Assay For Species Identification Research Articles

A screening assay based on PCR amplification of Cytochrome b for species identification of some animals in Cervus

Sambar deer (Cervus unicolor) is a protective animal in Thailand. Sambar deer meat has become popular among consumer. Therefore, the smuggling of sambar deer has increased. For the success of prosecution, species identification of meat origin is necessary. An easy-to-perform screening assay for species identification of sambar deer meat is described in this study. The detection of three species of cervids including sambar deer, sika deer (Cervus nippon) and rusa deer (Cervus timorensi) was achieved by using newly designed primers targeting sambar deer cytochrome b (Cyt b) region producing an amplicon of approximately 350 base pairs (bp). The possibility of cross-amplification was prevented by testing with other species of cervids including eld’s deer (Cervus eldi), muntjac (Muntiacus spp.), chital deer (Cervus axis), hog deer (Axis porcinus) and popular consumed meats (pork, beef, chicken, seabass). The limit of detection (LOD) on sambar deer DNA was 31.25 pg. Sensitivity of detection of possible sambar deer in mixed meat species was 2%. Suitability of the screening assay was confirmed on processed meats like frozen meats (-20 oC, -80 oC) and cooked meats including boiled, steamed, autoclaved, fried, microwave cooked and grilled. Hence, this screening assay can effectively be used for preliminary examination of sambar deer origin in questioned evidence.

Rapid Identification of Nontuberculous Mycobacterium Species from Respiratory Specimens Using Nucleotide MALDI-TOF MS.

We performed a prospective study to evaluate the diagnostic accuracy of nucleotide matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) in identifying nontuberculous mycobacterium (NTM) from clinical respiratory samples. A total of 175 eligible patients were prospectively enrolled, including 108 patients diagnosed with NTM pulmonary disease (NTM-PD) and 67 control patients with other diseases. All specimens were subjected to acid-fast staining, liquid culture combined with MPT64 antigen detection, and a nucleotide MALDI-TOF MS assay. NTM cultures were also subjected to the MeltPro Myco assay for species identification. Altogether, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of nucleotide MALDI-TOF MS were 77.8% (95% CI: 68.6-85.0%), 92.5% (82.8-97.2%), 94.4% (86.8-97.9%), and 72.1% (61.2-81.0%), respectively; these results were not statistically different from the results of culture + MPT64 antigen testing (75.0% [65.6-82.6%], 95.5% [86.6-98.8%], 96.4% [89.2-99.1%], and 70.3% [59.7-79.2%], respectively). In the identification of NTM species, of the 84 nucleotide MALDI-TOF MS positive samples, 77 samples (91.7%) were identified at the species level. Using culture + MeltPro Myco assay as the reference standard, nucleotide MALDI-TOF MS correctly identified 77.8% (63/81) of NTM species. Our results demonstrated that the nucleotide MALDI-TOF MS assay was a rapid single-step method that provided the reliable detection of NTM and identification of NTM species. This new method had the same sensitivity and specificity as the culture + MPT64 antigen method, but was much more rapid.

DNA analysis and validation for species identification of seized helmeted hornbill (Rhinoplax vigil) casques

Helmeted hornbills (Rhinoplax vigil, J.R. Forster, 1781) are ‘Critically Endangered’ due to illegal hunting for their casques which are carved and traded for ornamental purposes. DNA species identification techniques can aid enforcement efforts, and validated wildlife forensic techniques for the species identification of R. vigil are needed. Here we tested multiple methods for sampling and extracting DNA from R. vigil casques and a validated a previously published assay using cytochrome B (cytB) primers to identity species and origin of traded casques. Phenol-chloroform: isoamyl alcohol extractions resulted in samples with higher quantity and quality of DNA than those extracted using the commercial Qiagen DNeasy Blood and Tissue kit. Samples collected from the caudal side of the casque yielded higher DNA quantity and quality than rostral and lateral sides, regardless of sampling method. We then assessed the repeatability, reproducibility, robustness, sensitivity, specificity, and phylogenetic resolution of a previously published species identification assay. We confirm the ability of this method to phylogenetically distinguish between R. vigil and closely related hornbills with high bootstrap support (99%). We also report the first genetic evidence of illegally traded R. vigil in Hong Kong using confiscated casques and provide more reference samples of R. vigil for future work. Overall, we provide multiple protocols for sampling and extracting DNA, and a validated species identification assay for amplifying DNA from R. vigil casques with potential to aid law enforcement in illegal wildlife crimes.

Development of a nanoplate-based digital PCR assay for species identification with mixture deconvolution

In crime scenes, not all biological stains are human in origin. Some exhibits can be from pets living on the premises or from animal products used in food consumption. In addition, it could be necessary to test animal carcasses for other forensic purposes. Often such stains can include mixtures involving humans or other species. Thus, identifying and deconvoluting mixtures of species commonly found in and around a household can be crucial in forensic casework. Different molecular techniques have been employed for species identification such as immunoprecipitation, qPCR, and DNA sequencing.In this project, a nanoplate-based digital PCR assay for species identification was developed, targeting Homo sapiens, canine, feline, bovine swine, pisces, and gallus in two multiplexes. An internal positive control was included in the design. The assay is simple, rapid, and can determine a wide variety of different vertebrates from biological exhibits, as well as in mixtures. Because the assay utilizes digital PCR, the procedure shows sensitivity down to a few copies, even in the presence of larger amounts of a major contributor, making the assay particularly useful in mixture deconvolution. Overall, this assay presents the forensic community with a novel application in which digital PCR can provide a sensitive and specific determination of species.

A Novel Medium for Isolating Two Japanese Species in the Fusarium graminearum Species Complex and a Dipstick DNA Chromatography Assay for Species Identification and Trichothecene Typing.

Members of the Fusarium graminearum species complex (Fg complex) are the primary pathogens that cause Fusarium head blight in wheat and barley. Fg complex members grow poorly on Fusarium oxysporum-selective media, such as Komada and Fo-G2, that have also been used for the isolation of other Fusarium species. Therefore, Komada medium was modified as FG medium for the isolation of Fg complex members. However, the production of pentachloronitrobenzene that is the most effective component of FG medium is discontinued and new media is required for the selective isolation of Fg complex members. In addition, the rapid diagnosis of isolated fungi is useful for the disease control. Novel tools have been developed for isolating and characterizing Fg complex members. FG21, a semi-selective medium for isolating Fg complex members, was developed using potato dextrose agar. Furthermore, a dipstick DNA chromatography assay was developed both to identify Fusarium graminearum sensu stricto and Fusarium asiaticum in the Fg complex and their trichothecene mycotoxin types. The easier isolation and characterization of Fg complex members in Japan was attained by the combined use of FG21 medium and the dipstick DNA chromatography assay.

Characterization of simple sequence repeat loci for Peltigera membranacea (lichenized Ascomycota) and its Nostoc photobiont

AbstractTo facilitate population-genetic studies, we developed simple sequence repeat (SSR) markers and a molecular species identification assay for Peltigera membranacea (Ascomycota, Peltigerales), a common ground-dwelling lichen of forest and tundra ecosystems. Additional markers were developed for its Nostoc photobiont. Twenty-one fungal markers for P. membranacea were found to be polymorphic, with the number of alleles ranging from 3–21. Nei's unbiased gene diversity ranged from 0.588 to 0.640 in four significantly structured (FST = 0.059) mycobiont populations. For the Nostoc photobiont, 14 polymorphic SSR were developed, yielding 4–14 alleles each, with gene diversity ranging from 0.062 to 0.771 in four populations showing substantial population structure (FST = 0.278). The new markers developed are suitable for population genetic studies of Peltigera membranacea and of its cyanobiont, and at the same time allowed us to distinguish 98.5% of P. membranacea specimens from morphologically similar species of Peltigera.

Multiplex PCR assay for species identification of meat and dairy products from buffalo (Bubalus bubalis), cattle (Bos indicus and Bos taurus), goat (Capra hircus), and sheep (Ovis aries)

Cases of fraudulent meat and dairy products have increased worldwide, especially in developing countries. To determine the misrepresented animal species, appropriate tools in routine monitoring should be available for food inspections. In the present work, a multiplex polymerase chain reaction assay for species identification of products from ruminants including buffalo, cattle, goat, and sheep was developed. The primer set KUMUT_cFarmSp1 was composed of five species-specific primers and a pair of positive-control primers. The primer set amplified 106-, 163-, 232-, and 308-bp specific fragments from the cytochrome b (cyt b) gene of buffalo, cattle, goat, and sheep, respectively, and 370-bp positive-control fragment from 16S ribosomal RNA (16S rRNA). The detection limit of this PCR assay is 0.1 ng of DNA template. The developed primer set exhibited strong specificity, sensitivity, robustness, and simplicity for food verification, thus indicating its usefulness for species verification in food quality control and law enforcement.

Developmental validation of SpeID: A pyrosequencing-based assay for species identification

In crime scenes, biological exhibits are often human in origin, yet biological stains from other fauna may also be present at a crime scene, creating confusion during an investigation. Furthermore, identifying the source of a biological sample can be critical during an investigation. To identify the presence of biological material from non-human sources, it is common to use genetic markers within mitochondrial DNA such as cytochrome b, 16S rRNA, and 12S rRNA genes. This process usually requires DNA sequencing, a process that is neither quick nor easy. In general, a faster, more standardized method for species identification from tissue and body fluids is desirable.For this reason, we have developed a vertebrate specific real-time quantitation method that is followed by an automated pyrosequencing-based procedure that sequences a short fragment within the 12S rRNA gene. Using no more than 35 bases, the assay can distinguish between 32 different species commonly found in and around a household with a turnaround time of 6 h from extraction to sequencing. -Using this procedure, up to 48 samples can be run at a time without the need for expensive reagents or bioinformatic skills.

Usefulness of BioFire FilmArray BCID2 for Blood Culture Processing in Clinical Practice.

ABSTRACTRapid pathogen characterization from positive blood cultures (BC) can improve management of patients with bloodstream infections (BSI). The FilmArray blood culture identification (BCID) assay is a molecular test approved for direct identification of BSI causing pathogens from positive BC. A recently updated version of the panel (BCID2) comprises improved species identification characteristics and allows for the detection of one expanded-spectrum β-lactamase (ESBL)- and several carbapenemase-encoding genes. Here, the clinical performance of the BCID2 assay for species identification in 180 positive BCs was evaluated. BCID2 results were concordant with the standard of care (SOC) in 159/180 (88.3%) BCs; 68/74 (91.9%) and 71/74 (96.0%) of all samples growing monobacterial, Gram-positive or Gram-negative pathogens, respectively, were identified, in agreement with SOC results. Nonconcordance was related to the detection of additional pathogens by the BCID2 assay (n = 4), discrepant species identification (n = 4), or failure of BCID2 to detect on-panel pathogens (n = 1). A number (12/31; 38.7%) of discordant results became evident in polymicrobial BC specimens. BCID2 identified the presence of blaCTX-M-carrying species in 12 BC specimens but failed to predict third-generation cephalosporin resistance in four isolates exhibiting independent cephalosporin resistance mechanisms. Carbapenem resistance related to the presence of blaVIM-2 or blaOxa-48-like was correctly predicted in two isolates. In conclusion, the BCID2 assay is a reliable tool for rapid BC processing and species identification. Despite inclusion of common ESBL- or carbapenemase-encoding markers, the multifactorial nature of β-lactam resistance in Gram-negative organisms warrants combination of BCID2 with (rapid) phenotypic susceptibility assays.

A rapid multiplex PCR assay for species identification of Asian rice planthoppers (Hemiptera: Delphacidae) and its application to early-instar nymphs in paddy fields.

Rice (Oryza sativa L.) is the main cereal crop in many Asian countries. The Asian rice planthoppers, Nilaparvata lugens (Stål) (brown planthopper), Sogatella furcifera (Horváth) (white-backed planthopper), and Laodelphax striatellus (Fallén) (small brown planthopper) (Hemiptera: Delphacidae), are the most economically important pests of rice. These three rice planthopper species often co-occur in the same paddy field. Traditionally, species identification of individuals of the three rice planthopper species has relied on morphological characters, but accurate discrimination of early-instar nymphs is very difficult, even for expert researchers. In this study, we developed a rapid one-step multiplex PCR assay using conserved and species-specific 5.8S-ITS2 rDNA gene primers for simultaneous identification of individuals of the three rice planthopper species. The multiplex PCR results showed that the three rice planthopper species could be identified accurately based on the length of the resultant amplicon, regardless of the individual developmental stage. Furthermore, we applied this assay for the first accurate quantification of early-instar nymphs of each rice planthopper species in paddy fields. Notably, we found that the species composition of early-instar nymphs cannot be extrapolated from that of adults. Thus, the multiplex PCR assay developed here facilitates detection of each rice planthopper species at the beginning of outbreaks in paddy fields.

Simple Nested Allele-Specific approach with penultimate mismatch for precise species and sex identification of tiger and leopard.

Accurate species and sex identification of non-invasive and forensic samples of the tiger and leopard is still confusing when using the allele-specific methods. We designed allele-specific methods with penultimate nucleotide mismatch in a nested manner for the exact identification and double-checking of forensic samples. The mismatch design is a novel concept in species and sex identification, making the allele-specific targeting precise. We developed three sets of markers, a 365bp outer and a 98bp inner marker for nested tiger species identification assay, 136bp leopard specific marker, and carnivore sex identification markers. We validated the method with tissue/blood forensic samples of various felids and herbivorous available in our lab and on known fecal samples from Vandalur Zoo. We also collected 37 scat samples at diverse stages of deterioration from the Mudumalai Tiger Reserve, Tamil Nadu, India. The 365bp targeted markers resulted in 70.2% (n = 22; 22/37) amplification success, while the 98bp FAM-labelled marker amplified 89% (n=33; 33/37) scat samples independently. The 136bp leopard markers answered four scat samples (11%) unrequited by the tiger specific markers. We evaluated species and the sex identification with these markers in another 190 non-invasive samples provided by the Mudumalai Tiger Reserve authorities. Among which 56.3% (n= 107) of samples were recognized as tiger (64 male and 43 female) and 38.9% (n= 74) as leopard (41 male and 33 female). The method supersedes any other previous methods in this regard by its high accuracy and simplicity.

A new minibarcode assay to facilitate species identification from processed, degraded or historic ray (batoidea) samples

Rays (Batoidea) are among the most threatened groups of vertebrates. Slow growth and low fecundity make many species vulnerable to overfishing, but increased demand for gill rakers in traditional Chinese medicine and elasmobranch meat means exploitation continues. In response, protection has increased, with manta and devilrays (Mobulidae) and sawfishes (Pristidae) now listed on Convention on International Trade in Endangered Species (CITES). This protection requires an accurate assay for species identification, even when parts of the body have been removed or products have been processed. Therefore, we developed and tested a new COI minibarcode to identify ray species among processed samples. This assay was tested on 25 samples from across four batoid orders and showed consistent amplification. In 68% of cases, a correct top match was identified on GenBank and BOLD, but its accuracy should be much higher (particularly due to extensive taxonomic revisions in this group). Bioinformatic analysis of existing sequences also showed 81% of minibarcodes were matched back to a single species and 100% correctly back to all other taxonomic ranks. This increased to 91% when only including records published in the previous 2 years (that should help to account for recent taxonomic revisions). Analysis of skate ‘wings’ and cooked samples was highly successful, allowing the unambiguous identification of species. These results suggest this minibarcode will be highly useful in identifying ray species and could have a range of applications from the analysis of processed products to investigations of environmental DNA, making them an effective tool in the conservation of endangered batoids.

Novel real-time PCR species identification assays for British and Irish bats and their application to a non-invasive survey of bat roosts in Ireland

Detection and monitoring of extant bat populations are crucial for conservation success. Non-invasive genetic analysis of bat droppings collected at roosts could be very useful in this respect as a rapid, cost‐efficient monitoring tool. We developed species‐specific real-time PCR assays for 18 British and Irish bat species to enable non‐invasive, large‐scale distribution monitoring, which were then applied to a field survey in Ireland. One hundred and sixty-four DNA samples were collected from 95 bat roosts, of which 73% of samples were identified to species, and the resident bat species were identified at 89% of roosts. However, identification success varied between roost types, ranging from 22% for underground sites to 92% for bat boxes. This panel of DNA tests will be especially useful in cases where roosts contain multiple species, where the number of bats present is small, or bats are otherwise difficult to directly observe. The methodology could be applied to the surveillance of proposed development sites, post development mitigation measures, distribution surveys, bat box schemes and the evaluation of agri-environmental bat box schemes.

Characterization of Penicillium s.s. and Aspergillus sect. nigri causing postharvest rots of pomegranate fruit in Southern Italy

Most of diseases of pomegranate fruit are caused by fungal pathogens, which provoke postharvest yield and economical losses. Aspergillus and Penicillium sensu lato (s.l.) are the main wound pathogens of pomegranate fruit. In the present investigation, the populations of Aspergillus and Penicillium s.l. isolated from pomegranate fruit in Southern Italy were characterized. Since the morphological identification of species belonging to these genera is laborious, molecular approaches, such as PCR and High-Resolution Melting (HRM), were used. Particularly, a specific primer pair was designed to discriminate, within the Penicillium s.l. population, Penicillium sensu stricto (s.s.) from Talaromyces strains. Then, a new HRM assay for species identification within Penicillium s.s. according to SNPs present in a portion of the beta-tubulin gene was set up. Similarly, Aspergillus sect. nigri population was characterized arranging a HRM assay, whose primer pair was designed on a portion of the calmodulin gene. According to these assays, 10% of the Penicillium s.l. population proved to be made up of Talaromyces biverticillius strains. Furthermore, six species of Penicillium s.s. (P. adametzioides, P. brevicompactum, P. citrinum, P. glabrum, P. pagulum, and P. johnkrugii) and four of black aspergilli (A. tubingensis, A. welwitschiae, A. japonicus, and A. uvarum) were identified; all species belonging to both genera disclosed different incidences in postharvest rotted pomegranate fruit. Moreover, since Aspergillus and Penicillium are potentially producers of mycotoxins, like ochratoxin A and fumonisins, the presence/absence of genes involved in mycotoxin biosynthetic pathways was tested. Some Aspergillus strains belonging to species A. welwitschiae proved to possess fumonisin genes. The setup of molecular tools to characterize Penicillium s.l. and Aspergillus sect. nigri species infecting pomegranate fruit after harvest is of paramount importance for their effective control, even more considering the ability of these fungal genera to produce mycotoxins, which are hazardous for human health and potentially present also in by-products.

An application of PCR-RFLP species identification assay for environmental DNA detection.

Recent advancement of environmental DNA (eDNA) methods for surveying species in aquatic ecosystems has been used for various organisms and contributed to monitoring and conservation of species and environments. Amphibians are one of the promising taxa which could be monitored efficiently by applying quantitative PCR (qPCR) or next generation sequencing to eDNA. However, the cost of eDNA detection using these approaches can be quite high and requires instruments that are not usually installed in ecology laboratories. For aiding researchers in starting eDNA studies of amphibians, especially those not specialized in molecular biology, we developed a cost efficient protocol using PCR-RFLP method. We attempted to detect eDNA of three Japanese Rana species (Rana japonica, Rana ornativentris, and Rana tagoi tagoi) in various spatial scales including an area close to the Fukushima nuclear power plant where the environment is recovering after the disaster in 2011. Our PCR-RFLP protocol was successful in detecting Rana species in static water in both laboratory and field; however, it could not detect Rana species in non-static water samples from the field. Even a more sensitive detection method (standard qPCR) was unable to detect frogs in all non-static water samples. We speculate that our new protocol is effective for frogs living in lentic habitats, but not for lotic habitats which may still require the gold standard of field observation for detection approach.

Application of Mass Spectrometry-Based Immunoassays for the Species- and Tissue-Specific Quantification of Banned Processed Animal Proteins in Feeds

Processed Animal Proteins (PAPs) are considered as a sustainable protein source to improve the nutritional profile of feed for livestock and aquaculture. However, the use of these proteins is strongly regulated since the bovine spongiform encephalopathy (BSE) crisis. The reintroduction of nonruminant PAPs for use in aquaculture in 2013 has driven the need for alternative analytical methods to determine the species origin as well as the tissue source (legal or not). The current official methods, light microscopy and polymerase chain reaction, do not fulfill these requirements. Furthermore, future methods need to be quantitative, because the pending zero-tolerance-concept is planned to be replaced by accurate thresholds. Here, we developed a 7-plex mass spectrometry-based immunoassay that is capable of quantifying 0.1% (w/w) ruminant PAP in feed in a tissue- and species-specific way. The workflow comprises a 2 h tryptic digestion of PAPs in suspension, an immunoaffinity enrichment of peptides, and LC-MS/MS-based quantification. In combination with a previously published assay for species identification, we were able to confirm the species and tissue origin of sixring trial samplesobtained in former PCR and microscopy proficiency tests. The sensitive, quantitative, species- and tissue-specific character of the developed assays meets the requirements for new methods for PAP detection and can be used in future feed authentication studies.

ELEquant: a developmental framework and validation of forensic and conservation real-time PCR assays.

A framework for the development and validation of a qPCR assay for species identification and DNA quantification for conservation and forensic purposes is presented. Elephants are commonly poached for their ivory tusks, which is the primary driving force behind their endangered status. In addition to poaching and trade, habitat loss due to logging and mining has also resulted in loss of elephants. Crimes against animals can be deterred and/or further prosecution sought through testing with forensic genetic techniques. The creation of novel genetic assays can greatly impact wildlife forensic science investigations in identifying the species. Molecular genetic techniques can help enforce conservation efforts; however, they must be properly developed and validated to be of evidentiary quality for court systems. African and Asian elephant buccal cells were used as model in this work. The assay provides a method to differentiate biological fluids of both genera of elephants simultaneously. It can be used for identification of elephant derived products and presents valuable quantification for optimized further testing, such as microsatellite detection.

Communications of Staphylococcus aureus and non-aureus Staphylococcus species from bovine intramammary infections and teat apex colonization

The role of non-aureus staphylococci (NAS) in the risk of acquisition of intramammary infections with Staphylococcus aureus is vague and still under debate. The objectives of this study were to (1) investigate the distribution patterns of NAS species from milk and teat skin in dairy herds with automatic milking systems, and (2) examine if the isolated NAS influences the expression of S. aureus virulence factors controlled by the accessory gene regulator (agr) quorum sensing system. In 8 herds, 14 to 20 cows with elevated somatic cell count were randomly selected for teat skin swabbing and aseptic quarter foremilk samples from right hind and left front quarters. Teat skin swabs were collected using the modified wet-dry method and milk samples were taken aseptically for bacterial culture. Colonies from quarters with suspicion of having NAS in milk or teat skin samples (or both) were subjected to MALDI-TOF assay for species identification. To investigate the interaction between S. aureus and NAS, 81 isolates NAS were subjected to a qualitative β-galactosidase reporter plate assay. In total, 373 NAS isolates were identified representing 105 from milk and 268 from teat skin of 284 quarters (= 142 cows). Sixteen different NAS species were identified, 15 species from teat skin and 10 species from milk. The most prevalent NAS species identified from milk were Staphylococcus epidermidis (50%), Staphylococcus haemolyticus (15%), and Staphylococcus chromogenes (11%), accounting for 76%. Meanwhile, the most prevalent NAS species from teat skin were Staphylococcus equorum (43%), S. haemolyticus (16%), and Staphylococcus cohnii (14%), accounting for 73%. Using reporter gene fusions monitoring transcriptional activity of key virulence factors and regulators, we found that out of 81 supernatants of NAS isolates, 77% reduced expression of hla, encoding a-hemolysin, 70% reduced expression of RNAIII, the key effector molecule of agr, and 61% reduced expression of spa encoding protein A of S. aureus, respectively. Our NAS isolates showed 3 main patterns: (1) downregulation effect such as S. chromogenes (milk) and Staphylococcus xylosus (milk and teat), (2) no effect such as Staphylococcus sciuri (teat) and S. vitulinus (teat), and the third pattern (c) variable effect such as S. epidermidis (milk and teat) and S. equorum (milk and teat). The pattern of cross-talk between NAS species and S. aureus virulence genes varied according to the involved NAS species, habitat type, and herd factors. The knowledge of how NAS influences S. aureus virulence factor expression could explain the varying protective effect of NAS on S. aureus intramammary infections.

Double specific nested PCR and diagnostic SNP assay for species identification in lynx fecal critical samples

The single lynx species present in the Iberian Peninsula—Lynx pardinus (Temmink, 1827)—is one of the most threatened felines in the world so, any biological indication of its presence is significant. However, it is a very elusive species and physical evidence through direct visualization or camera trapping is difficult to obtain. Genetic noninvasive methods, in particular, molecular scatology, are a reliable alternative to unequivocally identify the presence of a species in a given geographical area. We have developed a highly specific and sensitive molecular method to identify the presence of this critically endangered lynx species from severely deteriorated fecal samples. The method consists in a double specific nested PCR of mitochondrial D-loop sequences and two diagnostic SNPs detected by a multiplex primer extension reaction.

An analysis of Echinacea chloroplast genomes: Implications for future botanical identification

Echinacea is a common botanical used in dietary supplements, primarily to treat upper respiratory tract infections and to support immune function. There are currently thought to be nine species in the genus Echinacea. Due to very low molecular divergence among sister species, traditional DNA barcoding has not been successful for differentiation of Echinacea species. Here, we present the use of full chloroplast genomes to distinguish between all 9 reported species. Total DNA was extracted from specimens stored at the National Museum of Natural History, Smithsonian Institution, which had been collected from the wild with species identification documented by experts in the field. We used Next Generation Sequencing (NGS) and CLC Genomics Workbench to assemble complete chloroplast genomes for all nine species. Full chloroplasts unambiguously differentiated all nine species, compared with the very few single nucleotide polymorphisms (SNPs) available with core DNA barcoding markers. SNPs for any two Echinacea chloroplast genomes ranged from 181 to 910, and provided robust data for unambiguous species delimitation. Implications for DNA-based species identification assays derived from chloroplast genome sequences are discussed in light of product safety, adulteration and quality issues.