Faecal genetic analysis to determine the presence and distribution of elusive carnivores: design and feasibility for the Iberian lynx

Noninvasive methods using genetic markers have been suggested as ways to overcome difficulties associated with documenting the presence of elusive species. We present and assess a novel, reliable and effective molecular genetic technique for the unequivocal genetic identification of faeces from the endangered Iberian lynx (Lynx pardinus). From mitochondrial DNA (mtDNA) cytochrome b and D-loop region sequences, we designed four species-specific primers (for products 130-161 bp long) that were considered to be likely to amplify degraded DNA. We compared two DNA extraction methods, various DNA amplification conditions and the robustness and specificity of the primer pairs with 87 lynx samples from 5 potentially different lynx populations and with 328 samples of other carnivore species. The utility of the identification technique was tested with faeces of different ages, with faeces from controlled field experiments, and with faeces collected from locales with possible lynx populations from throughout the state of Andalusia, Spain (8052 km2). Faecal mtDNA extraction was more efficient using PBS wash of the faeces instead of a faeces homogenate. Our assay increased from 92.6 to 99% efficiency with a second amplification and a reduction in template concentration to overcome polymerase chain reaction (PCR) inhibition. Our assay never produced false positives, and correctly identified all lynx faeces. Of 252 faeces samples of unknown species collected throughout Andalusia, 26.6% (from three different areas) were classified as Iberian lynx, 1.4% showed evidence of PCR inhibition and 1.2% were of uncertain origin. This method has proven to be a reliable technique that can be incorporated into large-scale surveys of Iberian lynx populations and exemplifies an approach that can easily be extended to other species.     

Fecal DNA analysis for identifying species and sex of sympatric carnivores: a noninvasive method for conservation on the Tsushima Islands, Japan

Fecal analysis is a useful tool for the investigation of food habits and species identity in mammals. However, it is generally difficult to identify the species based on the morphological features and contents of feces deposited by mammals of similar body size. Therefore we developed noninvasive DNA analysis methods using fecal samples for identification of the species and sex of four small sympatric carnivores living on the Tsushima Islands of Japan: the leopard cat (Felis bengalensis), Japanese marten (Martes melampus), Siberian weasel (Mustela sibirica), and feral cat (Felis catus). Based on DNA sequence data from previous phylogenetic studies, we designed species-specific primers for polymerase chain reaction (PCR) amplification of the partial mitochondrial cytochrome b gene (112-347 bp) to identify the species and primers for the partial SRY gene (135 bp) to determine the sex. Due to the adjustment of PCR conditions, those specific DNA fragments were successfully amplified and then applied for species and sex identification. Nucleotide sequences obtained from the PCR products corresponded with cytochrome b sequences of the carnivore species expected. The protocol developed could be a valuable tool in the management and conservation of the four carnivore species occurring on the Tsushima Islands.     

Genetic identification of mammalian carnivore species in the Kushiro Wetland, eastern Hokkaido, Japan, by analysis of fecal DNA

To identify mammalian carnivore species distributed in the Kushiro Wetland, eastern Hokkaido, Japan, we developed molecular-genetic methods for identification of the species from fecal samples collected from the field. Species-specific primers and PCR programs were established for five native and six alien species of carnivores: Martes zibellina, Mustela nivalis, Mustela erminea, Vulpes vulpes, and Nyctereutes procyonoides as native species, and Neovison vison, Martes melampus, Mustela itatsi, Canis familiaris, Felis catus, and Procyon lotor as alien species in Hokkaido. Touchdown PCR, in which the annealing temperature is decreased 1 degrees C every cycle, was more effective for some species from which fecal DNA was not amplified species-specifically with standard PCR programs. Of 405 fecal samples collected from the Kushiro Wetland, the species of origin of 246 samples were successfully identified: 88 samples for N. vison, 140 for M. zibellina, 13 for V. vulpes, four for C. familiaris and one for F. catus. The results show the particular applicability of this method to monitoring M. zibellina and N. vison. In addition, methods to PCR-amplify DNA from two crayfish species (Pacifastacus leniusculus and Cambaroides japonicus) were developed to determine whether the carnivore fecal samples contained detectable DNA from the prey crayfishes. DNA from P. leniusculus was amplified from feces of N. vison identified in the present study, but no DNA from C. japonicus was detected. This indicates that N. vison preys on the alien species P. leniusculus.     

Identification of the pathogenic Aspergillus species by nested PCR using a mixture of specific primers to DNA topoisomerase II gene

For PCR-based identification of Aspergillus species, a common primer of the DNA topoisomerase II genes of Candida, Aspergillus and Penicillium, and species-specific primers of the genomic sequences of DNA topoisomerase II of A. fumigatus, A. niger, A. flavus (A. oryzae), A. nidulans and A. terreus were tested for their specificities in PCR amplifications. The method consisted of amplification of the genomic DNA topoisomerase II gene by a common primer set, followed by a second PCR with a primer mix consisting of 5 species-specific primer pairs for each Aspergillus species. By using the common primer pair, a DNA fragment of approximately 1,200 bp was amplified from the Aspergillus and Penicillium genomic DNAs. Using each species-specific primer pair, unique sizes of PCR products were amplified, all of which corresponded to a species of Aspergillus even in the presence of DNAs of several fungal species. The sensitivity of A. fumigatus to the nested PCR was found to be 100 fg of DNA in the reaction mixture. In the nested PCR obtained by using the primer mix (PsIV), the specific DNA fragment of A. fumigatus was amplified from clinical specimens. These results suggest that this nested PCR method is rapid, simple and available as a tool for identification of pathogenic Aspergillus to a species level.     

Multiplex PCR assay for rapid identification of three endangered snake species of India

Species identification has been the core issue in all approaches of conservation of endangered wild life. In this regard molecular techniques for species authentication have proved indispensable. A novel multiplex PCR assay for the identification of three Indian snake species Python morulus, Ptyas mucosus, and Naja naja is successfully demonstrated using 16S rRNA gene. Three reverse primers and a common forward primer were designed to generate three different size species-specific PCR fragments. Absence of any PCR amplification in non-target species proves the specificity of the primers. These four primers were combined in a multiplex assay to enable identification of three snake species in a single reaction. The assay described here shows its utility in identifying unknown snake specimen and in case of samples yielding low quality DNA. This multiplex PCR technique using novel primers is an unprecedented approach offered for forensic identification of exhibits originating from three Indian snake species. It is expected that this endeavor will help strengthening conservation efforts for these species.     

Successful carnivore identification with faecal DNA across a fragmented Amazonian landscape

The use of scat surveys to obtain DNA has been well documented in temperate areas, where DNA preservation may be more effective than in tropical forests. Samples obtained in the tropics are often exposed to high humidity, warm temperatures, frequent rain and intense sunlight, all of which can rapidly degrade DNA. Despite these potential problems, we demonstrate successful mtDNA amplification and sequencing for faeces of carnivores collected in tropical conditions and quantify how sample condition and environmental variables influence the success of PCR amplification and species identification. Additionally, the feasibility of genotyping nuclear microsatellites from jaguar (Panthera onca) faeces was investigated. From October 2007 to December 2008, 93 faecal samples were collected in the southern Brazilian Amazon. A total of eight carnivore species was successfully identified from 71% of all samples obtained. Information theoretic analysis revealed that the number of PCR attempts before a successful sequence was an important negative predictor across all three responses (success of species identification, success of species identification from the first sequence and PCR amplification success), whereas the relative importance of the other three predictors (sample condition, season and distance from forest edge) varied between the three responses. Nuclear microsatellite amplification from jaguar faeces had lower success rates (15-44%) compared with those of the mtDNA marker. Our results show that DNA obtained from faecal samples works efficiently for carnivore species identification in the Amazon forest and also shows potential for nuclear DNA analysis, thus providing a valuable tool for genetic, ecological and conservation studies.     

Design of molecular markers for identification of species of the genus Chironomus (Diptera,Chironomidae)

Accurate identification and differentiation of species of the genus Chironomus based on their morphological features is a difficult problem. Unambiguous species identification by means of molecular markers is possible at any stage of the life cycle. Polymerase chain reaction (PCR) with species-specific primers was used to develop molecular markers (amplicons) for identification of Chironomus piger, Ch. dorsalis, and Ch. pseudothummi. Nucleotide sequences of the internal transcribed spacer region (ITS) of the locus coding for ribosomal RNA were used to design species-specific primers for these target species. Each of the species-specific primer pairs yielded species-specific amplicons (molecular markers) only with the DNA of target species: Ch. piger, Ch. dorsalis, and Ch. pseudothummi. Test PCRs with the DNA of eighteen Chironomus species confirmed the specificity of the primers obtained. The molecular markers produced in PCR with the designed species-specific primers permit reliable identification of Ch. piger, Ch. dorsalis, and Ch. pseudothummi and their differentiation from other species of the genus Chironomus.     

Discrimination of Six Pratylenchus Species Using PCR and Species-Specific Primers

A PCR-based assay for identification of six species of Pratylenchus common in California is described. In this assay, five forward species-specific primers were designed from the internal variable portion of the D3 expansion region of the 26S rDNA and were each used with a single, common reverse primer. The optimized species-specific primers produced unique amplicons from their respective target and did not amplify DNA from other Pratylenchus species. With this assay we were able to identify single females to species level. This method obviates the need for subsequent RFLP or sequence analysis of the PCR product and can be used as a rapid diagnostic tool in epidemiological and management studies.     

Characterization of a western North American carnivore community using PCR–RFLP of cytochrome <Emphasis Type="Italic">b</Emphasis> obtained from fecal samples

We developed a simple and reliable method to identify carnivore scats to species using PCR and RFLP of a portion of the mtDNA cytochrome b gene, which works for seven of the most common carnivores in western North America. We identified a short (196 bp) polymorphic region of cytochrome b which would be easily amplifiable even from degraded DNA, developed a primer set, and isolated a set of three restriction enzymes (HpaII, DdeI, HpyCH4V) that would identify the seven target species. In order to test whether this protocol would effectively identify scats obtained in the field we collected 243 carnivore scats from 12 sites in the San Francisco Bay area. Eighty five percent (206) of our samples successfully amplified and were subsequently identified to species using our RFLP protocol. We selected 108 of these samples to sequence; our species identifications based on sequencing were identical to those obtained using our PCR–RFLP method. Our PCR–RFLP method is a simple and efficient means to identify carnivore scats to species, eliminating the need for sequencing, which is costly and requires more laboratory equipment. The technique can also be modified depending on the species present at a particular site. It allows for rapid and noninvasive assessment of multiple carnivore taxa and is particularly useful for surveying populations across many sites.     

Faecal DNA amplification in Pacific walruses (Odobenus rosmarus divergens)

Dietary information is critical for assessing the population status of seals, sea lions and walruses—and is determined for most species of pinnipeds using non-invasive methods. However, diets of walruses continue to be described from the stomach contents of dead individuals. Our goal was to assess whether DNA could be extracted from the faeces of Pacific walruses (Odobenus rosmarus divergens) collected at haulout sites, and whether potential prey species or taxa could be amplified from that DNA. We extracted DNA from 70 faecal samples collected from ice pans in the Bering Sea during the spring of 2008 and 2009 (with between 4.6 and 308.9 ng/μl of DNA in every sample). We also extracted DNA from 12 potential prey species or taxa collected by bottom-grabs in 2009 to identify positive controls for primers and to test the ability of previously published taxon-specific and species-specific primers to correctly identify the prey using conventional PCR. We tested primers that successfully amplified DNA from the tissue of at least one potential prey species or taxon on all 70 walrus faecal samples. We found that two sets of primers successfully amplified many of the potential prey species or taxa using DNA from their tissue, and that one of these primer sets produced positive amplification in 4 of the 70 faecal samples. The band size that was produced for prey organisms and in the faecal samples was consistent with expectations, although prey identities were not verified with sequencing. Our pilot study demonstrates that DNA can be successfully extracted and amplified from walrus faeces, providing a stepping stone towards describing the diets of walruses from faecal DNA.     

Species-specific detection of hydrocarbon-utilizing bacteria

Rapid detection and quantitative assessment of specific microbial species in environmental samples is desirable for monitoring changes in ecosystems and for tracking natural or introduced microbial species during bioremediation of contaminated sites. In the interests of developing rapid tests for hydrocarbon-degrading bacteria, species-specific PCR primer sets have been developed for Pseudomonas aeruginosa, Stentrophomonas (Xanthomonas) maltophilia, and Serratia marsescens. Highly variable regions of the 16S rRNA gene were used to design these primer sets. The amplification products of these primer sets have been verified and validated with hemi-nested PCR and with ligase chain reaction (LCR) techniques, and have been applied to the analyses of environmental water samples. These species-specific primer sets were also chosen to amplify in conjunction with a universal set of PCR primers chosen from highly conserved neighboring sequences in the same gene. These multiplex or competitive PCR procedures enable testing with an internal marker and/or the quantitative estimation of the relative proportion of the microbial community that any one of these species occupies. In addition, this universal PCR primer set amplified the same size amplicon from a wide spectrum of procaryotic and eucaryotic organisms and may have potential in earth biota analyses.     

PCR detection of dinoflagellate cysts in field sediment samples from tropic and temperate environments

Species-specific primers were constructed for Scrippsiella trochoidea, Protoceratium reticulatum and Lingulodinium polyedrum, which all are common cosmopolitan cyst forming dinoflagellates. The designed primers amplified a product of expected size from cultured planktonic cells of the three species, and did not yield any product with a wide range of other algal species used as negative controls. The PCR method for detection and identification of dinoflagellate cysts from the three species was applied on field samples. Undisturbed surface sediment was collected along the southwest coast of India and the west coast of Sweden. DNA extract from sediment including DNA from dinoflagellate cysts could be obtained after repeated grinding with mortar and pestle under liquid nitrogen followed by microwave boiling. All sediment samples that contained any of the target species as confirmed by microscopy, were also positive for PCR. Field samples negative for any of the target species by microscopy, were also negative by PCR. Restriction enzyme digestion and/or DNA sequencing confirmed the specificity of all the PCR products from field samples. The yield of DNA from sediment extraction was low, and therefore nested PCR was necessary for accurate species-specific detection of the three species in most of the field samples.     

A novel specific DNA marker in Saccharomyces bayanus for species identification of the Saccharomyces sensu stricto complex

This study was based on RAPD fingerprinting for species identification of the Saccharomyces sensu stricto complex. 40 random primers were used for RAPD analysis. The results showed that one of these primers, OPT-18, produced a 974 bp species-specific band, which was only found in the tested S. bayanus. Afterward this specific fragment was isolated from agarose gel and ligated into vector for DNA sequencing. A pair of primer SpeOPT18Sbay-F2 and SpeOPT18Sbay-R2 were designed according to the cloned species-specific sequence, which was employed for PCR with the template DNA of the S. sensu stricto strains, single 779 bp species-specific band was only found in S. bayanus. Therefore, we conclude that our novel species DNA marker could be used to rapidly and accurately identify the species of S. bayanus from S. sensu stricto complex by direct PCR.     

A new cultivation independent approach to detect and monitor common Trichoderma species in soils

A set of primers was developed for the detection, identification and quantification of common Trichoderma species in soil samples. Based on a broad range master alignment primers were derived to amplify an approximate 540 bp fragment comprising the internal transcribed spacer region 1 (ITS 1), 5.8S rDNA and internal transcribed spacer region 2 (ITS 2) from all taxonomic Clades of the genus Trichoderma. The primer set was applied to test strains as well as community DNA isolated from arable and forest soil. For all tested isolates the corresponding internal transcribed spacer regions of Trichoderma spp. strains were amplified, but none of non-Trichoderma origin. PCR with community DNA from soil yielded products of the expected size. Analysis of a clone library established for an arable site showed that all amplified sequences originated exclusively from Trichoderma species mainly being representatives of the Clades Hamatum, Harzianum and Pachybasioides and comprising most of the species known for biocontrol ability. In a realtime PCR approach the primer set uTf/uTr also proved to be a suitable system to quantify DNA of Trichoderma spp. in soils.     

Development of species-specific PCR primer sets for the detection of Leptospira

Spirochetes of the genus Leptospira infect animals and humans and are the causative agents for the emerging infectious disease leptospirosis. Rapid and simple assays for the identification of individual Leptospira species are currently not available. For identification of individual Leptospira species, PCR primers that detect the ompL1 gene sequence for the majority of pathogenic leptospires were developed in this study. The primer pairs detect Leptospira interrogans, Leptospira borgpetersenii, Leptospira kirschneri, Leptospira santarosai, Leptospira weilii and Leptospira noguchii, without cross-reacting with other Leptospira species. The development of the primers revealed a divergence of the ompL1 gene within L. interrogans, splitting this species into two separate groups. The species-specific primers will be especially useful in epidemiological studies and disease outbreak investigations for the detection of Leptospira species in human, animal and environmental samples.     

Species identification using a small nuclear gene fragment: application to sympatric wild carnivores from South-western Europe

Species identification is essential for non-invasive studies of elusive and rare animals, and for detecting illegal harvest or trade of wildlife species. However, most molecular tests identify only a limited number of species or require multiple laboratory steps to distinguish many taxa. Additionally, most protocols use mitochondrial DNA being, therefore, especially prone to problems such as nuclear insert copies, high intraspecific diversity or heteroplasmy. Here, we developed a molecular test based on the polymorphisms detected on a small nuclear gene fragment (221 bp of the IRBP -Interphotoreceptor Retinoid-Binding Protein- exon 1). This fragment revealed 51 variable sites (including 12 non-synonymous and 19 species-specific sites), which enabled the successful distinction of all 16 carnivore species native to South-western Europe. A SSCP (Single-Strand Conformational Polymorphism) gel electrophoresis technique was also optimized to allow the simple and inexpensive application of this molecular test. Sequences and SSCP profiles were consistent in identifying a total of 387 samples, including faeces (172) and hairs (17) collected non-invasively in the field. Due to its low cost, simplicity, and wide range of identifiable species, this test shows great promise to facilitate studies in molecular ecology, conservation genetics, and forensic analysis, as well as DNA bar-coding projects.     

Molecular identification of vaginal lactobacilli isolated from Bulgarian women

Lactobacilli play an important role in maintaining the vaginal health of women. The development of suitable bacterial replacement therapies for the treatment of vaginosis requires knowledge of the vaginal lactobacilli species representation. The aim of this study was to identify at the species level vaginal Lactobacillus isolates obtained from Bulgarian women in childbearing age by using different molecular methods. Twenty-two strains of lactobacilli isolated from vaginal samples were identified and grouped according to their genetic relatedness. A combined approach, which included amplified ribosomal DNA restriction analysis (ARDRA), ribotyping and polymerase chain reaction (PCR) with species-specific oligonucleotide primers was applied. All vaginal isolates were grouped into 5 clusters in␣comparison with a set of 21 reference strains based␣on the initial ARDRA results, which was then confirmed by ribotyping. Finally, the strains were subjected to PCR analysis with eight different species-specific primer pairs, which allowed most of␣them to be classified as belonging to one of␣the␣following species: Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus and Lactobacillus plantarum. In conclusion, this study suggests that the most straightforward identification strategy for vaginal lactobacilli would be grouping by ARDRA or ribotyping, followed by PCR specific primers identification at species level.     

Species-specific PCR for identification of common contaminant mollicutes in cell culture.

Mycoplasma arginini, M. fermentans, M. hyorhinis, M. orale, and Acholeplasma laidlawii are the members of the class Mollicutes most commonly found in contaminated cell cultures. Previous studies have shown that the published PCR primer pairs designed to detect mollicutes in cell cultures are not entirely specific. The 16S rRNA gene, the 16S-23S rRNA intergenic spacer region, and the 5' end of the 23S rRNA gene, as a whole, are promising targets for design of mollicute species-specific primer pairs. We analyzed the 16S rRNA genes, the 16S-23S rRNA intergenic spacer regions, and the 5' end of the 23S rRNA genes of these mollicutes and developed PCR methods for species identification based on these regions. Using high melting temperatures, we developed a rapid-cycle PCR for detection and identification of contaminant mollicutes. Previously published, putative mollicute-specific primers amplified DNA from 73 contaminated cell lines, but the presence of mollicutes was confirmed by species-specific PCR in only 60. Sequences of the remaining 13 amplicons were identified as those of gram-positive bacterial species. Species-specific PCR primers are needed to confirm the presence of mollicutes in specimens and for identification, if required.     

Differentiation of sourdough yeast species by a novel species-specific PCR assay

The objective of this study was to develop species-specific primer pairs based on the internal transcribed spacer region (ITS) of the ribosome DNA for species identification of the frequently found sourdough yeast species. Species-specific primers were designed by the alignment of sourdough yeast ITS sequences, which were then employed for PCR using the template DNA of sourdough yeast strains. PCR primers were shown to be specific for the following species: Issatchenkia orientalis (Candida krusei), C. humilis, Kazachstania exigua (C. holmii), Pichia anomala and P. subpelliculosa. Therefore, we conclude that our novel species-specific primers could be used to rapidly and accurately identify the most frequent sourdough yeast species using a PCR-based assay.     

Gel‐free species identification using melt‐curve analysis

DNA‐based identification of organisms is an important tool in biosecurity, ecological monitoring and wildlife forensics. Current methods usually involve post‐polymerase chain reaction (PCR) manipulations (e.g. restriction digest, gel electrophoresis), which add to the expense and time required for processing samples, and may introduce error. We developed a method of species identification that uses species‐specific primers and melt‐curve analysis, and avoids post‐PCR manipulation of samples. The method was highly accurate when trialled on DNA from six large carnivore species from Tasmania, Australia. Because of its flexibility and cost‐effectiveness, this method should find wide use in many areas of applied biological science.