Method For Identification Of Species Research Articles

Pragmatic applications of DNA barcoding markers in identification of fish species – A review

Abstract DNA barcoding and mini-barcoding involve Cytochrome Oxidase Subunit I (COI) gene in mitochondrial genome and are used for accurate identification of species and biodiversity. The basic goal of the current study is to develop a complete reference database of fishes. It also evaluates the applicability of COI gene to identify fish at the species level with other aspects i.e., as Kimura 2 parameter (K2P) distance. The mean observed length of the sequence was ranging from 500 to 700 base pairs for fish species in DNA barcoding and from 80 to 650 base pairs for DNA mini-barcoding. This method describes the status of known to unknown samples but it also facilitates the detection of previously un-sampled species at distinct level. So, mini-barcoding is a method which focuses on the analysis of short-length DNA markers and has been demonstrated to be effective for species identification of processed food containing degraded DNA. While DNA metabarcoding refers to the automated identification of multiple species from a single bulk sample. They may contain entire organisms or a single environmental sample containing degraded DNA. Despite DNA barcoding, mini-barcoding and metabarcoding are efficient methods for species identification which are helpful in conservation and proper management of biodiversity. It aids researchers to take an account of genetic as well as evolutionary relationships by collecting their morphological, distributional and molecular data. Overall, this paper discusses DNA barcoding technology and how it has been used to various fish species, as well as its universality, adaptability, and novel approach to DNA-based species identification.

Assessing the potential use of environmental DNA for multifaceted genetic monitoring of cetaceans: Example of a wandering whale in a highly disturbed bay area

Environmental DNA (eDNA) sampling of water is a powerful method for comprehensive and noninvasive monitoring of aquatic animal species. However, there have been few reports of its application to cetacean species. On June 29, 2021, a whale (nicknamed Xiaobu) appeared in Dapeng Bay off Guangdong Province, China. We used eDNA technology to obtain information related to this whale (e.g., species identification and food resources) and to trace its possible origin. Fragments of four whale mitochondrial sequences (12S rDNA, 16S rDNA, cytochrome c oxidase subunit 1, and control region) were obtained from amplicons of eDNA collected in Dapeng Bay; sequence barcoding showed that this was an Eden’s whale (Balaenoptera edeni edeni Anderson 1879). Analysis of potential prey species (PPS) suggested that this whale might enter Dapeng Bay while tracking prey, mainly sardines (Sardinella lemuru, Sardinella gibbosa and Sardinella jussieui) and anchovies (Thryssa dussumieri, Thryssa vitrirostris and Thryssa kammalensis). Retrieval of eDNA metabarcoding data from samples collected in waters adjacent to Dapeng Bay (i.e. Lingding Bay and Daya Bay) revealed that Eden’s whale had appeared outside Lingding Bay up to 2 months prior to the appearance of this whale in Dapeng Bay (early April 2021). Overall, this study showed that eDNA is a highly effective noninvasive survey method for the accurate identification of target cetacean species and prey composition; it can be used to monitor megafauna that are under strict legal protection or to monitor megafauna with unknown conditions.

Call repertoire of Ptychadena uzungwensis (Anura: Ptychadenidae) to complement molecular and morphological identification of the species from the Soutpansberg, South Africa.

ABSTRACT The identity of adults and tadpoles of Ptychadena uzungwensis from the Soutpansberg range in northern South Africa was confirmed using a combination of morphological characters and molecular analysis. Detailed vocalisation analysis was made and used to complement existing species identification methods. This study confirms the presence of P. uzungwensis in South Africa. Sound recordings obtained by way of directional manual recordings were used to analyse call structure and call type while Passive Acoustic Monitoring with Song Meter® technology were used to survey circadian call patterns. Following confirmation of species identity, the detailed vocalisation analysis indicated a much more diverse repertoire than previously known. Three call types were observed with Type A consisting of two or three compressed notes; Type B consisting of a multi-note vocalisation, containing multiple pulses and notes produced in rapid succession; and Type C being a multi-note vocalisation consisting of rapidly released, randomly-spaced single notes. Detailed descriptions of call repertoires, including patterns and call types are useful for species identification and for use in comparison studies.

Diversity of aquatic plants in the Rote Dead Sea area, East Nusa Tenggara, Indonesia, based on rbcL marker

Abstract. Lonthor DW, Miftahudin, Kayat, Julzarika A, Subehi L, Iswandono E, Dima AOM, Dianto A, Setiawan F, Nugraha MFI. 2023. Diversity of aquatic plants in the Rote Dead Sea area, East Nusa Tenggara, Indonesia, based on rbcL marker. Biodiversitas 24: 810-818. The Rote Dead Sea area in Rote Islands has two freshwater and 24 saltwater lakes. The saltwater lakes have salinity levels varied between 0-100 ppt. Every lake has specific aquatic plant species. Species identification through morphological analysis only has its limitations; therefore, DNA barcoding is a preference due to an effective, accurate, and faster method for species identification. In this study, we used the rbcL marker to identify and analyze the diversity of the aquatic plant species in lakes Ledulu, Oenduy, Oemasapoka, Oesotimori, and Landu Leko. Plants samples' DNA was amplified using rbcL primers, and the amplicons were sequenced. Species identification was based on morphological study and the rbcL sequence database. Diversity analyses were performed based on pairwise genetic distances, analysis of molecular variance, and genetic relationships based on the Kimura 2 parameter. The results showed that all 20 aquatic plants were correctly identified, and high genetic diversity was discovered. AMOVA analysis showed that genetic variations within the population were higher than among the population. All plant species were grouped into four groups based on the phylogenetic tree and fractional component analyses. The species belonging to the certain family were grouped in the same clade and the closely related plant families. The rbcL marker can be used to discriminate aquatic plant species and analyze their genetic diversity and relationships.

Benchmarking and Validation of a Bioinformatics Workflow for Meat Species Identification Using 16S rDNA Metabarcoding

DNA-metabarcoding is becoming more widely used for routine authentication of meat-based food and feed products. Several methods validating species identification methods through amplicon sequencing have already been published. These use a variety of barcodes and analysis workflows, however, no methodical comparison of available algorithms and parameter optimization are published hitherto for meat-based products' authenticity. Additionally, many published methods use very small subsets of the available reference sequences, thereby limiting the potential of the analysis and leading to over-optimistic performance estimates. We here predict and compare the ability of published barcodes to distinguish taxa in the BLAST NT database. We then use a dataset of 79 reference samples, spanning 32 taxa, to benchmark and optimize a metabarcoding analysis workflow for 16S rDNA Illumina sequencing. Furthermore, we provide recommendations as to the parameter choices, sequencing depth, and thresholds that should be used to analyze meat metabarcoding sequencing experiments. The analysis workflow is publicly available, and includes ready-to-use tools for validation and benchmarking.

Conservation-relevant plant species indicate arthropod richness across trophic levels: Habitat quality is more important than habitat amount

The massive declines in terrestrial arthropods reported across Europe call for effective methods to monitor and promote biodiversity in human-dominated landscapes. Previous studies vary in their support for the suitability of plants as indicators of arthropod diversity, while the potential of subsets of conservation-relevant plant species to estimate arthropod richness remains to be tested. Moreover, the relative importance of plant species richness compared to other factors driving arthropod richness, such as land-use intensity, habitat amount and landscape configuration, is poorly understood. We conducted a multi-scale field study in Southern Germany, sampling vascular plants and terrestrial arthropods in four local land-use types (forest, grassland, arable field, settlement) across large-scale gradients of climate and land-use intensity. We obtained an extensive arthropod dataset using Malaise traps as sampling method and DNA metabarcoding for species identification. We compared the correlation of three sets of plant species richness (total, red-listed and biotope-indicator plants) with arthropod richness, including different trophic and taxonomic groups. Using mixed-effect models, we assessed the partial effects of plant species richness, habitat amount and landscape configuration on local arthropod richness while controlling for climate and land-use effects, and explored the environmental drivers of plant species richness. Arthropod species numbers of all trophic groups strongly responded to species numbers of plants that indicate protected habitats, while red-listed plant species richness was a key determinant for the richness of red-listed arthropods and butterflies. In most cases, plant species richness and temperature were stronger drivers of arthropod richness than the amount of surrounding semi-natural habitat and landscape configuration. While total plant species richness was highest in settlements, the richness of biotope-indicator plants was highest in forests and positively influenced by the amount of semi-natural habitat and edge density. The number of red-listed plant species increased with edge density at the landscape scale but was independent of habitat amount. We conclude that the richness of conservation-relevant plant species provides a powerful indicator of arthropod diversity, and underpins the potential of such plant lists for monitoring habitat quality. Our study highlights the importance of openings in forests for conservation of plants and arthropods as well as of preserving and restoring fragments of high-quality biotopes in agricultural and urban areas where the amount of semi-natural habitat is limited.

Successful identification of the species of the semipetrified amber medicinal resin benzoin using molecular diagnostic technology

Benzoin is an incomplete lithified resin secreted from the trunk of the Styrax Linn. that is known as "semipetrified amber" and has been widely used in medicine due to its blood circulation-promoting and pain-relieving properties. However, the lack of an effective species identification method due to the numerous sources of benzoin resin and the difficulty of DNA extraction has led to the uncertainty of species of benzoin in the trade process. Here, we report the successful extraction of DNA from benzoin resin containing bark-like residues and the evaluation of commercially available benzoin species using molecular diagnostic techniques. By performing a BLAST alignment of ITS2 primary sequences and homology prediction analysis of ITS2 secondary structures, we found that commercially available benzoin species were derived from Styrax tonkinensis (Pierre) Craib ex Hart. and Styrax japonicus Sieb. et Zucc. of the genus Styrax Linn. In addition, some of the benzoin samples were mixed with plant tissues from other genera, accounting for 29.6%. Therefore, this study provides a new method to solve the problem of species identification of semipetrified amber benzoin using information from bark residues.

Using a Madurella mycetomatis-specific PCR on grains obtained via non-invasive fine-needle aspirated material is more accurate than cytology.

Eumycetoma is a chronic subcutaneous inflammatory fungal infection most often caused by the fungus Madurella mycetomatis. Using a species-specific PCR on DNA directly isolated from grains is currently the most reliable method for species identification. However, so far, PCR has been performed on grains obtained through deep-seated surgical biopsies, which are invasive procedures. Grains can also be obtained via ultrasound-guided fine-needle aspiration (US-FNA). Here we determined the diagnostic performance of species-specific PCRs performed on samples obtained through US-FNA. From 63 patients, US-FNA was performed to obtain eumycetoma grains; 34 patients also underwent a deep-seated biopsy. From the grains, DNA was isolated, and one pan-fungal and two M. mycetomatis-specific PCRs were performed. The sensitivity and specificity were determined. Of the 63 patients who underwent US-FNA, 78% (49/63) had evidence of eumycetoma based on cytology and 93.7% (59/63) based on species-specific PCRs. In the 34 patients for whom surgical biopsies were performed as well, 31 patients had a positive PCR for M. mycetomatis when DNA was isolated from the deep-seated biopsy, and 30 had a positive PCR when DNA was obtained from the US-FNA material. This resulted in a 96.8% sensitivity, and 100% specificity with 97.1% diagnostic accuracy for PCR performed on US-FNA. PCR performed on the US-FNA material has a similar sensitivity and specificity as PCR performed on deep-seated biopsies. Therefore, when using PCR, a deep-seated biopsy may not be necessary to obtain grains.

Pathogen detection in RNA-seq data with Pathonoia

BackgroundBacterial and viral infections may cause or exacerbate various human diseases and to detect microbes in tissue, one method of choice is RNA sequencing. The detection of specific microbes using RNA sequencing offers good sensitivity and specificity, but untargeted approaches suffer from high false positive rates and a lack of sensitivity for lowly abundant organisms.ResultsWe introduce Pathonoia, an algorithm that detects viruses and bacteria in RNA sequencing data with high precision and recall. Pathonoia first applies an established k-mer based method for species identification and then aggregates this evidence over all reads in a sample. In addition, we provide an easy-to-use analysis framework that highlights potential microbe-host interactions by correlating the microbial to the host gene expression. Pathonoia outperforms state-of-the-art methods in microbial detection specificity, both on in silico and real datasets.ConclusionTwo case studies in human liver and brain show how Pathonoia can support novel hypotheses on microbial infection exacerbating disease. The Python package for Pathonoia sample analysis and a guided analysis Jupyter notebook for bulk RNAseq datasets are available on GitHub.

DNA signaturing derived from the internal transcribed spacer 2 (ITS2): a novel tool for identifying Desmodesmus species (Scenedesmaceae, Chlorophyta)

The freshwater green algal genus <i>Desmodesmus</i> contains many cryptic species that are difficult to identify using morpho-anatomy. This study analyzed 310 ITS2 sequences related to <i>Desmodesmus</i> species from GenBank and developed a new tool named DNA signaturing. DNA signaturing is a species identification method using signature sequences specific for each species or strain group within the genus <i>Desmodesmus</i>. These signature sequences had 18-33 nucleotides in length. This tool could exactly identify 19 species and 24 strain groups of <i>Desmodesmus</i>. In addition, a signature sequence, AGA GGC TTA AAC TGG GAC C, was specific for almost 90% of <i>Desmodesmus</i> strains and could represent the genus <i>Desmodesmus</i>. A strain MLC1 isolated from freshwater in Da Nang, Vietnam, was identified as <i>Desmodesus pseudoserratus</i> by this tool. DNA signaturing tool described here showed the highest efficacy for identifying strains and a remarkable ability to accurately classify cryptic species in the genus<i> Desmodesmus</i>.

Proteomic analysis of nontuberculous bacteria protein spectra as the element of subtyping of strains.

For the present, matrix-assisted laser desorption/ionization-time-of-flight (MALDI-ToF) mass spectrometry is the fastest and the most correct method for species identification of microorganisms. Apart from species-level identification, it allows to use a variety of approaches for the analysis and comparison of protein spectra of microorganisms of the same species, which are isolated from a patient at various disease states, that can be used in routine microbiological practice in laboratories fitted with mass analyzers. Two strains of Mycobacterium fortuitum and two strains of Mycobacterium peregrinum were isolated from sputum samples, which were obtained from patients with different clinical aspects of mycobacteriosis, whereat were reinoculated on the universal chromogenic culture medium "UriSelect 4." Further, the MALDI-ToF mass spectrometry method was used, aiming to obtain protein profiles, which were analyzed using the FlexAnalysis 3.0 software package. Results of the statistical proteomic comparison of mass spectra were visualized using MALDI Biotyper 3.0 Offline Classification software. Presented clinical examples demonstrate that strains of the same species, which are isolated from the same patient at different times of infection, change their cultural properties. Dynamic changes in cultural properties are reflected in changes in protein profiles by comparison spectra of isolates at different stages of colonization, which is reflected in the correlation with the clinical condition of the patient. Thus, the mentioned examples of proteomic analysis, using MALDI-ToF mass spectrometry, demonstrate the possibility of subtyping of strains, that are isolated on a universal chromogenic culture medium, in case of detection in the culture signs of population's heterogeneity, based on cultural properties.

Identification and Differentiation of Closely Related Members of Bacillus cereus Group by Multiplex PCR

Highly similar genetic and phenotypic traits of at least eight bacterial species forming the ‘Bacillus cereus group’ create their precise identification and differentiation quite difficult. The present study explores the applicability of a previously suggested multiplex-PCR method for the accurate identification of the candidate Bacillus species. Out of the 257 Bacillus isolates collected from soil, 44 were identified as B. thuringiensis and 39 as B. cereus by chromogenic cultural method using Bacillus agar, although few of them shared similar colony characteristics. Identification by the multiplex PCR in a thermo cycler using 5 different sets of primer-pairs, however produced distinct amplification corresponding to the bacterial species. Four of those pairs, named BMSH, BCJH, BTJH and BASH were designed based on gyrB gene that produced amplicons of four different sizes: 604, 475, 299 and 253 bp and were specific for B. mycoides, B. cereus, B. thuringiensis, and B. anthracis respectively. The remaining one of the sets was used as an internal control which was a universal primerpair, BCGSH, designed targeting a housekeeping gene, groEL that could produce an amplicon of 400 bp in polymerase chain reactions for all members of the B. cereus group, When probing the chromosomes extracted from 257 Bacillus isolates by multiplex PCR; 48, 39 and 5 were identified as B. thuringiensis, B. cereus, and B. anthracis respectively, however the rest of the isolates did not any amplification. Interestingly, the phylogenetic tree, constructed based on partial sequences of 16S rRNA genes of selected isolates including the reference strain of B. thuringiensis (HD-73, sotto) could not differentiate the species, instead posited those in a single cluster. The multiplex PCR, therefore, proved to be a sensitive and reliable method for the identification of the bacterial candidates of Bacillus cereus group than that of cultural and rRNA gene sequence analyses.&#x0D; Bangladesh J Microbiol, Volume 39, Number 1, June 2022, pp 21-29

Evaluation of nematode DNA extraction methods for species identification of root rice nematode (Hirschmanniella spp.)

The species-level identification of rice root nematode (Hirschmanniella spp.) is an important effort in pest management. Meanwhile, the selection of DNA extraction methods is the first important step in the molecular identification. Thereby, this study aimed to obtain the DNA extraction method and optimal quantity in the identification of Hirschmanniella spp. to the species level. The DNA extraction methods used were Cetyltrimethylammonium bromide (CTAB) and Commercial Kit (Geneaid; Genomic DNA Mini Kit Tissue), with variations in the number of nematodes: 1, 5, 10, 15 and 20 nematodes. There were no significant differences on DNA concentration and purity between CTAB and commercial kit method, but the DNA extraction of Hirschmaniella spp. with the CTAB method reached the highest DNA concentration of 189.57 ng/µL. DNA purity level in the ratio (A260/A280) ranging from 1.80 to 1.99. The PCR test obtained the amplification of the extracted DNA at ∼766 bp.

Comparing the performance of the common used eumycetoma diagnostic tests.

Mycetoma is a neglected tropical implantation disease caused by 70 different infectious agents. Identifying the causative organism to the species level is essential for appropriate patient management. Ultrasound, histopathology, culture and two species-specific PCRs are most the commonly used methods for species identification in endemic regions. The aim of this study was to compare the diagnostic performance of these commonly used assays using sequencing of barcoding genes as the gold standard. This descriptive cross-sectional study was conducted at the Mycetoma Research Centre, University of Khartoum, Sudan. It included 222 patients suspected of fungal mycetoma caused by Madurella mycetomatis. 154 (69.3%) were correctly identified by ultrasound, histology, culture and both species-specific PCRs. In 60 patients, at least one of the diagnostic tests failed to identify M. mycetomatis. Five patients had no evidence of eumycetoma, and for three, only the ultrasound was indicative of mycetoma. The two species-specific PCRs were the most sensitive and specific methods, followed by culture and histology. Ultrasound was the least specific as it only allowed differentiation between actinomycetoma and eumycetoma. The time to result was 9.38 minutes for ultrasound, 3.76 hours for PCR, 8.5 days for histopathology and 21 days for grain culturing. Currently, PCR directly on DNA isolated from grains is the most rapid and reliable diagnostic tool to identify M. mycetomatis eumycetoma.

Application of spectral image processing with different dimensions combined with large-screen visualization in the identification of boletes species.

Boletes are favored by consumers because of their unique flavor, rich nutrition and delicious taste. However, the different nutritional values of each species lead to obvious price differences, so shoddy products appear on the market, which affects food safety. The aim of this study was to find a rapid and effective method for boletes species identification. In this paper, 1,707 samples of eight boletes species were selected as the research objects. The original Mid-Infrared (MIR) spectroscopy data were adopted for support vector machine (SVM) modeling. The 11,949 spectral images belong to seven data sets such as two-dimensional correlation spectroscopy (2DCOS) and three-dimensional correlation spectroscopy (3DCOS) were used to carry out Alexnet and Residual network (Resnet) modeling, thus we established 15 models for the identification of boletes species. The results show that the SVM method needs to process complex feature data, the time cost is more than 11 times of other models, and the accuracy is not high enough, so it is not recommended to be used in data processing with large sample size. From the perspective of datasets, synchronous 2DCOS and synchronous 3DCOS have the best modeling results, while one-dimensional (1D) MIR Spectrum dataset has the worst modeling results. After comprehensive analysis, the modeling effect of Resnet on the synchronous 2DCOS dataset is the best. Moreover, we use large-screen visualization technology to visually display the sample information of this research and obtain their distribution rules in terms of species and geographical location. This research shows that deep learning combined with 2DCOS and 3DCOS spectral images can effectively and accurately identify boletes species, which provides a reference for the identification of other fields, such as food and Chinese herbal medicine.

Gauging DNA degradation among common insect trap preservatives

AbstractGenetic methods for species identification are becoming increasingly popular and can accelerate insect monitoring. However, obtaining good DNA quality and quantity from insect traps remains a challenge for field studies. Ethylene glycol, propylene glycol, and Renner solution have been previously suggested as suitable preservatives for the collection of genetic material, but a systematic overview of their performance under compromising field conditions is lacking. Here we experimentally test whether and how different preservatives affect DNA quality under different conditions and evaluate how choice of preservative may affect metabarcoding and more demanding downstream applications (e.g., RADseq). For this, we used the house cricket, Acheta domesticus (L.) (Orthoptera: Gryllidae), and tested propylene glycol, ethylene glycol, and Renner solution for their ability to preserve DNA over 27 days in various dilutions and temperatures. DNA quality was measured as DNA fragmentation and success rates in PCR amplifying a COI fragment of 658, 313, or 157 bp. Undiluted propylene glycol and ethylene glycol always retained high molecular weight DNA at room temperature. No high molecular weight DNA was preserved at 37 °C or in any dilution. Nevertheless, the COI sequence could be amplified from samples at every condition. Renner solution did not preserve high molecular weight DNA and fragmentation increased over time at 37 °C until amplification was impossible. The results suggest that propylene glycol and ethylene glycol are suitable preservatives for collecting both genetic and morphological material, but dilution or high temperatures compromise their ability to preserve high molecular weight DNA. For genomic approaches requiring high DNA quality, additional preservatives may need to be tested.

Morphological characterization of some poorly known populations of the genus Spalerosophis Jan, 1865 (Reptilia: Colubridae): is the geometric morphometrics useful to identify them as taxonomic units?

Despite the use of traditional morphology as a standard method for species identification, additional methods like image-based analysis can be desirable to differentiate species and populations. In this study, we used traditional morphology and geometric morphometrics (GM) to describe three distinct populations of the genus Spalerosophis and test the hypothesis that all populations are morphologically distinct. A total of 132 specimens were analysed and based on morphological traits (morphometric, meristic, and colour pattern) subcaudal scales was the only distinguishable trait between Spalerosophis diadema cliffordii and Spalerosophis diadema schirasianus. In addition, Spalerosophis atriceps showed significant differences in most studied characters compared to the other two populations. In the geometric morphometric analysis, we placed 2D Landmarks on the dorsal view images of the head of 93 specimens. Canonical variate analysis (CVA) showed significant differences among the head shape of all three populations. There was an increase in the head width, snout length, and curvature in the posterior part of parietals from west Africa in S. d. cliffordii to the north of India in S. atriceps. This may be the result of variations in their environmental factors, diet and the amount of time that has passed since their separation.

A report on occurrence of coccidial infection in sheep

An investigation was carried out in a sheep farm from Bengaluru North region to identify the presence of coccidia species. In a total of 70 sheep, about twenty faecal samples were randomly collected from animals and subjected to faecal examination by concentration methods. All the 20 samples were found positive for oocysts. The oocysts were subjected for sporulation by invitro methods for identification of species respectively. Based on the morphometric studies it revealed five different Eimeria oocysts viz., Eimeriaarkhari (42x33.6 μm), E. ovina (25.65x33.6 μm), E. pallida (39.9x28.5 μm), E. parva (22.8x19.95 μm) and E. faurei (34.2x22.8 μm).

MORPHOLOGIC OBSERVATION AND FIRST MOLECULAR CHARACTERIZATION OF ENCYCLOMETRA JAPONICA (DIGENEA: ENCYCLOMETRIDAE) IN THE DIGESTIVE TRACT OF WILD TIGER KEELBACKS (RHABDOPHIS TIGRINUS) IN JAPAN.

Encyclometra japonica Yoshida and Ozaki, 1929 is a fluke that parasitizes the digestive tract of snakes. When the species was first reported, it was originally characterized on the basis of morphologic features, namely, the characteristics of the 2 testes located obliquely in the anterior-posterior direction, and the position of the ventral sucker one-fourth to one-third from the anterior extremity. Thereafter, more specimens with other morphologic variations were reported, and a new morphologic feature for species discrimination was proposed. However, the proposal is uncertain, and the criteria for morphologic discrimination between E. japonica and other Encyclometra species remain debatable. Thus, morphologically discriminable characteristics on the basis of a sufficient number of specimens are required, and molecular methods for species identification that allow for a more objective assessment need to be established. Therefore, in the present study, nucleotide sequences of 18S, 28S ribosomal deoxyribonucleic acid and cytochrome c oxidase subunit I (COI) of E. japonica were determined along with detailed morphologic observations for the first time. Key characteristics were observed in all specimens, whereas some morphologic variations were observed in the symmetry of the cecum. The COI sequences, which are known to be variable genetic regions, were identical among all specimens; therefore, all of them were considered the same species. This result clarifies the high morphologic variation in E. japonica. The 18S and 28S sequences were 99.78% and 99.11-99.19% similar to those of Encylometra colubrimurorum. The high degree of homology between these genes indicates that E. japonica and E. colubrimurorum are closely related but independent species. The present data will be used for the identification of E. japonica and to evaluate the relationships within the genus Encyclometra.

Validation of PCR methods for confirmation and species identification of thermotolerant Campylobacter as part of EN ISO 10272 - Microbiology of the food chain - Horizontal method for detection and enumeration of Campylobacter spp.

This article describes the outline and organisation of the validation of three multiplex PCR methods for species identification and/or confirmation of thermotolerant Campylobacter spp. The three PCR methods were validated against the reference method described in the EN ISO standard 10272:2017. The results of the PCR methods were compared against the reference method in a method comparison study and an interlaboratory study based on EN ISO 16140-6:2019.The performance, in terms of inclusivity and exclusivity, of each of the eight PCR targets were comparable to the performance of the reference method: close, equal, or better depending on the target. In total, all three PCR methods were concluded to be equally qualified as the reference method for molecular identification and/or confirmation of thermotolerant Campylobacter spp., C. jejuni, C. coli and C. lari isolated from the food chain and have been included in Amendment 1 of ISO 10272:2017.