfully achieved an ultrafast diagnosis of Monkeypox virus (MPXV), capable of detecting as few as 10 copies (0.67copies/μL) of pseudovirus within 20 min using real-time fluorescence assay or within 30 min using a colorimetric assay, suggesting that the proposed TLAMP offers a sensitive, specific, reliable, and, most importantly, ultrafast DNA detection method when facing the challenges posed by infectious diseases.

Optimization of CRISPR/Cas12a detection assay and its application in the detection of Echinococcus granulosus

Cystic echinococcosis, resulting from infection with Echinococcus granulosus, poses a significant challenge as a neglected tropical disease owing to the lack of any known effective treatment. Primarily affecting under-resourced, remote, and conflict-ridden regions, the disease is compounded by the limitations of current detection techniques, such as microscopy, physical imaging, ELISA, and qPCR, which are unsuitable for application in these areas. The emergence of CRISPR/Cas12a as a promising tool for nucleic acid detection, characterized by its unparalleled specificity, heightened sensitivity, and rapid detection time, offers a potential solution. In this study, we present a one-pot CRISPR/Cas12a detection method for E. granulosus (genotype G1, sheep strain) integrating recombinase polymerase amplification (RPA) with suboptimal protospacer adjacent motif (PAM) and structured CRISPR RNA (crRNA) to enhance reaction efficiency. The evaluation of the assay's performance using hydatid cyst spiked dog feces and the examination of 62 dog fecal samples collected from various regions of Western China demonstrate its efficacy. The assay permits visual observation of test results about 15 minutes under blue light and displays superior portability and reaction speed relative to qPCR, achieving a sensitivity level of 10 copies of standard plasmids of the target gene. Analytic specificity was verified against four tapeworm species (E. multilocularis, H. taeniaeformis, M. benedeni, and D. caninum) and two other helminths (T. canis and F. hepatica), with negative results also noted for Mesocestoides sp. This study presents a rapid, sensitive, and time-efficient DNA detection method for E. granulosus of hydatid cyst spiked and clinical dog feces, potential serving as an alternative tool for field detection. This novel assay is primarily used to diagnose the definitive host of E. granulosus. Further validation using a larger set of clinical fecal samples is warranted, along with additional exploration of more effective approaches for nucleic acid release.

Development of a Multiplex PCR Assay for the Detection of Tomato Wilt Caused by Coinfection of Fusarium brachygibbosum, Fusarium oxysporum, and Ralstonia solanacearum Based on Comparative Genomics.

Tomato is consumed worldwide as fresh or processed food products. However, soilborne diseases of tomato plants caused by coinfection of various pathogens result in great economic losses to the tomato industry. It is difficult to accurately identify and diagnose soilborne diseases of tomato plants caused by pathogen complexes. In this study, we investigated field diseases of tomato plants by pathogen isolation and molecular identification and found that tomato wilt was caused by coinfection of Fusarium brachygibbosum, F. oxysporum, and Ralstonia solanacearum. Therefore, developing a method for simultaneous detection of DNA from F. brachygibbosum, F. oxysporum, and R. solanacearum is of great importance to efficiently and accurately monitor disease development at different growth stages of tomato plants. In this study, we performed a comparative genomic analysis of F. brachygibbosum, F. oxysporum, and R. solanacearum and determined the primer sets for simultaneous detection of DNA from these target pathogens. Then, we tested the reagent and condition parameters of multiplex PCR, including primers, dNTP and Mg2+ concentrations, and annealing temperatures, to determine the optimal parameters of a multiplex PCR system. We evaluated the specificity, sensitivity, and stability of the multiplex PCR system based on the optimized reaction conditions. The multiplex PCR system can specifically identify 13 target pathogens from 57 different fungal and bacterial pathogens, at the lower detection limit of the three target pathogens at concentrations of 100 pg/μl. In addition, we can accurately identify the three pathogens in tomato plants using the optimized multiplex PCR method. These results demonstrated that the multiplex PCR method developed in this study can simultaneously detect DNA from F. brachygibbosum, F. oxysporum, and R. solanacearum in a single PCR system to accurately identify and diagnose the pathogen causing tomato wilt.

Domestic dogs in indigenous Amazonian communities: Key players in Leptospira cycling and transmission?

Leptospirosis is the world's most common zoonotic disease. Mitigation and control rely on pathogen identification and understanding the roles of potential reservoirs in cycling and transmission. Underreporting and misdiagnosis obscure the magnitude of the problem and confound efforts to understand key epidemiological components. Difficulties in culturing hamper the use of serological diagnostics and delay the development of DNA detection methods. As a result, especially in complex ecosystems, we know very little about the importance of different mammalian host species in cycling and transmission to humans. We sampled dogs from five indigenous Kichwa communities living in the Yasuní National Park in the Ecuadorian Amazon basin. Blood and urine samples from domestic dogs were collected to assess the exposure of these animals to Leptospira and to identify the circulating species. Microscopic Agglutination Tests with a panel of 22 different serovars showed anti-leptospira antibodies in 36 sampled dogs (75%), and 7 serogroups were detected. Two DNA-based detection assays revealed pathogenic Leptospira DNA in 18 of 19 dog urine samples (94.7%). Amplicon sequencing and phylogenetic analysis of 16S rRNA and SecY genes from 15 urine samples revealed genetic diversity within two of three different Leptospira species: noguchii (n = 7), santarosai (n = 7), and interrogans (n = 1). The high prevalence of antibodies and Leptospira DNA provides strong evidence for high rates of past and current infections. Such high prevalence has not been previously reported for dogs. These dogs live in the peridomestic environment in close contact with humans, yet they are free-ranging animals that interact with wildlife. This complex web of interactions may explain the diverse types of pathogenic Leptospira observed in this study. Our results suggest that domestic dogs are likely to play an important role in the cycling and transmission of Leptospira. Future studies in areas with complex ecoepidemiology will enable better parsing of the significance of genotypic, environmental, and host characteristics.

Simple closed-system combining direct PCR and fluorescence for all types of milk and dairy product authentication

Cow's milk is a popular food choice due to its nutritional value. However, some people have cow milk protein allergy. Alternative milk and dairy products are available, but they may be adulterated or fraudulently substituted with cow's milk to lower the production cost. Current conventional methods for cow DNA detection suffer from laboratory-based instruments and settings, which can be expensive, limit on-site usage, lack sensitivity, or cannot be used with highly degraded samples. Here, a simple closed-system based on direct PCR, which eliminates the need to extract DNA from samples, and SYBR Green I detection of PCR amplicons using the naked eye was developed and validated. Fluorescence detection was enhanced with the use of a self-designed portable LED lightbox, which mitigated color interference, enabling detection in products with diverse colorations without any sample preparation. Validation studies revealed 100% specificity when tested with 13 other species, over 99% accuracy with 208 cow’s milk and dairy products in both liquid and solid forms, 1×10−5 ng limit of detection, and 100% accuracy with blind testing. Market testing of 156 samples showed a fraud ratio of 0.0% in cow’s milk and dairy products, while non-cow’s milk and dairy products showed a fraud ratio of 10.7%, emphasizing the assay's capacity to identify fraudulent substitutions. Overall, this is the first time a cow-specific simple closed-system direct PCR with fluorescence has been used to authenticate fresh milk and dairy products, with one of the quickest turn-around time from sampling to detection of 50 min. It could be useful in a semi-laboratory setting or in the field with a portable PCR machine.

Bioluminescent Intercalating Dyes for Ratiometric Nucleic Acid Detection.

Rapid and sensitive DNA detection methods that can be conducted at the point of need may aid in disease diagnosis and monitoring. However, translation of current assays has proven challenging, as they typically require specialized equipment or probe-specific modifications for every new target DNA. Here, we present Luminescent Multivalent Intercalating Dye (LUMID), off-the-shelf bioluminescent sensors consisting of intercalating dyes conjugated to a NanoLuc luciferase, which allow for nonspecific detection of double-stranded DNA through a blue-to-green color change. Through the incorporation of multiple, tandem-arranged dyes separated by positively charged linkers, DNA-binding affinities were improved by over 2 orders of magnitude, detecting nanomolar DNA concentrations with an 8-fold change in green/blue ratio. We show that LUMID is easily combined with loop-mediated isothermal amplification (LAMP), enabling sequence-specific detection of viral DNA with attomolar sensitivity and a smartphone-based readout. With LUMID, we have thus developed a tool for simple and sensitive DNA detection that is particularly attractive for point-of-need applications.

A CRISPR/LbCas12a-based method for detection of bacterial fruit blotch pathogens in watermelon.

Acidovorax citrulli is the main pathogen causing bacterial fruit blotch, which seriously threatens the global watermelon industry. At present, rapid, sensitive, and low-cost detection methods are urgently needed. The established CRISPR/LbCas12a visual detection method can specifically detect A. citrulli and does not cross-react with other pathogenic bacteria such as Erwinia tracheiphila, Pseudomonas syringae, and Xanthomonas campestris. The sensitivity of this method for genomic DNA detection is as low as 0.7 copies/μL, which is higher than conventional PCR and real-time PCR. In addition, this method only takes 2.5 h from DNA extraction to quantitative detection and does not require complex operation and sample treatment. Additionally, the technique was applied to test real watermelon seed samples for A. citrulli, and the results were contrasted with those of real-time fluorescence quantitative PCR and conventional PCR. The high sensitivity and specificity have broad application prospects in the rapid detection of bacterial fruit blotch bacterial pathogens of watermelon.IMPORTANCEBacterial fruit blotch, Acidovorax citrulli, is an important seed-borne bacterial disease of watermelon, melon, and other cucurbits. The lack of rapid, sensitive, and reliable pathogen detection methods has hampered research on fruit spot disease prevention and control. Here, we demonstrate the CRISPR/Cas12a system to analyze aspects of the specificity and sensitivity of A. citrulli and to test actual watermelon seed samples. The results showed that the CRISPR/Cas12a-based free-amplification method for detecting bacterial fruit blotch pathogens of watermelons was specific for A. citrulli target genes and 100-fold more sensitive than conventional PCR with quantitative real-time PCR. This method provides a new technical tool for the detection of A. citrulli.

MOF-mediated dual energy transfer nanoprobe integrated with exonuclease III amplification strategy for highly sensitive detection of DNA.

Accurate quantitative detection of DNA is an advanced strategy in various fields (such as disease diagnosis and environmental monitoring), but the classical DNA detection method usually suffers from low sensitivity, expensive thermal cyclers, or strict annealing conditions. Herein, a MOF-ERA platform for ultrasensitive HBV-DNA detection is constructed by integrating metal-organic framework (MOF)-mediated double energy transfer nanoprobe with exonuclease III (Exo III)-assisted target recycling amplification. The proposed double energy transfer containing a donor and two receptors is simply composed of MOFs (UiO-66-NH2, a well-studied MOF) modified with a signal probe formed by the hybridization of carboxyuorescein (FAM)-labeled DNA (FDNA) and black hole quencher (BHQ1)-terminated DNA (QDNA), resulting in low fluorescence signal. After the addition of HBV-DNA, Exo III degradation to FDNA is activated, leading to the liberation of the numerous FAM molecules, followed by the generation of a significant fluorescence signal owing to the negligible binding of MOFs with free FAM molecules. The results certify that the MOF-ERA platform can be successfully used to assay HBV-DNA in the range of 1.0-25.0 nM with a detection limit of 97.2 pM, which is lower than that without BHQ1 or Exo III. The proposed method with the superiorities of low background signal and high selectivity holds promise for early disease diagnosis and clinical biomedicine applications.

Development of a Nanoplate-Based Digital PCR Test Method for Quantitative Detection of Human Adenovirus DNA.

Digital polymerase chain reaction (dPCR) assay is an advanced PCR technique that allows for the simultaneous detection and absolute quantification of diverse pathogens.Commercially validated kits available for detecting all subtypes of human adenovirus (HAdV) are limited. This study aimed to demonstrate the development of an in-house nanoplate-based dPCR assay with high sensitivity, even at low copy numbers. In this methodological study, the standardized HAdV DNA was prepared by amplifying the specific hexon gene region with real-time PCR and purifying the HAdV DNA using magnetic beads from HAdV-positive extractions. Dilutions were tested in triplicate during three independent runs to determine the dynamic range, the limit of detection (LoD), the limit of quantification (LoQ), precision, and reproducibility. The primer and probe sequences used in the study were selected based on a literature review to ensure the detection of all HAdV serotypes in a single run. The selected primers were verified using the US National Center for Biotechnology Information (NBCI) nBLAST tools, and the target sequence was determined using the BioEdit software. The DNA concentration of the stock solution was measured using a Qubit fluorometer. The estimated copy number of the stock solution per milliliter was calculated based on the length of the amplified base sequence and fluorometer measurement. The dynamic range of the test was determined to be from 770.4 to 0.9476 cp/μl, with the LoD and LoQ values both being 0.9476 cp/μl. The coefficient of determination (r 2) value of the test was 0.9986. The results demonstrated that the dPCR method could be an ideal tool for the diagnosis and absolute quantification of human adenoviruses, especially in low copy numbers. In order to determine the reproducibility of the test and validate the method for field use, it needs to be developed and adapted in various laboratories and supported by clinical studies.

Electrochemiluminescence of hot exciton nanomaterial with boosted efficiency for visual bioanalysis

The electrochemiluminescence (ECL) efficiency (ΦECL) of organic ECL emitters is greatly influenced by the excited state property and component. Here we design a hot exciton molecule (BCzP-BT) with hybridized local and charge-transfer (HLCT) excited state property to prepare the first hot exciton organic nanorods (BB NRs). The BB NRs exhibit both sensitive annihilation and intense coreactant ECL emissions with a band gap emission model. Due to the reverse intersystem crossing among high-lying excited states (hRISC), the HLCT excited state property leads to highly efficient utilization of triplet excitons and thus high photoluminescence quantum yields (ΦPL) of more than 80% at 554 nm, which is demonstrated by solvatochromic experiments and quantum chemical calculations. The high ΦPL endows BB NRs with superior ΦECL over other nanoemitters, even the thermally activated delayed fluorescence materials, and thus offers highly-efficient nanoemitters for the design of ECL imaging strategy. As a proof of concept, an ECL probe is constructed by assembling BHQ2-ssDNA on BB NRs to integrate CRISPR/Cas12a system for target recognition, which produces a rapid and high-throughput ECL imaging platform. The proposed imaging method for HPV16 DNA detection shows excellent performance with a detection limit of 0.6 fM. This work broadens the application of hot exciton materials in ECL field, and opens a new avenue for developing next-generation ECL devices.

Self- and non-self-DNA on hands and sleeve cuffs.

Studying DNA transfer and persistence has become increasingly important over the last decade, due to the impressive sensitivity of modern DNA detection methods in forensic genetics. To improve our understanding of background DNA that could also potentially be transferred, we analyzed the DNA composition on the outside of sleeve cuffs and sampled DNA directly from the hands of four different collaborators upon their arrival at work during 25 working days. Sampling of their hands was repeated after several hours working in our department. The shedder status of the participants, as assumed from previous internal studies, was well re-produced in the study. However, we noticed that the DNA shedding capacity could also change drastically during the day, with one participant showing a more than sixfold increase between hands sampled in the morning and hands sampled in the afternoon. As expected, poor DNA shedders carry more relative amounts of non-self-DNA on their hands than good shedders. Non-self-alleles were detected in 95% of the samples. We also observed potential effects of hand washing and the mode of transport to get to work on the DNA amount. People living with family members occasionally carried their DNA on their hands and more frequently on their sleeve cuffs. Sleeve cuffs, as being close to our hands, have a large potential to transfer DNA from one place to another, yet they have sparsely been studied as DNA transfer intermediates so far. In general, we collected consistently more DNA from the sleeve cuffs than from the hands of the participants, demonstrating their importance as potential transfer vectors. More DNA was recovered from sleeve cuffs made of synthetic fabric than from cuffs made of cotton or leather. In the afternoon, DNA from co-habitant family members could not be detected on the hands anymore and the detection of profiles from colleagues became more frequent. From two out of 100 analyzed sleeve cuffs and two out of 200 sampled hands, we established unknown major DNA profiles that would have been suitable for an entry in the national DNA database. This finding demonstrates the possibility to transfer DNA that has most likely been picked up somewhere in the public space.

Human schistosomiasis in Nigeria: present status, diagnosis, chemotherapy, and herbal medicines.

Schistosomiasis is a neglected tropical disease caused by a parasitic, trematode blood fluke of the genus Schistosoma. With 20 million people infected, mostly due to Schistosoma haematobium, Nigeria has the highest burden of schistosomiasis in the world. We review the status of human schistosomiasis in Nigeria regarding its distribution, prevalence, diagnosis, prevention, orthodox and traditional treatments, as well as snail control strategies. Of the country's 36 states, the highest disease prevalence is found in Lagos State, but at a geo-political zonal level, the northwest is the most endemic. The predominantly used diagnostic techniques are based on microscopy. Other methods such as antibody-based serological assays and DNA detection methods are rarely employed. Possible biomarkers of disease have been identified in fecal and blood samples from patients. With respect to preventive chemotherapy, mass drug administration with praziquantel as well as individual studies with artemisinin or albendazole have been reported in 11 out of the 36 states with cure rates between 51.1 and 100%. Also, Nigerian medicinal plants have been traditionally used as anti-schistosomal agents or molluscicides, of which Tetrapleura tetraptera (Oshosho, aridan, Aidan fruit), Carica papaya (Gwanda, Ìbẹ́pẹ, Pawpaw), Borreria verticillata (Karya garma, Irawo-ile, African borreria), and Calliandra portoricensis (Tude, Oga, corpse awakener) are most common in the scientific literature. We conclude that the high endemicity of the disease in Nigeria is associated with the limited application of various diagnostic tools and preventive chemotherapy efforts as well as poor knowledge, attitudes, and practices (KAP). Nonetheless, the country could serve as a scientific base in the discovery of biomarkers, as well as novel plant-derived schistosomicides and molluscicides.

Label free optofluidic DNA hybridization detection based on suspended core fiber whispering gallery mode resonator

An optofluidic DNA biosensor based on a suspended core fiber (SCF) has been proposed for the detection of specific DNA sequence fragments. The sensor is formed by coupling a micro/nano tapered optical fiber to the cladding of an SCF, producing a whispering gallery mode (WGM) effect. The WGM resonance is sensitive to samples within the channels of the SCF. The diameter of the SCF is reduced by etching to increase refractive index sensitivity, up to 274 nm/RIU. Biomarkers are captured on the inner surface of the microfluidic channel leading to a change in the local refractive index. The experimental results show that the sensor can quantitatively detect complementary DNA (cDNA) by immobilizing probe DNA (pDNA) on the surface of the microfluidic channel with a limit of detection of 1.5 nM. The proposed DNA biosensor provides a label-free method for DNA detection, which has great potential for multiplexed detection and realizing disease diagnostics.

Digital assay for rapid electronic quantification of clinical pathogens using DNA nanoballs.

Fast and accurate detection of nucleic acids is key for pathogen identification. Methods for DNA detection generally rely on fluorescent or colorimetric readout. The development of label-free assays decreases costs and test complexity. We present a novel method combining a one-pot isothermal generation of DNA nanoballs with their detection by electrical impedance. We modified loop-mediated isothermal amplification by using compaction oligonucleotides that self-assemble the amplified target into nanoballs. Next, we use capillary-driven flow to passively pass these nanoballs through a microfluidic impedance cytometer, thus enabling a fully compact system with no moving parts. The movement of individual nanoballs is detected by a change in impedance providing a quantized readout. This approach is flexible for the detection of DNA/RNA of numerous targets (severe acute respiratory syndrome coronavirus 2, HIV, β-lactamase gene, etc.), and we anticipate that its integration into a standalone device would provide an inexpensive (<$5), sensitive (10 target copies), and rapid test (<1 hour).