Successful PCR Research Articles

Developing a Machine-Learning 'Smart' PCR Thermocycler, Part 1: Construction of a Theoretical Framework.

The use of PCR is widespread in biological fields. Some fields, such as forensic biology, push PCR to its limits as DNA profiling may be required in short timeframes, may be produced from minute amounts of starting material, and may be required to perform in the presence of inhibitory compounds. Due to the extreme high-throughput of samples using PCR in forensic science, any small improvement in the ability of PCR to address these challenges can have dramatic effects for the community. At least part of the improvement in PCR performance could potentially come by altering PCR cycling conditions. These alterations could be general, in that they are applied to all samples, or they could be tailored to individual samples for maximum targeted effect. Further to this, there may be the ability to respond in real time to the conditions of PCR for a sample and make cycling parameters change on the fly. Such a goal would require both a means to track the conditions of the PCR in real time, and the knowledge of how cycling parameters should be altered, given the current conditions. In Part 1 of our work, we carry out the theoretical groundwork for the ambitious goal of creating a smart PCR system that can respond appropriately to features within individual samples in real time. We approach this task using an open qPCR instrument to provide real-time feedback and machine learning to identify what a successful PCR 'looks like' at different stages of the process. We describe the fundamental steps to set up a real-time feedback system, devise a method of controlling PCR cycling conditions from cycle to cycle, and to develop a system of defining PCR goals, scoring the performance of the system towards achieving those goals. We then present three proof-of-concept studies that prove the feasibility of this overall method. In a later Part 2 of our work, we demonstrate the performance of the theory outlined in this paper on a large-scale PCR cycling condition alteration experiment. The aim is to utilise machine learning so that throughout the process of PCR automatic adjustments can be made to best alter cycling conditions towards a user-defined goal. The realisation of smart PCR systems will have large-scale ramifications for biological fields that utilise PCR.

Designing A Novel Primer Set for GAPDH Gene to Enhance Taxonomic and Phylogenetic Studies of Golovinomyces Species

Golovinomyces (Erysiphaceae, Ascomycota) is an obligate plant pathogenic group causing powdery mildew on diverse angiosperm plants, including economically significant crops. Despite advancements in the taxonomy and phylogeny of Golovinomyces species using ribosomal DNA markers (ITS and LSU), several taxonomic issues remain unresolved. Previously, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, which exhibits higher nucleotide variation, has been proposed as an additional marker for powdery mildew species. In this study, we designed a new primer set (GoGPD-F and GoGPD-R) to improve the PCR success and efficiency of the GAPDH gene across various Golovinomyces species and dried herbarium specimens. The primers were successful in amplifying and sequencing the GAPDH gene in sixteen Golovinomyces species, including six species not previously registered in GenBank and two undescribed species. This development is a significant contribution to future research on the identification, taxonomy, and phylogeny of Golovinomyces species, offering a more robust tool for resolving existing taxonomic issues.

Distribution of eukaryotic environmental DNA in global subseafloor sediments

The analysis of eukaryotic environmental DNA (eDNA) in sediment has the potential for understanding past ecosystems, even for taxa lacking skeletons or preserved only as a part of necromass. Despite the paleoenvironmental and ecological importance of eukaryotic eDNA in marine sediment, the duration of remaining eDNA and the species of eDNA present in marine sediment has not been well investigated. Here, we analyzed eDNA extracted from 299 sediment samples down to 678 m below the seafloor at 40 geologically distinct sites. The results showed that eukaryotic eDNA was amplified from more than 80% of the sediments with a depositional age of less than 100,000 years. The eDNA was well conserved in anoxic sediments than in oxic sediments, with PCR success rates of 48% and 18%, respectively. The eukaryotic communities include non-benthic organisms such as marine plankton, including diatoms, dinoflagellates, and coccolithophores. A freshwater diatom genus Aulacoseira was detected in the Baltic Sea sediments from the last glacial lacustrine environment. These results provide new insights into the global-scale distribution of the past eukaryotic eDNA preserved in marine sediment.

Analysis of markers for forensic plant species identification

While plant species identification in forensics can be useful in cases involving poisonous, psychoactive, or endangered plant species, it can also become quite challenging, especially, when dealing with processed, decaying, colonized or infected material of plant origin. The Animal Plant and Soil Traces expert working group of the European Network of Forensic Science Institutes in their best practice manual has recommended several markers for plant species identification. Current study is a part of implementation of method in a forensic laboratory and its aim is to evaluate four of the recommended markers (ITS, matK, rbcL, and trnH-psbA) for species identification of forensically important plant species including medicinal, poisonous, psychoactive, and other plants. Such parameters as PCR and sequencing success, sequence length, species resolution rate and species cover in GenBank were analysed. Blind testing was performed to evaluate use of the markers for identification of forensically more complicated samples.According to results, a combination of ITS, matK and trnH-psbA is the best choice for plant species identification. The best results with fresh plant material can be achieved with ITS, trnH-psbA, and matK, while ITS and matK are the best choice when working with low quality plant material. rbcL due to its low species discrimination rate can be used only as an indicative marker.

Quantitative and qualitative analysis of three DNA extraction methods from soybean, maize, and canola oils and investigation of the presence of genetically modified organisms (GMOs)

The objective of this study was to develop a DNA-based method for the identification and tracking of edible oils, which is important for health management. Three different DNA extraction methods (CTAB, MBST kit, and manual hexane-based method) were used to obtain high-purity DNA from crude and refined soybean, maize, and canola oils. PCR was then conducted using specific primers to identify the presence of genes related to each oil type and to assess transgenicity. The results showed that DNA was present in crude and refined oils, but in very low amounts. However, using method 3 for DNA extraction provided sufficient quantity and quality of DNA for successful PCR amplification. The study concluded that the main challenge in DNA extraction from oils is the presence of PCR inhibitors, which can be overcome using the manual hexane-based method. Also, the examination of protein presence in the oils using SDS-PAGE did not indicate any protein bands.

Optimizing waterborne eDNA capture from waterholes in savanna systems under remote field conditions.

Environmental DNA (eDNA) is used for biodiversity assessments in a variety of ecosystems across the globe, whereby different eDNA concentration, preservation and extraction methods can outperform others depending on the sampling conditions and environment. Tropical and subtropical ecosystems in Africa are among the less studied systems concerning eDNA-based monitoring. Waterholes in arid parts of southern Africa represent important agglomeration points for terrestrial mammals, and the eDNA shed into such waterbodies provides a powerful source of information for monitoring mammalian biodiversity in the surrounding area. However, the applied methods for eDNA sampling, preservation and filtering in different freshwater systems vary greatly, and rigorous protocol testing in African freshwater systems is still lacking. This study represents the first attempt to examine variations in eDNA concentration, preservation and extraction methods under remote field conditions using waterborne eDNA in a savanna system. Collected samples were heavily affected by microalgal and bacterial growth, impeding eDNA capture and PCR success. We demonstrate clear effects of the methodological choices, which also depend on the state of eDNA. A preliminary metabarcoding run showed little taxonomic overlap in mammal species detection between two metabarcoding primers tested. We recommend water filtering (using filters with pore sizes >1 μm) over centrifugation for eDNA concentration, Longmire's solution for ambient temperature sample preservation and Qiagen's DNeasy PowerSoil Pro Kit for DNA extraction of these inhibitor-prone samples. Furthermore, at least two independent metabarcoding markers should be utilized in order to maximize species detections in metabarcoding studies.

ONTbarcoder 2.0: rapid species discovery and identification with real-time barcoding facilitated by Oxford Nanopore R10.4.

Most arthropod species are undescribed and hidden in specimen-rich samples that are difficult to sort to species using morphological characters. For such samples, sorting to putative species with DNA barcodes is an attractive alternative, but needs cost-effective techniques that are suitable for use in many laboratories around the world. Barcoding using the portable and inexpensive MinION sequencer produced by Oxford Nanopore Technologies (ONT) could be useful for presorting specimen-rich samples with DNA barcodes because it requires little space and is inexpensive. However, similarly important is user-friendly and reliable software for analysis of the ONT data. It is here provided in the form of ONTbarcoder 2.0 that is suitable for all commonly used operating systems and includes a Graphical User Interface (GUI). Compared with an earlier version, ONTbarcoder 2.0 has three key improvements related to the higher read quality obtained with ONT's latest flow cells (R10.4), chemistry (V14 kits) and basecalling model (super-accuracy model). First, the improved read quality of ONT's latest flow cells (R10.4) allows for the use of primers with shorter indices than those previously needed (9 bp vs. 12-13 bp). This decreases the primer cost and can potentially improve PCR success rates. Second, ONTbarcoder now delivers real-time barcoding to complement ONT's real-time sequencing. This means that the first barcodes are obtained within minutes of starting a sequencing run; i.e. flow cell use can be optimized by terminating sequencing runs when most barcodes have already been obtained. The only input needed by ONTbarcoder 2.0 is a demultiplexing sheet and sequencing data (raw or basecalled) generated by either a Mk1B or a Mk1C. Thirdly, we demonstrate that the availability of R10.4 chemistry for the low-cost Flongle flow cell is an attractive option for users who require only 200-250 barcodes at a time.

A comparison of live versus kill pitfall traps to assess the diet of carabids through a metabarcoding approach

AbstractMetabarcoding approaches are powerful tools to unravel trophic relationships between predators and prey. To apply metabarcoding analyses on invertebrate gut contents, specimens must be well preserved from DNA degradation, thus the trapping method should be selected accordingly. Dry pitfall traps are commonly assumed to provide a better DNA preservation than traps that use a killing agent. However, this assumption has never been specifically tested for gut content analyses. In our study, we compared how two types of pitfall trapping, dry vs. with brine, affect the conservation of prey DNA contained in the digestive tract of predators and subsequent metabarcoding analyses. We placed dry and ‘classic’ pitfall traps in oilseed rape fields within an intensive agricultural area in the French Nouvelle‐Aquitaine region. Traps were set up in autumn and compared for carabid trapping efficiency as well as our capacity to retrieve dietary information from the digestive tract of the main carabid species, Nebria salina (Fairmaire & Laboulbène) and Calathus fuscipes (Goeze) (both Coleoptera: Carabidae). The PCR success rate was higher in dry pitfall traps compared to classic ones for N. salina. We hypothesise that this was due to the presence of PCR inhibitors in the gut of this species. The ability to sequence prey DNA did not differ between specimens caught in both trap types. The list of preyed species was similar between both trap types. However, sequencing yielded more prey operational taxonomic units (OTUs) from specimens caught in dry pitfall traps, leading to differences in prey community composition and a greater ability to reconstruct prey community. Our analyses also shed light on the prey spectrum of C. fuscipes and N. salina in oilseed rape in autumn.

A New qPCR Assay for the Rapid Diagnosis of Anthonomus grandis Subspecies.

Rapid and accurate identification of Anthonomus grandis subspecies is crucial for effective management and eradication. Current diagnostic methods have limitations in terms of time to diagnosis (up to seven days) and can yield ambiguous results. Here, we present the validation of a custom TaqMan SNP Genotyping Assay for the rapid and accurate identification of A. grandis grandis (boll weevil) and A. g. thurberiae (thurberia weevil) subspecies. To validate the assay, we conducted three main experiments: (1) a sensitivity test to determine the DNA concentration range at which the assay performs, (2) a non-target specificity test to ensure no amplification in non-target weevils (false positives), and (3) an accuracy test comparing the results of the new assay to previously established methods. These experiments were carried out in parallel at three independent facilities to confirm the robustness of the assay to variations in equipment and personnel. We used DNA samples from various sources, including field-collected specimens, museum specimens, and previously isolated DNA. The assay demonstrated high sensitivity (PCR success with ≥0.05 ng/µL DNA template), specificity (0.02 false positive rate), and accuracy (97.7%) in diagnosing boll weevil and thurberia weevil subspecies. The entire workflow, including DNA extraction, assay preparation, PCR run time, and data analysis, can be completed within a single workday (7-9 h) by a single technician. The deployment of this assay as a diagnostic tool could benefit boll weevil management and eradication programs by enabling same-day diagnosis of trap-captured or intercepted weevil specimens. Furthermore, it offers a more reliable method for identifying unknown specimens, contributing to the overall effectiveness of boll weevil research and control efforts.

UPrimer: A Clade-Specific Primer Design Program Based on Nested-PCR Strategy and Its Applications in Amplicon Capture Phylogenomics.

Amplicon capture is a promising target sequence capture approach for phylogenomic analyses, and the design of clade-specific nuclear protein-coding locus (NPCL) amplification primers is crucial for its successful application. In this study, we developed a primer design program called UPrimer that can quickly design clade-specific NPCL amplification primers based on genome data, without requiring manual intervention. Unlike other available primer design programs, UPrimer uses a nested-PCR strategy that greatly improves the amplification success rate of the designed primers. We examined all available metazoan genome data deposited in NCBI and developed NPCL primer sets for 21 metazoan groups with UPrimer, covering a wide range of taxa, including arthropods, mollusks, cnidarians, echinoderms, and vertebrates. On average, each clade-specific NPCL primer set comprises ∼1,000 NPCLs. PCR amplification tests were performed in 6 metazoan groups, and the developed primers showed a PCR success rate exceeding 95%. Furthermore, we demonstrated a phylogenetic case study in Lepidoptera, showing how NPCL primers can be used for phylogenomic analyses with amplicon capture. Our results indicated that using 100 NPCL probes recovered robust high-level phylogenetic relationships among butterflies, highlighting the utility of the newly designed NPCL primer sets for phylogenetic studies. We anticipate that the automated tool UPrimer and the developed NPCL primer sets for 21 metazoan groups will enable researchers to obtain phylogenomic data more efficiently and cost-effectively and accelerate the resolution of various parts of the Tree of Life.

The Water‐Exclusion Trap (WET): A 3D printable window trap collector that prevents DNA degradation

Abstract Sampling is one of the most important and fundamental processes in field biology. Among many sampling methods in entomology, window traps have long been considered one of the most effective and standardized methods. Unfortunately, the DNA quality of samples collected by window traps is typically low due to incursion by water, mainly via rainfall. Water cannot be avoided in long‐term surveys, even with a roof, due to wind and condensation. As we move into the genomics era, this shortfall in DNA quality has become problematic for molecular biomonitoring, and solutions are urgently needed. In this study, we developed the Water‐Exclusion Trap (WET), a 3D printable long‐term storable bottom collector for window or pitfall traps. The collection medium in WETs does not dilute because water is excluded and evaporation is limited, maximizing DNA preservation. Our field tests show that the WET outperforms conventional traps in preserving DNA quality. Insect samples preserved in WETs in the field had significantly higher PCR success rates and DNA Integrity Numbers compared to conventional traps. The WET addresses a critical challenge in the use of window traps, DNA degradation caused by water intrusion, and represents a significant advancement in fieldwork for genomic research in entomology. We provide the 3D printing template of the WET, enabling immediate, free use for all. Furthermore, it can be easily customized for different environments and purposes, and can be adapted to a variety of other traps, including pitfall traps, funnel traps and more.

Development of PCR based SSR markers for Wilsonomyces carpophilus and a PCR based diagnosis protocol for the early detection of shot hole disease in stone fruit crops.

The conidial Ascomycota fungus Wilsonomyces carpophilus causing shot hole in stone fruits is a major constraint in the production of stone fruits worldwide. Shothole disease symptoms appear on leaves, fruits, and twigs. Successful isolation of the pathogen from different hosts on synthetic culture medium is a time consuming and tedious procedure for identification of the pathogen based on morpho-cultural characterization. The present research was carried out to develop a successful PCR based early detection protocol for the shot hole disease of stone fruits, viz., peach, plum, apricot, cherry, and almond using the pathogen specific SSR markers developed from the Wilsonomyces carpophilus genome using Genome-wide Microsatellite Analysing Tool package (GMATA) software. Diseased leaf samples of different stone fruits were collected from the SKUAST-K orchard and the pathogen was isolated on potato dextrose agar (PDA) medium and maintained on Asthana and Hawkers' medium with a total of 50 pathogen isolates comprised of 10 isolates each from peach, plum, apricot, cherry and almond. The DNA was extracted from both healthy and infected leaf samples of different stone fruits. The DNA was also extracted from the isolated pathogen cultures (50 isolates). Out of 2851 SSR markers developed, 30 SSRs were used for the successful amplification of DNA extracted from all the 50 pathogen isolates. These SSRs were used for the amplification DNA from shot hole infected leaf samples of different stone fruits, but the amplification was not observed in the control samples (DNA from healthy leaves), thus confirming the detection of this disease directly from the shot hole infected samples using PCR based SSR markers. To our knowledge, this forms the first report of SSR development for the Wilsonomyces carpophilus and their validation for the detection of shot hole disease directly from infected leaves. PCR based SSR makers were successfully developed and used for the detection of Wilsonomyces carpophilus causing shot hole disease in stone fruits including almond in nuts for the first time. These SSR markers could successfully detect the pathogen directly from the infected leaves of stone fruits namely peach, plum, apricot and cherry including almond from the nuts.

The quality of DNA isolated from autopsy formalin-fixed and formalin-fixed paraffin-embedded tissues: study of 1662 samples.

There are enormous formalin-fixed paraffin-embedded tissue archives and a constantly growing number of methods for molecular analyses but, the isolation of DNA from this tissue is still challenging due to the damaging effect of formalin on DNA. To determine the extent to which DNA purity, yield and integrity depend on the process of fixation in formalin, and to what extent on the process of tissue paraffin embedding, we compared the quality of DNA isolated from fixed tissues and DNA isolated from tissues embedded in paraffin blocks after fixation. Heart, liver and brain tissues obtained from healthy people who suddenly died a violent death were fixed in 10% buffered formalin as well as in 4% unbuffered formalin for 6h, 1-7days (every 24h), 10, 14, 28days and 2months. Additionally, the same tissues were fixed in 4% unbuffered formalin embedded in a paraffin block and stored from a few months to 30years. The yield and purity of the DNA samples isolated from these tissues were measured using spectrophotometry. PCR amplification of the hTERT gene was performed to evaluate the degree of DNA fragmentation. Although the purity of the DNA isolated from almost all tissue samples was satisfactory, the DNA yields changed significantly. There was a decrease in successful PCR amplification of the hTERT gene in DNA samples isolated from tissue fixed in buffered and unbuffered formalin for up to 2months from 100% to 8.3%. Archiving the tissue in paraffin blocks for up to 30years also impacts the integrity of DNA, so there was a decrease in PCR amplification of the hTERT gene from 91% success to 3%. The largest decrease in DNA yield was observed after tissue formalin fixation after 14days of fixation in buffered and unbuffered formalin. DNA integrity depends on the time of tissue formalin fixation, especially after 6days for tissue fixed in unbuffered formalin, while for tissue fixed in buffered formalin the time is prolonged up to 28days. The age of paraffin blocks also impacted DNA integrity, after 1year and 16years of archiving the paraffin blocks of tissues, there was a decrease in the success of PCR amplification.

PPE Barcoding Identifies Biclonal Mycobacterium ulcerans Buruli Ulcer, Côte d'Ivoire.

Mycobacterium ulcerans, an environmental opportunistic pathogen, causes necrotic cutaneous and subcutaneous lesions, named Buruli ulcers, in tropical countries. PCR-derived tests used to detect M. ulcerans in environmental and clinical samples do not allow one-shot detection, identification, and typing of M. ulcerans among closely related Mycobacterium marinum complex mycobacteria. We established a 385-member M. marinum/M. ulcerans complex whole-genome sequence database by assembling and annotating 341 M. marinum/M. ulcerans complex genomes and added 44 M. marinum/M. ulcerans complex whole-genome sequences already deposited in the NCBI database. Pangenome, core genome, and single-nucleotide polymorphism (SNP) distance-based comparisons sorted the 385 strains into 10 M. ulcerans taxa and 13 M. marinum taxa, correlating with the geographic origin of strains. Aligning conserved genes identified one PPE (proline-proline-glutamate) gene sequence to be species and intraspecies specific, thereby genotyping the 23 M. marinum/M. ulcerans complex taxa. PCR sequencing of the PPE gene correctly genotyped nine M. marinum/M. ulcerans complex isolates among one M. marinum taxon and three M. ulcerans taxa in the African taxon (T2.4). Further, successful PPE gene PCR sequencing in 15/21 (71.4%) swabs collected from suspected Buruli ulcer lesions in Côte d'Ivoire exhibited positive M. ulcerans IS2404 real-time PCR and identified the M. ulcerans T2.4.1 genotype in eight swabs and M. ulcerans T2.4.1/T2.4.2 mixed genotypes in seven swabs. PPE gene sequencing could be used as a proxy for whole-genome sequencing for the one-shot detection, identification, and typing of clinical M. ulcerans strains, offering an unprecedented tool for identifying M. ulcerans mixed infections. IMPORTANCE We describe a new targeted sequencing approach that characterizes the PPE gene to disclose the simultaneous presence of different variants of a single pathogenic microorganism. This approach has direct implications on the understanding of pathogen diversity and natural history and potential therapeutic implications when dealing with obligate and opportunistic pathogens, such as Mycobacterium ulcerans presented here as a prototype.

DNA barcoding of exuviae for species identification of Central European damselflies and dragonflies (Insecta: Odonata)

Monitoring of odonates has become an important instrument for ecological status assessment of (semi-)aquatic habitats. Besides information on presence and abundance, knowledge about a species´ autochthony at the surveyed waterbody is a significant information within the assessment process. Here, the finding of exuviae represents the ultimate proof of successful reproduction. Although feasible for most odonate species, morphological identification of exuviae is often time consuming, as it relies on small, fragile structures. To facilitate species identification of exuviae, a DNA barcoding approach was developed, including (1) non-destructive extraction of DNA using whole exuviae or their tracheal tubes, and (2) primer systems for long (< 600 bp) and short (< 200 bp) CO1 fragments. A total of 85 exuviae from 33 species were analysed and compared to results of morphological identification. Additionally, factors potentially influencing DNA quality and quantity, as well as PCR and sequencing success were investigated. Eighty-two exuviae matched the morphologically identified genus, and 60 matched at species level. Of the 33 species present in the data set, 82% could be identified to species level via DNA barcoding. The results show how DNA-based approaches can support fast and accurate species identification and therefore enhance monitoring of an ecologically important taxonomic group, with high relevance for conservation and habitat restoration. Moreover, the use of exuviae as DNA resource once more shows that non-invasive sampling offers great potential for molecular species identification, which is essential when studying rare and endangered species.Implications for insect conservationOur results show how molecular tools, here DNA barcoding of odonate exuviae, can support species monitoring without the need of catching individuals, harming, or even killing them. Obtaining DNA from non-invasive sources can thus be a direct advantage to the conservation of insects, especially when dealing with rare and endangered species and/or populations. Using the example of odonates as bioindicator organisms for aquatic and semi-aquatic habitats, we highlight the importance of non-invasive genetic approaches for population studies and monitoring of insect species and/or species communities for ecosystem assessments and conservation management.

A simple alkali lysis method for Plasmodium falciparum DNA extraction from filter paper blood samples

A fast, simple, easy, efficient, and inexpensive method for DNA extraction from malaria parasites collected on filter paper would be very useful for molecular surveillance. The quality and quantity of DNA are critical to molecular diagnosis and analysis. Here, we developed a simple alkali lysis method for DNA extraction from blood samples on filter paper. The results showed that 10–50 mM NaOH and deionized water all effectively isolated parasite DNA at higher parasitemia, as witnessed by successful PCR amplification, while at a parasitemia of 0.01%, the 10 mM NaOH lysis condition generated the best results. Furthermore, DNA extracted by this method was successfully used to amplify a fragment of > 2000 bp. This method successfully extracted DNA from 1 µl of blood at a parasitemia as low as 0.0001% (equivalent to 5 parasites /µl). The DNA isolated by the 10 mM NaOH lysis method was stable to yield PCR products after storage at 4 °C or − 20 °C for 12 months. These results indicate that this alkali lysis method is simple, effective, sensitive, and inexpensive for isolating stable Plasmodium DNA from dried blood spots on filter paper.

Evolutionary Trend in Passiflora Species Revealed by RAPD-PCR, Gene Specific ITS and rbcl Markers

Background: The present study represents a preliminary analysis of genetic diversity among Passiflora species using amplified genotypic data of specific ITS and rbcL sequences and non-specific RAPD-PCR markers for investigation of the molecular phylogeny.&#x0D; Methods: The PCR-RAPD uses ten primers for polymorphic DNA, which are compiled on NTSYS software to construct dendrogram. The gene specific ITS and rbcL primers are used for specific amplification from genomic. The amplified ITS and rbcL markers assembled using Maximum Parsimony (MP) and Maximum likelihood (ML) methods. The BLAST, CLUSTAL W, and MEGA 6.0 have been used to conclude final genetic relation tree.&#x0D; Results: The PCR-RAPD primers translate 133 random amplified polymorphic DNA. NTSYS dendrogram placed P. vitifolia from Ramdaspeth and Shankar Nagar Nagpur, India in same clade (similarity coefficient 0.609) confirming same origin Nagpur India. Moreover, P. foetida from England is not coming in same clade with Indian P. foetida showing geographically intra-specific variation. In addition, the change in a constructed tree was observed with respect to change in phylogeny methods MS/ML. The ITS MP consensus tree is supported by strong 100 bootstrap value, clusters P. vitifolia (HNI and RNI) and P. foetida (UAI and UGI) in equivalent clade. However, no single species have been recovered using rbcL in MP and ML method. Thus, it is inference that rbcL have tendency to differentiate Passiflora species not allowing clustering around same species in same clade and the ITS region having parsimony informative sites that provide valid resolution and identification at inter-intra species level.&#x0D; Conclusions: The evaluation of properties of RAPD indicates 100% PCR success, sometimes with low rate of amplification. The ITS region found to be best for identification at inter-intra species, On the contrary, rbcL region is good to distinguished inter species, making it best as local DNA barcode for marking a Passiflora species in phylogenetic community.

A Comparison of Feathers and Oral Swab Samples as DNA Sources for Molecular Sexing in Companion Birds.

The early age determinism of the sex in case of monomorphic birds is very important, because most companion birds have no distinct sexual dimorphic traits. Molecular genetic sexing was proved to be one of the most accurate sex determinations in monomorphic birds. The aim of this study was to compare the results obtained by PCR performed on isolate genomic DNA from paired samples of feathers and oral swabs collected from the same individuals. Samples of oral swabs (n = 101) and feathers (n = 74) were collected from 101 companion birds from four different species (Columba livia domestica, Psittacula krameri, Neophema splendida and Agapornis spp.). The PCR was performed for the amplification of the CHD1W and CHD1Z genes in females and the CHD1Z gene in males. The overall PCR success rate of sex determination was significantly higher from oral swabs than from feathers. The PCR success rate from oral swabs was higher in juveniles and from feathers was significantly higher in adults. The similarity between the oral swab and feathers was obtained in 78.38% of the birds. Oral swabs proved to be a more reliable sample for genetic sex determination in the species tested in this study.

Hidden Pitfalls of Using Onion Pollen in Molecular Research

There is little information on the use of pollen in molecular research, despite the increased interest in genome editing by pollen-mediated transformation. This paper presents an essential toolbox of technical procedures and observations for molecular studies on onion (Allium cepa L.) pollen. PCR is a useful tool as an express method to evaluate editing results before pollination. A direct PCR protocol for pollen suspension has been adapted without needing DNA pre-extraction. We showed that the outer layer of lipids known as pollenkitt is a limiting factor for successful PCR on pollen. A simple pre-washing step of pollen suspension was able to eliminate the pollenkitt and enormously affect the PCR results. Additionally, our pollenkitt study helped us develop a simple and effective pollination method using wetted onion pollen grains. Classical manual pollination usually is conducted by intact pollen without wetting. Most existing methods of the editing system delivery into pollen are carried out in a wet medium with consequent drying before pollination, which adversely affects the viability of pollen. The optimal medium for wet pollination was 12% sucrose water solution. Our method of using wetted pollen grains for pollination might be very beneficial for pollen genetic manipulation.

Cost-Effective PCR-Based Identification of Tunga penetrans (Siphonaptera) Larvae Extracted from Soil Samples Containing PCR Inhibitor-Rich Material

Tungiasis is a neglected tropical disease caused by skin-penetrating female Tunga penetrans fleas. Although tungiasis causes severe health problems, its ecology is poorly understood and morphological descriptions of the larvae are unavailable. To identify T. penetrans immature stages and sites where they develop, diagnostic PCRs are required. However, flea larvae feed on soil organic matter rich in PCR inhibitors. Here, three DNA preparation methods, including a soil DNA kit that removes inhibitors, a simple ammonium acetate precipitation approach (AmAcet) and a crude lysate of larvae (CL), were combined with amplification by the highly processive FIREPol® Taq or the inhibitor-resistant Phusion® polymerase. Independent of the polymerase used, the frequency of successful amplification, Cq values and PCR efficacies for the low-cost CL and AmAcet methods were superior to the commercial kit for amplification of a 278 bp partial internal transcribed spacer-2 (ITS-2) and a 730 bp pan-Siphonaptera cytochrome oxidase II PCR. For the CL method combined with Phusion® polymerase, the costs were approximately 20-fold lower than for the methods based on the soil DNA kit, which is a considerable advantage in resource-poor settings. The ITS-2 PCR did not amplify Ctenocephalides felis genomic or Tunga trimammilata ITS-2 plasmid DNA, meaning it can be used to specifically identify T. penetrans.