High-resolution molecular identification of smalltooth sawfish prey.
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ABSTRACT:
SUBMITTER: Hancock TL
PROVIDER: S-EPMC6892823 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Diet analysis is an important aspect when investigating the ecology of fish-eating animals and essential for assessing their functional role in food webs across aquatic and terrestrial ecosystems. The identification of fish remains in dietary samples, however, can be time-consuming and unsatisfying using conventional morphological analysis of prey remains. Here, we present a two-step multiplex PCR system, comprised of six assays, allowing for rapid, sensitive and specific detection of fish DNA in dietary samples. This approach encompasses 78 fish and lamprey species native to Central European freshwaters and enables the identification of 31 species, six genera, two families, two orders and two fish family clusters. All targeted taxa were successfully amplified from 25 template molecules, and each assay was specific when tested against a wide range of invertebrates and vertebrates inhabiting aquatic environments. The applicability of the multiplex PCR system was evaluated in a feeding trial, wherein it outperformed morphological prey analysis regarding species-specific prey identification in faeces of Eurasian otters. Additionally, a wide spectrum of fish species was detected in field-collected faecal samples and regurgitated pellets of Common Kingfishers and Great Cormorants, demonstrating the broad applicability of the approach. In conclusion, this multiplex PCR system provides an efficient, easy to use and cost-effective tool for assessing the trophic ecology of piscivores in Central Europe. Furthermore, the multiplex PCRs and the primers described therein will be applicable wherever DNA of the targeted fish species needs to be detected at high sensitivity and specificity.
| S-EPMC4744964 | biostudies-literature
Project description:BackgroundGenotyping of human papillomarvirus (HPV) is crucial for patient management in a clinical setting. This study accesses the combined use of broad-range real-time PCR and high-resolution melting (HRM) analysis for rapid identification of HPV genotypes.MethodsGenomic DNA sequences of 8 high-risk genotypes (HPV16/18/39/45/52/56/58/68) were subject to bioinformatic analysis to select for appropriate PCR amplicon. Asymmetric broad-range real-time PCR in the presence of HRM dye and two unlabeled probes specific to HPV16 and 18 was employed to generate HRM molecular signatures for HPV genotyping. The method was validated via assessment of 119 clinical HPV isolates.ResultsA DNA fragment within the L1 region was selected as the PCR amplicon ranging from 215-221 bp for different HPV genotypes. Each genotype displayed a distinct HRM molecular signature with minimal inter-assay variability. According to the HRM molecular signatures, HPV genotypes can be determined with one PCR within 3 h from the time of viral DNA isolation. In the validation assay, a 91% accuracy rate was achieved when the genotypes were in the database. Concomitantly, the HRM molecular signatures for additional 6 low-risk genotypes were established.ConclusionsThis assay provides a novel approach for HPV genotyping in a rapid and cost-effective manner.
| S-EPMC3423390 | biostudies-literature
Project description:Mosquitoes are a diverse group of invertebrates, with members that are among the most important vectors of diseases. The correct identification of mosquitoes is paramount to the control of the diseases that they transmit. However, morphological techniques depend on the quality of the specimen and often unavailable taxonomic expertise, which may still not be able to distinguish mosquitoes among species complexes (sibling and cryptic species). High resolution melting (HRM) analyses, a closed-tube, post-polymerase chain reaction (PCR) method used to identify variations in nucleic acid sequences, has been used to differentiate species within the Anopheles gambiae and Culex pipiens complexes. We validated the use of PCR-HRM analyses to differentiate species within Anopheles and within each of six genera of culicine mosquitoes, comparing primers targeting cytochrome b ( cyt b), NADH dehydrogenase subunit 1 (ND1), intergenic spacer region (IGS) and cytochrome c oxidase subunit 1 ( COI) gene regions. HRM analyses of amplicons from all the six primer pairs successfully differentiated two or more mosquito species within one or more genera ( Aedes ( Ae. vittatus from Ae. metallicus), Culex ( Cx. tenagius from Cx. antennatus, Cx. neavei from Cx. duttoni, cryptic Cx. pipiens species), Anopheles ( An. gambiae s.s. from An. arabiensis) and Mansonia ( Ma. africana from Ma. uniformis)) based on their HRM profiles. However, PCR-HRM could not distinguish between species within Aedeomyia ( Ad. africana and Ad. furfurea), Mimomyia ( Mi. hispida and Mi. splendens) and Coquillettidia ( Cq. aurites, Cq. chrysosoma, Cq. fuscopennata, Cq. metallica, Cq. microannulatus, Cq. pseudoconopas and Cq. versicolor) genera using any of the primers. The IGS and COI barcode region primers gave the best and most definitive separation of mosquito species among anopheline and culicine mosquito genera, respectively, while the other markers may serve to confirm identifications of closely related sub-species. This approach can be employed for rapid identification of mosquitoes.
| S-EPMC5031131 | biostudies-literature
Project description:Broomrapes are holoparasitic plants spreading through seeds. Each plant produces hundreds of thousands of seeds which remain viable in the soils for decades. To limit their spread, drastic measures are being taken and the contamination of a commercial seed lot by a single broomrape seed can lead to its rejection. Considering that broomrapes species identification from a single seed is extremely difficult even for trained botanists and that among all the described species, only a few are really noxious for the crops, numerous seed lots are rejected because of the contamination by seeds of non-noxious broomrape species. The aim of this study was to develop and evaluate a High Resolution Melting assay identifying the eight most noxious and common broomrape species (Phelipanche aegyptiaca, Orobanche cernua, O. crenata, O. cumana, O. foetida, O. hederae, O. minor, and P. ramosa) from a single seed. Based on trnL and rbcL plastidial genes amplification, the designed assay successfully identifies O. cumana, O. cernua, O. crenata, O. minor, O. hederae, and O. foetida; P. ramosa, and P. aegyptiaca can be differentiated from other species but not from each other. Tested on 50 seed lots, obtained results perfectly matched identifications performed by sequencing. Through the analysis of common seed lots by different analysts, the reproducibility of the assay was evaluated at 90%. Despite an original sample preparation process it was not possible to extract enough DNA from some seeds (10% of the samples). The described assay fulfills its objectives and allows an accurate identification of the targeted broomrape species. It can be used to identify contaminants in commercial seed lots or for any other purpose. The assay might be extended to vegetative material.
| S-EPMC5149549 | biostudies-literature
Project description:The prey range of the invasive Asian paper wasp, Polistes chinensis antennalis, was studied using molecular diagnostics. Nests of paper wasps were collected from urban residential and salt marsh habitats, larvae were removed and dissected, and DNA in the gut of the paper wasp larvae was amplified and sequenced with cytochrome c oxidase subunit I (COI). Seventy percent of samples (211/299) yielded medium-to high-quality sequences, and prey identification was achieved using BLAST searches in BOLD. A total of 42 taxa were identified from 211 samples. Lepidoptera were the majority of prey, with 39 taxa from 91% of samples. Diptera was a relatively small component of prey (three taxa, 19 samples). Conclusive species-level identification of prey was possible for 67% of samples, and genus-level identification, for another 12% of samples. The composition of prey taken was different between the two habitats, with 2.5× more native prey species being taken in salt marsh compared with urban habitats. The results greatly extend the prey range of this invasive species. The technique is a more effective and efficient approach than relying on the collection of "prey balls", or morphological identification of prey, for the study of paper wasps.
| S-EPMC3856741 | biostudies-literature
Project description:Verticillium wilt, caused by the soilborne fungus, Verticillium alfalfae, is one of the most serious diseases of alfalfa (Medicago sativa L.) worldwide. To identify loci associated with resistance to Verticillium wilt, a bulk segregant analysis was conducted in susceptible or resistant pools constructed from 13 synthetic alfalfa populations, followed by association mapping in two F1 populations consisted of 352 individuals. Simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers were used for genotyping. Phenotyping was done by manual inoculation of the pathogen to replicated cloned plants of each individual and disease severity was scored using a standard scale. Marker-trait association was analyzed by TASSEL. Seventeen SNP markers significantly associated with Verticillium wilt resistance were identified and they were located on chromosomes 1, 2, 4, 7 and 8. SNP markers identified on chromosomes 2, 4 and 7 co-locate with regions of Verticillium wilt resistance loci reported in M. truncatula. Additional markers identified on chromosomes 1 and 8 located the regions where no Verticillium resistance locus has been reported. This study highlights the value of SNP genotyping by high resolution melting to identify the disease resistance loci in tetraploid alfalfa. With further validation, the markers identified in this study could be used for improving resistance to Verticillium wilt in alfalfa breeding programs.
| S-EPMC4275272 | biostudies-literature
Project description:Molecular approaches to prey identification are increasingly useful in elucidating predator-prey relationships, and we aimed to investigate the feasibility of these methods to document the species identities of prey consumed by invasive Burmese pythons in Florida. We were particularly interested in the diet of young snakes, because visual identification of prey from this size class has proven difficult. We successfully extracted DNA from the gastrointestinal contents of 43 young pythons, as well as from several control samples, and attempted amplification of DNA mini-barcodes, a 130-bp region of COX1. Using a PNA clamp to exclude python DNA, we found that prey DNA was not present in sufficient quality for amplification of this locus in 86% of our samples. All samples from the GI tracts of young pythons contained only hair, and the six samples we were able to identify to species were hispid cotton rats. This suggests that young Burmese pythons prey predominantly on small mammals and that prey diversity among snakes of this size class is low. We discuss prolonged gastrointestinal transit times and extreme gastric breakdown as possible causes of DNA degradation that limit the success of a molecular approach to prey identification in Burmese pythons.
| S-EPMC4662595 | biostudies-literature
Project description:Accurate identification of mycobacterial species and subspecies is essential to evaluate their significance and to perform epidemiological studies. The subspecies of Mycobacterium avium have different attributes but coincide in their zoonotic potential. Our knowledge about M. avium subsp. silvaticum is limited, since its identification is uncertain. Mycobacterium avium subsp. avium and M. avium subsp. silvaticum can be discriminated from each other based only on phenotypic characteristics, as they have almost identical genome sequences. Here we describe the development of a diagnostic method which enables the molecular identification of M. avium subsp. silvaticum and discrimination from M. avium subsp. avium based on genomic differences in a duplex high-resolution melt and M. avium subsp. silvaticum-specific mismatch real-time PCR. The developed assay was tested on reference strains and 199 field isolates, which were analyzed by phenotypic methods previously. This assay not only identified all 63 M. avium subsp. silvaticum and 138 M. avium subsp. avium strains correctly but also enabled the detection of mixed M. avium subsp. avium-M. avium subsp. silvaticum cultures. This is the first time that such a large panel of strains has been analyzed, and we also report the first isolation of M. avium subsp. silvaticum from red fox, red deer, wild boar, cattle, and badger. This assay is reliable, rapid, simple, inexpensive, and robust. It eliminates the long-existing problem of ambiguous phenotypic identification and opens up the possibility for detailed and comprehensive strain studies.
| S-EPMC4400758 | biostudies-literature
Project description:BACKGROUND:Chromoblastomycosis (CBM) is a chronic fungal disease. In China, the principle etiologic agent was a group of dematiaceous fungi, including Fonsecaea monophora, Fonsecaea nubica, and Cladophialophora carrionii. Although the Fonsecaea species have similar morphology, their pathogenicity is quite different. This study aims to establish a new solution for early identification of Fonsecaea species because of their distinctive potential infection risk. METHODS:Five reference strains and 35 clinical isolates from patients with CBM, preserved in our laboratory, were used in this study. The universal primer ITS1 and ITS2 were chosen to amplify the highly conserved regions of rDNA. High-resolution melting (HRM) analysis was performed using the LIGHTCYCLER® 96 System. All the amplicons were verified by direct sequencing and the sequence were aligned with those in GenBank by BLAST analysis. RESULTS:We successfully differentiated the five strains according to their different Tm values and curve shapes. The 35 clinical isolates from patients were identified as 24 strains for F. monophora and 11 strains for F. nubica, which is consistent with the DNA sequencing results. CONCLUSION:It is the first time to use HRM analysis for identification of Fonsecaea species. Since the CBM etiologic agent in South China is mainly F. monophora and F. nubica, this strategy is sufficient to be applied in the clinical examination with high accuracy, speed, and throughput.
| S-EPMC6817294 | biostudies-literature
Project description:Infections caused by non-tuberculous mycobacteria (NTM) is increasing wordwide. Due to the difference in treatment of NTM infections and tuberculosis, rapid species identification of mycobacterial clinical isolates is necessary for the effective management of mycobacterial diseases treatment and their control strategy. In this study, a cost-effective technique, real-time PCR coupled with high-resolution melting (HRM) analysis, was developed for the differentiation of Mycobacterial species using a novel rpoBC sequence. A total of 107 mycobacterial isolates (nine references and 98 clinical isolates) were subjected to differentiation using rpoBC locus sequence in a real-time PCR-HRM assay scheme. From 98 Mycobacterium clinical isolates, 88 species (89.7%), were identified at the species level by rpoBC locus sequence analysis as a gold standard method. M. simiae was the most frequently encountered species (41 isolates), followed by M. fortuitum (20 isolates), M. tuberculosis (15 isolates), M. kansassi (10 isolates), M. abscessus group (5 isolates), M. avium (5 isolates), and M. chelonae and M. intracellulare one isolate each. The HRM analysis generated six unique specific groups representing M. tuberculosis complex, M. kansasii, M. simiae, M. fortuitum, M. abscessus-M. chelonae group, and M. avium complex. In conclusion, this study showed that the rpoBC-based real-time PCR followed by HRM analysis could differentiate the majority of mycobacterial species that are commonly encountered in clinical specimens.
| S-EPMC5660063 | biostudies-literature