Multiple Primers Research Articles

A rapid and visual detection assay for Senecavirus A based on recombinase-aided amplification and lateral flow dipstick.

Senecavirus A (SVA) is a newly pathogenic virus correlated with the acute death of piglets and vesicular lesions in pigs. The further prevalence of SVA will cause considerable economic damage to the global pig farming industry. Therefore, rapid and accurate diagnostic tools for SVA are crucial for preventing and controlling the disease. We designed multiple primer pairs targeting the most conserved region of the SVA 3D gene and selected those with the highest specificity. Nfo-probes were subsequently developed based on the highest specificity primer pairs. Subsequently, the recombinase-assisted amplification (RAA) reaction was completed, and the reaction temperature and duration were optimized. The RAA amplicons were detected using a lateral flow device (LFD). Finally, a rapid and intuitive RAA-LFD assay was established against SVA. The SVA RAA-LFD assay can be performed under reaction conditions of 35°C within 17 minutes, with results observable to the naked eye. We then evaluated the performance of this method. It exhibited high specificity and no cross-reaction with the other common swine pathogens. The lowest detectable limits of this method for the plasmid of pMD18-SVA-3D, DNA amplification product, and viral were 3.86×101 copies/µL, 8.76×10-7 ng/µL, and 1×100.25 TCID50/mL, respectively. A total of 44 clinical samples were then tested using the RAA-LFD, PCR, and RT-qPCR methods. The results demonstrated a consistent detection rate between the RAA-LFD and RT-qPCR assays. The SVA RAA-LFD assay developed in our study exhibits excellent specificity, sensitivity, and time-saving attributes, making it ideally suited for utilization in lack-instrumented laboratory and field settings.

Deep learning assisted, smartphone based universal Multi-RPA-CRISPR/Cas12a-G4 portable chip for simultaneous detection of CaMV35S and NOS

Portable and fast genetically modified organisms (GMOs) detection can achieve accurate labeling and precise regulation. Here, we build a universal and intelligent Multi-RPA-CRISPR/Cas12a-G4 detection chip platform for simultaneous CaMV35S and NOS detection. The use of multiple primer pairs for RPA enables one-tube amplification of dual targets. Independent cleavage of Cas12a for dual targets was controlled by flow amplification products into the respective detection chambers containing CaMV35S crRNA and NOS crRNA. The detection results are obtained based that uncut G4 in solution can produce peroxidase activity to catalyze ABTS2− making the negative result approaching dark green. The multi-RPA-CRISRP/Cas12a-G4 colorimetric platform achieves a 1 aM LOD for CaMV35S and NOS in 55 min. The Yolov5 deep learning algorithm and the analysis of gray values was trained and developed for colorimetric detection that is more sensitive than the naked eye. These algorithms are integrated into a smartphone app to automate the reading of test results. The 100% accuracy of colorimetry detection and the 93.3% accuracy of portable chip detection for rice and soy sauce samples demonstrate our method can rapidly and inexpensively detect samples of GMOs across species. Overall, our efficient, low-cost portable Multi-RPA-CRISPR/Cas12a-G4 detection platform coupled with deep learning algorithms enables portable GMOs detection with automated signal readout.

In silico PCR analysis: a comprehensive bioinformatics tool for enhancing nucleic acid amplification assays.

Nucleic acid amplification assays represent a pivotal category of methodologies for targeted sequence detection within contemporary biological research, boasting diverse utility in diagnostics, identification, and DNA sequencing. The foundational principles of these assays have been extrapolated to various simple and intricate nucleic acid amplification technologies. Concurrently, a burgeoning trend toward computational or virtual methodologies is exemplified by in silico PCR analysis. In silico PCR analysis is a valuable and productive adjunctive approach for ensuring primer or probe specificity across a broad spectrum of PCR applications encompassing gene discovery through homology analysis, molecular diagnostics, DNA profiling, and repeat sequence identification. The prediction of primer and probe sensitivity and specificity necessitates thorough database searches, accounting for an optimal balance of mismatch tolerance, sequence similarity, and thermal stability. This software facilitates in silico PCR analyses of both linear and circular DNA templates, including bisulfited treatment DNA, enabling multiple primer or probe searches within databases of varying scales alongside advanced search functionalities. This tool is suitable for processing batch files and is essential for automation when working with large amounts of data.

Development of machine learning algorithm for loop-mediated isothermal amplification including influence of temperature

To predict amplification in the Loop-Mediated Isothermal Amplification (LAMP) method, a binary model has been developed. This model assesses whether the combination of partial template binding facilitated by multiple primers aligns with a specific signature indicating amplification. For this model, 1512 settings were adjusted, encompassing variations in the extraction oligomer position from the primer, the oligomer length, the number of allowed mismatched bases, and the stringency of the homology position. The optimal set was determined based on the F1 value at a reaction temperature of 60°C, resulting in a value of 0.58. To implement machine learning by amalgamating multiple models, duplicate models were eliminated through cluster analysis. Machine learning using AutoML was performed on 108 distinct models. Consequently, the F1 values were 0.7119 at a reaction temperature of 60°C and 0.7210 at 68°C. The weak correlation between feature importance and the F1 value of the model alone suggests that machine learning utilizing this model incorporates numerous models to enhance prediction accuracy.

Refining Sampling Efforts for Fish Diversity Assessment in Subtropical Urban Estuarine and Oceanic Waters Using Environmental DNA With Multiple Primers

ABSTRACTThe environmental DNA (eDNA) approach is an emerging tool for monitoring marine biodiversity. However, the sampling effort needs optimization according to the site characteristics and target taxonomic groups. In this study, we optimized the eDNA sampling effort in terms of sample volume and number of replicates to monitor the diversity of marine vertebrates (mainly fish) in Hong Kong's subtropical waters that show a gradient of estuarine to oceanic waters. To maximize detection, we used three pairs of metabarcoding primers (12S‐v5, MiFish‐U, and MiFish‐E). We compared vertebrate diversity in 78 water samples, ranging from 1 to 10 L, collected from oceanic and estuarine sites. Metabarcoding yielded a total of 140 vertebrate species, of which 18 were unique to the estuarine site, 66 unique to the oceanic site, and 56 shared between both sites. The detected species were predominantly ray‐finned fish (136 species), and the three primer pairs exhibited differential sensitivity toward different taxa, especially cartilaginous fish and cetaceans. Increasing sampling volume per replicate generally increased the total detected species, average species per replicate, and species coverage, and sampling 3 or 4 × 4 L represented the most efficient sampling effort for the estuarine and oceanic sites, respectively. The diversity analysis revealed that sampling >2 L per replicate reduced variability and improved diversity analysis. The results also showed that a larger sampling volume per replicate increased the probability of detecting endangered, indicator, invasive, and elusive species, with 4 L representing the most efficient volume. This study recommended sampling 4 L per replicate and 3 replicates for estuarine and 4 for oceanic sites, respectively for effectively monitoring marine fish in subtropical waters using the eDNA approach.

Effect of Long-Term Tai Chi Therapy on the Immune-Inflammatory Pathway in Patients with Schizophrenia with Antipsychotic-Stabilized.

The primary objective of this study was to explore the influence of prolonged (24weeks) supplementary Tai Chi therapy on cognitive capabilities and immune-inflammatory pathways in subjects diagnosed with schizophrenia. A total of 90 individuals who have been clinically diagnosed with schizophrenia were assigned to two treatment groups, namely the Tai Chi treatment (TT) group and the routine treatment (RT) group. Following a 24-week duration of intervention, the data obtained from 32 patients in the TT group and 30 patients in the RT group were meticulously analyzed. At the commencement of the investigation and upon completion of the 24-week intervention, blood samples were gathered, and clinical evaluations were executed. In plasma, the identification of nine cytokines (IL-10, IFN-γ, IL-5, GM-CSF, TNF-α, IL-13, IL-4, IL-2, and IL-12) was conducted using the multiple primer suspension chip method. The clinical evaluations encompassed CGI, WHOQUOL-BREF, SOFS, PSS, BPRS, SAPS, SANS, and RBANS. In comparison to the RT group, the patients in the TT group demonstrated decreased levels of TNF-α and IL-5 (P < 0.05). Moreover, they encountered more pronounced advancements in SAPS, SANS, PSS, SOFS, and RBANS scores (P < 0.05). Additionally, a positive connection was detected between the plasma TNF-α level in the TT group and both the SANS score and the SPFS score (P < 0.05). Tai Chi has been shown to improve clinical symptoms in patients with schizophrenia as an add-on therapy, potentially through its effects on immunomodulatory pathways.

Sequence-specific nanoparticle barcode strategy for multiplex human enterovirus typing

Human enteroviruses (HEV) can cause a range of diseases from mild to potentially life-threatening. Identification and genotyping of HEV are crucial for disease management. Existing typing methods, however, have inherent limitations. Developing alternative methods to detect HEV with more virus types, high accuracy, and sensitivity in an accessible manner presents a technological and analytical challenge. Here, a sequence-specific nanoparticle barcode (SSNB) method is presented for simultaneous detection of 10 HEV types. This method significantly increases sensitivity, enhancing detection by 10-106 times over the traditional multiplex hybrid genotyping (MHG) method, by resolving cross-interference between the multiple primer sets. Furthermore, the SSNB method demonstrates a 100% specificity in accurately distinguishing between 10 different HEV types and other prevalent clinical viruses. In an analysis of 70 clinical throat swab samples, the SSNB method shows slightly higher detection rate for positive samples (50%) compared to the RT-PCR method (48.6%). Additionally, further assessment of the typing accuracy for samples identified as positive by SSNB using sequencing method reveals a concordance rate of 100%. The combined high sensitivity and specificity level of the methodology, together with the capability for multiple type analysis and compatibility with clinical workflow, make this approach a promising tool for clinical settings.

Enhancing efficiency in detection of COVID-19 through AI-driven colorimetric isothermal detection with multiplex primers

COVID-19 has afflicted millions of lives worldwide. Although there are many rapid methods to detect it based on colorimetric loop-mediated isothermal amplification, there remains room for improvement. This study aims to 1) integrate multiple primers into a singleplex assay to enhance the diagnostic sensitivity, and 2) utilize a high-throughput smartphone-operatable AI-driven color reading tool to enable a rapid result analysis. This setup can improve the sensitivity by 10–100 times and can analyze approximately 6700 samples per minute. The assay is simpler than RT-qPCR, with a turnaround time of less than 75 min. It can detect various types of SARS-CoV-2 by targeting 3 genes, increasing the likelihood that it will remain effective even if the virus undergoes mutations in any single target gene. In summary, it affords potential for adaptation to detection of new/re-emerging diseases with the visual readout for maximum assay simplicity and AI-operated mode for large-scale testing.

Neuropathologically directed profiling of PRNP somatic and germline variants in sporadic human prion disease.

Creutzfeldt-Jakob Disease (CJD), the most common human prion disease, is associated with pathologic misfolding of the prion protein (PrP), encoded by the PRNP gene. Of human prion disease cases,<1% were transmitted by misfolded PrP, ~ 15% are inherited, and ~ 85% are sporadic (sCJD). While familial cases are inherited through germline mutations in PRNP, the cause of sCJD is unknown. Somatic mutations have been hypothesized as a cause of sCJD, and recent studies have revealed that somatic mutations accumulate in neurons during aging. To investigate the hypothesis that somatic mutations in PRNP may underlie sCJD, we performed deep DNA sequencing of PRNP in 205 sCJD cases and 170 age-matched non-disease controls. We included 5 cases of Heidenhain variant sporadic CJD (H-sCJD), where visual symptomatology and neuropathology implicate localized initiation of prion formation, and examined multiple regions across the brain including in the affected occipital cortex. We employed Multiple Independent Primer PCR Sequencing (MIPP-Seq) with a median depth of > 5000× across the PRNP coding region and analyzed for variants using MosaicHunter. An allele mixing experiment showed positive detection of variants in bulk DNA at a variant allele fraction (VAF) as low as 0.2%. We observed multiple polymorphic germline variants among individuals in our cohort. However, we did not identify bona fide somatic variants in sCJD, including across multiple affected regions in H-sCJD, nor in control individuals. Beyond our stringent variant-identification pipeline, we also analyzed VAFs from raw sequencing data, and observed no evidence of prion disease enrichment for the known germline pathogenic variants P102L, D178N, and E200K. The lack of PRNP pathogenic somatic mutations in H-sCJD or the broader cohort of sCJD suggests that clonal somatic mutations may not play a major role in sporadic prion disease. With H-sCJD representing a localized presentation of neurodegeneration, this serves as a test of the potential role of clonal somatic mutations in genes known to cause familial neurodegeneration.

DNA, databases and diet: a case study on the parrotfish Scarus rivulatus

DNA metabarcoding is increasingly used in dietary studies, but this molecular technique has limitations including DNA extraction, primer choice suitability and completeness of DNA reference databases. Together, these limitations can create successive biases; some of which can be identified by careful data interrogation. Here, we examine some of the influential factors impacting DNA metabarcoding by focusing on the diet of the grazing coral reef parrotfish Scarus rivulatus (Labridae). The 16S V3-4 and 18S V4 gene regions were targeted to detect a wide range of taxa in the pharyngeal content, but genus-level amplicon sequence variant (ASV) assignments were ≤ 40%. Multiple universal 18S V4 primer sets, which amplified the same portion of the gene region, highlighted disparities that impacted resulting datasets and dietary data interpretation. Approximately 50% of ASVs overlapped among these 18S V4 datasets. Overall, our results show that protein-rich epilithic and endolithic microscopic photoautotrophs were consistently present and that S. rivulatus appears to rely on protein-rich taxa, especially dinoflagellates and cyanobacteria. Even if all qualitative limitations could be addressed, our data emphasise the difficulty of making straightforward quantitative conclusions about the diet composition of grazing fishes using DNA metabarcoding alone. Thus, until DNA metabarcoding reaches a state of maturity where quantitative data can be gained, additional support is required from other dietary analysis methods to provide robust interpretations and conclusions.

Neuropathologically-directed profiling of PRNP somatic and germline variants in sporadic human prion disease.

Creutzfeldt-Jakob Disease (CJD), the most common human prion disease, is associated with pathologic misfolding of the prion protein (PrP), encoded by the PRNP gene. Of human prion disease cases, ~1% were transmitted by misfolded PrP, ~15% are inherited, and ~85% are sporadic (sCJD). While familial cases are inherited through germline mutations in PRNP, the cause of sCJD is unknown. Somatic mutations have been hypothesized as a cause of sCJD, and recent studies have revealed that somatic mutations accumulate in neurons during aging. To investigate the hypothesis that somatic mutations in PRNP may underlie sCJD, we performed deep DNA sequencing of PRNP in 205 sCJD cases and 170 age-matched non-disease controls. We included 5 cases of Heidenhain variant sporadic CJD (H-sCJD), where visual symptomatology and neuropathology implicate focal initiation of prion formation, and examined multiple regions across the brain including in the affected occipital cortex. We employed Multiple Independent Primer PCR Sequencing (MIPP-Seq) with a median depth of >5,000X across the PRNP coding region and analyzed for variants using MosaicHunter. An allele mixing experiment showed positive detection of variants in bulk DNA at a variant allele fraction (VAF) as low as 0.2%. We observed multiple polymorphic germline variants among individuals in our cohort. However, we did not identify bona fide somatic variants in sCJD, including across multiple affected regions in H-sCJD, nor in control individuals. Beyond our stringent variant-identification pipeline, we also analyzed VAFs from raw sequencing data, and observed no evidence of prion disease enrichment for the known germline pathogenic variants P102L, D178N, and E200K. The lack of PRNP pathogenic somatic mutations in H-sCJD or the broader cohort of sCJD suggests that clonal somatic mutations may not play a major role in sporadic prion disease. With H-sCJD representing a focal presentation of neurodegeneration, this serves as a test of the potential role of clonal somatic mutations in genes known to cause familial neurodegeneration.

Development of Highly Efficient Universal Pneumocystis Primers and Their Application in Investigating the Prevalence and Genetic Diversity of Pneumocystis in Wild Hares and Rabbits.

Despite its ubiquitous infectivity to mammals with strong host specificity, our current knowledge about Pneumocystis has originated from studies of merely 4% of extant mammalian species. Further studies of Pneumocystis epidemiology across a broader range of animal species require the use of assays with high sensitivity and specificity. To this end, we have developed multiple universal Pneumocystis primers targeting different genetic loci with high amplification efficiency. Application of these primers to PCR investigation of Pneumocystis in free-living hares (Lepus townsendii, n = 130) and rabbits (Oryctolagus cuniculus, n = 8) in Canada revealed a prevalence of 81% (105/130) and 25% (2/8), respectively. Genotyping analysis identified five and two variants of Pneumocystis from hares and rabbits, respectively, with significant sequence divergence between the variants from hares. Based on phylogenetic analysis using nearly full-length sequences of the mitochondrial genome, nuclear rRNA operon and dihydropteroate synthase gene for the two most common variants, Pneumocystis in hares and rabbits are more closely related to each other than either are to Pneumocystis in other mammals. Furthermore, Pneumocystis in both hares and rabbits are more closely related to Pneumocystis in primates and dogs than to Pneumocystis in rodents. The high prevalence of Pneumocystis in hares (P. sp. 'townsendii') suggests its widespread transmissibility in the natural environment, similar to P. oryctolagi in rabbits. The presence of multiple distinct Pneumocystis populations in hares contrasts with the lack of apparent intra-species heterogeneity in P. oryctolagi, implying a unique evolution history of P. sp. 'townsendii' in hares.

Validation and development of eDNA metabarcoding primers for comprehensive assessment of Chinese amphibians.

Environmental DNA (eDNA) metabarcoding has emerged as a powerful, non-invasive tool for biodiversity assessments. However, the accuracy and limitations of these assessment techniques are highly dependent on the choice of primer pairs being used. Although several primer sets have been used in eDNA metabarcoding studies of amphibians, there are few comparisons of their reliability and efficiency. Here, we employed lab- and field-tested sets of publicly available and de novo-designed primers in amplifying 83 species of amphibian from all three orders (Anura, Caudata, and Gymnophiona) and 13 families present in China to evaluate the versatility and specificity of these primers sets in amphibian eDNA metabarcoding studies. Three pairs of primers were highly effective, as they could successfully amplify all the major clades of Chinese amphibians in our study. A few non-amphibian taxa were also amplified by these primers, which implies that further optimization of amphibian-specific primers is still needed. The simultaneous use of three primer sets can completely cover all the species obtained by conventional survey methods and has even effectively distinguished quite a number of species (n = 20) in the Wenshan National Nature Reserve. No single primer set could individually detect all of the species from the studied region, indicating that multiple primers might be necessary for a comprehensive survey of Chinese amphibians. Besides, seasonal variations in amphibian species composition were also revealed by eDNA metabarcoding, which was consistent with traditional survey methods. These results indicate that eDNA metabarcoding has the potential to be a powerful tool for studying spatial and temporal community changes in amphibian species richness.

Pathogenicity, Host Resistance, and Genetic Diversity of Fusarium Species under Controlled Conditions from Soybean in Canada.

Fusarium spp. are commonly associated with the root rot complex of soybean (Glycine max). Previous surveys identified six common Fusarium species from Manitoba, including F. oxysporum, F. redolens, F. graminearum, F. solani, F. avenaceum, and F. acuminatum. This study aimed to determine their pathogenicity, assess host resistance, and evaluate the genetic diversity of Fusarium spp. isolated from Canada. The pathogenicity of these species was tested on two soybean cultivars, 'Akras' (moderately resistant) and 'B150Y1' (susceptible), under greenhouse conditions. The aggressiveness of the fungal isolates varied, with root rot severities ranging from 1.5 to 3.3 on a 0-4 scale. Subsequently, the six species were used to screen a panel of 20 Canadian soybean cultivars for resistance in a greenhouse. Cluster and principal component analyses were conducted based on the same traits used in the pathogenicity study. Two cultivars, 'P15T46R2' and 'B150Y1', were consistently found to be tolerant to F. oxysporum, F. redolens, F. graminearum, and F. solani. To investigate the incidence and prevalence of Fusarium spp. in Canada, fungi were isolated from 106 soybean fields surveyed across Manitoba, Saskatchewan, Ontario, and Quebec. Eighty-three Fusarium isolates were evaluated based on morphology and with multiple PCR primers, and phylogenetic analyses indicated their diversity across the major soybean production regions of Canada. Overall, this study contributes valuable insights into host resistance and the pathogenicity and genetic diversity of Fusarium spp. in Canadian soybean fields.

Autonomous enzymatic synthesis of functional nucleic acids for sensitive measurement of long noncoding RNA in human lung tissues

Aberrant long noncoding RNA (lncRNA) expression is linked to varied pathological processes and malignant tumors, and lncRNA can serve as potential disease biomarkers. Herein, we demonstrate the autonomous enzymatic synthesis of functional nucleic acids for sensitive measurement of lncRNA in human lung tissues on the basis of multiple primer generation-mediated rolling circle amplification (mPG-RCA). This assay involves two padlock probes that act as both a detection probe for recognizing target lncRNA and a domain for producing complementary DNAzyme. Two padlock probes can hybridize with target lncRNA at different sites, followed by ligation to form a circular template with the aid of RNA ligase. The circular template can initiate mPG-RCA to generate abundant Mg2+-dependent DNAzymes that can specifically cleave signal probes to induce the recovery of Cy3 fluorescence. The inherent characteristics of ligase-based ligation reaction and DNAzymes endow this assay with excellent specificity, and the introduction of multiple padlock probes endows this assay with high sensitivity. This strategy can rapidly and sensitively measure lncRNA with a wide linear range of 1 fM − 1 nM and a detection limit of 678 aM within 1.5 h, and it shows distinct advantages of simplicity and immobilization-free without the need of precise temperature control and tedious procedures of nanomaterial preparation. Moreover, it enables accurate measurement of lncRNA level in normal cells and malignant tumor cells as well as differentiation of lncRNA expressions in tissues of non-small cell lung cancer (NSCLC) patients and normal individuals, with promising applications in biomedical studies and disease diagnosis.

Using a Handful of Transcriptomes to Detect Sex-Linked Markers and Develop Molecular Sexing Assays in a Species with Homomorphic Sex Chromosomes.

To understand the biology of a species, it is often crucial to be able to differentiate males and females. However, many species lack easily identifiable sexually dimorphic traits. In those that possess sex chromosomes, molecular sexing offers a good alternative, and molecular sexing assays can be developed through the comparison of male and female genomic sequences. However, in many nonmodel species, sex chromosomes are poorly differentiated, and identifying sex-linked sequences and developing sexing assays can be challenging. In this study, we highlight a simple transcriptome-based procedure for the detection of sex-linked markers suitable for the development of sexing assays that circumvents limitations of more commonly used approaches. We apply it to the spotted snow skink Carinascincus ocellatus, a viviparous lizard with homomorphic XY chromosomes that has environmentally induced sex reversal. With transcriptomes from three males and three females alone, we identify thousands of putative Y-linked sequences. We confirm linkage through alignment of assembled transcripts to a distantly related lizard genome and readily design multiple single locus polymerase chain reaction primers to sex C. ocellatus and related species. Our approach also facilitates valuable comparisons of sex determining systems on a broad taxonomic scale.

Multiplex PCR method for MinION sequencing of Bagaza virus isolated from wild caught mosquitoes in South Africa

Bagaza virus (BAGV) is a mosquito-borne orthoflavivirus known to occur in regions of southern Europe, Africa, India and the Middle East. The virus has been associated with neurological disease and fatalities in various wild bird species. Association with human disease is not confirmed although limited serological evidence has suggested human infection. Surveillance programs for screening mosquitoes for evidence of arbovirus infection play an important role in providing information regarding the circulation and spread of viruses in specific regions. BAGV was detected in a mosquito pool during surveillance of mosquitoes collected in central South Africa between November 2019 and March 2023. Homogenized mosquito pools were screened for flaviviral RNA using conventional RT-PCR and virus isolation was attempted on positive samples. BAGV was detected and subsequently isolated using cell culture. A multiplex tiling PCR method for targeted enrichment using a PCR based or amplicon sequencing approach of the complete genome of BAGV was developed and optimized. Primers were designed using alignment of complete genome sequence data retrieved from GenBank to identify suitable primer sites that would generate overlapping fragments spanning the complete genome. Six forward primers and eight reverse primers were identified that target the complete genome and amplified nine overlapping fragments, that ranged in length from 1954 to 2039 with an overlap ranging from 71 to 711 base pairs. The design strategy included multiple forward and reverse primer pairs for the 5’ and 3’ ends. Phylogenetic analysis with other isolates was performed and BAGV isolate VBD 74/23/3 was shown to share high similarity with previous BAGV isolates from all regions, with genetic distance ranging from 0.026 to 0.083. VBD 74/23/3 was most closely related to previous isolates from southern Africa, ZRU96/16/2 isolated from a post-mortem sample from a pheasant in 2016 and MP-314-NA-2018 isolated from mosquitoes in northwestern Namibia with genetic distance 0.0085 and 0.016 respectively. Currently there is limited complete genome sequence data available for many of the arboviruses circulating in Africa. The multiplex tiling method provided a simple and cost-effective method for obtaining complete genome sequence. This method can be readily applied to other viruses using sequence data from publicly available databases and would have important application facilitating genomic surveillance of arboviruses in low resource countries.

Sex identification of birds in Taipei Zoo.

As a conservation and breeding institution for birds, Taipei Zoo plays an important role in restoring endangered species. As approximately half of all bird species are monomorphic, precisely confirming the sex of individuals is critical for the management of ex-situ conservation breeding populations, as well as for understanding the sex ratio of those in the wild. Generally, PCR is used more reliably for sex determination versus traditional methods such as plumage, behavior or hormone levels. Nevertheless, the various primer sets and annealing temperatures vary between species, and so inaccurate sexing can occasionally happen due to inadequate PCR conditions. To reduce the probability of misidentification, and to establish a PCR condition database for sex determination across the diverse range of avian taxa, we tested multiple primer sets and annealing temperatures for amplification of the bird sex-specific gene fragments (CHD1) for each captive or rescued avian species held at Taipei Zoo since 2014. A total of 162 species across 22 orders were tested using one or two primer sets. One hundred and fifty-five species were successfully sexed by the primer set 2550F/2718R and the success rate of sex typing reached over 90% of species tested in each order. Most species have suitable PCR annealing temperatures between 45°C and 55°C, and the species in the same avian taxa showed similar results in temperature. This indicates that it is possible to select the annealing temperature of other species in the same family when the species had not been tested before. We expect this study will improve the success rate of identifying sex by using applicable PCR conditions and reduce the time for searching references every time before attempts to PCR sex birds.

Using DNA metabarcoding to separate natural and human provided food in wild boar diet at the northern distribution range of Europe

Wild boar Sus scrofa is increasing in numbers and extending its distribution across Europe and is difficult to control due to high reproductive potential. Dietary quality is a main determinant of wild boar population dynamics, and the extent to which they rely on human‐provided food provide a key to limit their distribution. Yet, we lack data on wild boar diet from northern Europe. Here we use DNA‐metabarcoding of faecal samples (n = 50) to determine wild boar diet during fall and winter in Norway. We mainly aimed to quantify the extent to which wild boar relies on natural or human‐provided food sources. A secondary aim was to determine whether diet varies with individual characteristics (sex, age or weight), season (winter or fall), and between the two regions with wild boar in Norway. We found a high degree of diet variability between individuals. Individuals consuming high amounts of edible fungi consumed low amounts of plant material. The (heavier) male wild boars consumed 50% more human provided food than the (lighter) female wild boars. There was no clear effect of age, season (winter versus fall), or region on diet with the sample size available. The negative correlation between plants and fungi in each sample suggests that using multiple primers targeting different taxa can provide quantitative diet information, and points to an important role of fungi (truffles) during winter and fall. The large individual variation in diet may reflect opportunistic feeding tactics in Scandinavian boreal forests, driven by a lack of acorns and few crops. Our study has relevance for understanding limitations of wild boars at their northern distribution range in Europe, and thus also provides important information for management.

Enhancing amphibian biomonitoring through eDNA metabarcoding.

Surveying biodiversity has taken a quantum leap with environmental DNA (eDNA) metabarcoding, an immensely powerful approach lauded for its efficiency, sensitivity, and non-invasiveness. This approach emerges as a game-changer for the elusive realm of endangered and rare species-think nocturnal, environmentally elusive amphibians. Here, we have established a framework for constructing a reliable metabarcoding pipeline for amphibians, covering primer design, performance evaluation, laboratory validation, and field validation processes. The Am250 primer, located on the mitochondrial 16S gene, was optimal for the eDNA monitoring of amphibians, which demonstrated higher taxonomic resolution, smaller species amplification bias, and more extraordinary detection ability compared to the other primers tested. Am250 primer exhibit an 83.8% species amplification rate and 75.4% accurate species identification rate for Chinese amphibians in the in silico PCR and successfully amplified all tested species of the standard samples in the invitro assay. Furthermore, the field-based mesocosm experiment showed that DNA can still be detected by metabarcoding even days to weeks after organisms have been removed from the mesocosm. Moreover, field mesocosm findings indicate that eDNA metabarcoding primers exhibit different read abundances, which can affect the relative biomass of species. Thus, appropriate primers should be screened and evaluated by three experimental approaches: in silico PCR simulation, target DNA amplification, and mesocosm eDNA validation. The selection of a single primer set or multiple primers' combination should be based on the monitoring groups to improve the species detection rate and the credibility of results.