Method For Identification Of Species Research Articles

Evaluation of the MALDI-TOF MS method for routine identification of Candida species in a tertiary care hospital in Thailand

Evaluation of the MALDI-TOF MS method for routine identification of Candida species in a tertiary care hospital in Thailand

The Threat Called Candida haemulonii Species Complex in Rio de Janeiro State, Brazil: Focus on Antifungal Resistance and Virulence Attributes.

Although considered rare, the emergent Candida haemulonii species complex, formed by C. haemulonii sensu stricto (Ch), C. duobushaemulonii (Cd) and C. haemulonii var. vulnera (Chv), is highlighted due to its profile of increased resistance to the available antifungal drugs. In the present work, 25 clinical isolates, recovered from human infections during 2011–2020 and biochemically identified by automated system as C. haemulonii, were initially assessed by molecular methods (amplification and sequencing of ITS1-5.8S-ITS2 gene) for precise species identification. Subsequently, the antifungal susceptibility of planktonic cells, biofilm formation and susceptibility of biofilms to antifungal drugs and the secretion of key molecules, such as hydrolytic enzymes, hemolysins and siderophores, were evaluated by classical methodologies. Our results revealed that 7 (28%) isolates were molecularly identified as Ch, 7 (28%) as Chv and 11 (44%) as Cd. Sixteen (64%) fungal isolates were recovered from blood. Regarding the antifungal susceptibility test, the planktonic cells were resistant to (i) fluconazole (100% of Ch and Chv, and 72.7% of Cd isolates), itraconazole and voriconazole (85.7% of Ch and Chv, and 72.7% of Cd isolates); (ii) no breakpoints were defined for posaconazole, but high MICs were observed for 85.7% of Ch and Chv, and 72.7% of Cd isolates; (iii) all isolates were resistant to amphotericin B; and (iv) all isolates were susceptible to echinocandins (except for one isolate of Cd) and to flucytosine (except for two isolates of Cd). Biofilm is a well-known virulence and resistant structure in Candida species, including the C. haemulonii complex. Herein, we showed that all isolates were able to form viable biofilms over a polystyrene surface. Moreover, the mature biofilms formed by the C. haemulonii species complex presented a higher antifungal-resistant profile than their planktonic counterparts. Secreted molecules associated with virulence were also detected in our fungal collection: 100% of the isolates yielded aspartic proteases, hemolysins and siderophores as well as phospholipase (92%), esterase (80%), phytase (80%), and caseinase (76%) activities. Our results reinforce the multidrug resistance profile of the C. haemulonii species complex, including Brazilian clinical isolates, as well as their ability to produce important virulence attributes such as biofilms and different classes of hydrolytic enzymes, hemolysins and siderophores, which typically present a strain-dependent profile.

Quality of MALDI-TOF mass spectra in routine diagnostics: results from an international external quality assessment including 36 laboratories from 12 countries using 47 challenging bacterial strains

ObjectivesMatrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) is a widely used method for bacterial species identification. Incomplete databases and mass spectral quality (MSQ) still represent major challenges. Important proxies for MSQ are the number of detected marker masses, reproducibility, and measurement precision. We aimed to assess MSQs across diagnostic laboratories and the potential of simple workflow adaptations to improve it. MethodsFor baseline MSQ assessment, 47 diverse bacterial strains, which are challenging to identify by MALDI-TOF MS, were routinely measured in 36 laboratories from 12 countries, and well-defined MSQ features were used. After an intervention consisting of detailed reported feedback and instructions on how to acquire MALDI-TOF mass spectra, measurements were repeated and MSQs were compared. ResultsAt baseline, we observed heterogeneous MSQ between the devices, considering the median number of marker masses detected (range = [2–25]), reproducibility between technical replicates (range = [55%–86%]), and measurement error (range = [147 parts per million (ppm)–588 ppm]). As a general trend, the spectral quality was improved after the intervention for devices, which yielded low MSQs in the baseline assessment as follows: for four out of five devices with a high measurement error, the measurement precision was improved (p-values <0.001, paired Wilcoxon test); for six out of ten devices, which detected a low number of marker masses, the number of detected marker masses increased (p-values <0.001, paired Wilcoxon test). DiscussionWe have identified simple workflow adaptations, which, to some extent, improve MSQ of poorly performing devices and should be considered by laboratories yielding a low MSQ. Improving MALDI-TOF MSQ in routine diagnostics is essential for increasing the resolution of bacterial identification by MALDI-TOF MS, which is dependent on the reproducible detection of marker masses. The heterogeneity identified in this external quality assessment (EQA) requires further study.

An Inexpensive CRISPR-Based Point-of-Care Test for the Identification of Meat Species and Meat Products.

The growing demand for and supply of meat and meat products has led to a proportional increase in cases of meat adulteration. Adulterated meat poses serious economic and health consequences globally. Current laboratory methods for meat species identification require specialized equipment with limited field applications. This study developed an inexpensive, point-of-care Loop-Mediated Isothermal Amplification (LAMP)-CRISPR/Cas12a colorimetric assay to detect meat species using a Texas Red-labelled single-strand (ssDNA) reporter. As low as 1.0 pg/µL of the porcine NADH4, the chicken NADH dehydrogenase subunit 2 (ND2) and the duck D-loop genes was detectable under white, blue and ultraviolet light. The test turnaround time from DNA extraction to visualization was approximately 40 min. The assay accurately detected pure and mixed-meat products in the laboratory (n = 15) and during a pilot point-of-care test (n = 8) in a food processing factory. The results are 100% reproducible using lateral flow detection strips and the real-time PCR detection instrument. This technology is fully deployable and usable in any standard room. Thus, our study demonstrates that this method is a straightforward, specific, sensitive, point-of-care test (POCT) adaptable to various outlets such as customs, quarantine units and meat import/export departments.

Harmonisation of Pharmacopoeial Requirements for Identification of Closely Related Species in Herbal Medicinal Products

Global interest in herbal medicinal products is growing, thus increasing the demand for medicinal plant raw materials. However, the growth in sales of raw materials entails the risk of a wider spread of counterfeit and substandard medicinal products. The use of such products can lead to serious complications and even death. This brings to the forefront the studies aiming at identification and quality control of herbal drugs. In addition, the relevance of these studies stems from the need for harmonisation of test methods for identification of related plant species as acceptable foreign elements in herbal medicinal products. The aim of the study was to determine the most widely used methods for identification of closely related plant species and to analyse the possibility of aligning the requirements of Russian standards and foreign pharmacopoeial monographs. According to the analysis of identification requirements, the main test method for identification of related plant species is thin-layer chromatography; morphological and anatomical characteristics of plants are also used. Methodological approaches of the European Pharmacopoeia to identification of closely related species in herbal medicinal products have found application in updating compendial standards of the Russian Pharmacopoeia. Russian standards are harmonised with the requirements described in monographs of leading pharmacopoeias and provide for the detection of unacceptable impurities in herbal medicinal products by morphological, anatomical and chromatographic characteristics.

Resolution of housekeeping gene sequences used in MLSA for the genus Streptomyces and reclassification of Streptomyces anthocyanicus and Streptomyces tricolor as heterotypic synonyms of Streptomyces violaceoruber.

Although 16S rRNA gene sequences are conventionally analysed in bacterial systematics, their resolution is not sufficient for species identification. Multilocus sequence analysis (MLSA) is a powerful method for species identification as well as the elucidation of phylogenetic relationships in the genus Streptomyces. Gene sequences of atpD, gyrB, recA, rpoB and trpB are generally used in MLSA for Streptomyces species. This study aims to evaluate the sequence analysis of one gene instead of all five genes to be employed for species identification. The resolution of atpD gene sequences was not necessarily able to distinguish closely related species. In contrast, trpB gene sequence similarities correlated to the MLSA-based evolutionary distances, especially among closely related strains. A pairwise similarity of 97.9 % in trpB gene sequences was proposed as the threshold for species delineation based on the feasibility examined using strain pairs that shared >99.93 % pairwise 16S rRNA gene sequence similarities. Resequencing the five housekeeping genes followed by MLSA suggested that Streptomyces anthocyanicus and Streptomyces tricolor are synonyms of Streptomyces violaceoruber.

Interspecific and intraspecific analysis of Selinum spp. collected from Indian Himalayas using DNA barcoding

BackgroundDNA barcoding is a powerful method for phylogenetic mapping and species identification. However, recent research has come to a consistent conclusion about the universality of DNA barcoding. We used matK and rbcL markers to test the universality of twelve accessions from different locations belonging to two Selinum species, Selinum tenuifolium Wall. C. B. Clarke and Selinum vaginatum C. B. Clarke, keeping in mind their ability to identify species and establish phylogenetic relationships within and between the accessions. ResultsThe success rates of PCR amplification using matK and rbcL were 75.26% ± 3.65% and 57.24% ± 4.42%, and the rate of DNA sequencing was 63.84% ± 4.32% and 50.82% ± 4.36%, respectively, suggesting that success rates of species identification of the two fragments were higher than 41.00% (matK, 41.50% ± 2.81%; rbcL, 42.88% ± 2.59%), proving that these fragments might be used to identify species. The best evolutionary tree with good supporting values was produced utilizing combinations of matK + rbcL markers when phylogenetic relationships were built with random fragment combinations. The twelve accessions of Selinum collected from different locations and their molecular sequences of matK and rbcL markers were blasted with other genera of Apiaceae family, and it was found that Selinum is most closely related to Angelica species of Apiaceae family. ConclusionThe present study has grouped twelve accessions of Selinum species using molecular markers into phylogenies, which is first-of-its-kind report that established interrelationships within different species of Apiaceae with respect to Selinum. Graphical abstract▪

Clinico-bacteriological profile and antibiogram of Staphylococcus epidermidis with special emphasis on Methicillin resistance and hospital acquired infections in a tertiary care center south India

is a normal commensal of the skin and mucous membrane of humans and animals. Despite the growing importance of its pathogenesis especially in neonatal septicemia and device associated infections, it is still considered as insignificant isolate in the clinical practice. Hence, the present study is taken up to analyze the sources and risk factors of the isolates and to know their antibiogram along with occurrence of Methicillin resistant (MRSE).150 clinically significant isolates from various clinical specimens were considered in this study. Species identification was done by phenotypic methods. The antimicrobial susceptibility test and detection of Methicillin resistance were performed by Kirby-Bauer's disc diffusion method as per CLSI guidelines.Among 150 isolates, 78% were recovered from hospital acquired infections. They were commonly isolated in pediatric age group (30%) and among males (60.67%). Total of 34.67% were isolated from pus samples followed by blood (25.33%). Most of the isolates were associated with multiple risk factors like hospitalization, prior antibiotic administration, foreign body in situ and ICU admission. Majority of the isolates expressed resistance towards Penicillin (93.33%), followed by Amoxicillin-Clavulanic acid (76%), Cotrimoxazole (71.33%), Fluoroquinolones (64%), Gentamicin (60%) and Erythromycin (55.33%). Resistance against Amikacin (16.67%), Tetracycline (9.3%) and Linezolid (0.67%) was low. All isolates were sensitive to Vancomycin. Inducible Clindamycin resistance was 18% and MRSE was 68%.: Clinical importance and emergence of drug resistance among infections is growing with the advent of advanced medicine. This warrants the need to implement simple laboratory methods for species identification of the and to determine the antibiotic resistant patterns on routine basis. Clinical correlation of the isolate is crucial to rule out the colonizers and contaminants.

Exogenous Melatonin Activating Nuclear Factor E2-Related Factor 2 (Nrf2) Pathway via Melatonin Receptor to Reduce Oxidative Stress and Apoptosis in Antler Mesenchymal Stem Cells.

Antler growth depends on the proliferation and differentiation of mesenchymal stem cells (MSCs), and this process may be adversely affected by oxidative stress. Melatonin (MLT) has antioxidant functions, but its role in Cervidae remains largely unknown. In this article, flow cytometry, reactive oxygen species (ROS) identification, qPCR, and other methods were used to investigate the protective mechanism of MLT in H2O2-induced oxidative stress of antler MSCs. The results showed that MLT significantly increases cell viability by relieving the oxidative stress of antler MSCs. MLT inhibits cell apoptosis by protecting mitochondrial function. We blocked the melatonin receptor with luzindole (Luz) and found that the receptor blockade significantly increases H2O2-induced hyperoxide levels and causes significant inhibition of mitochondrial function. MLT treatment activates the nuclear factor E2-related factor 2 (Nrf2) antioxidant signaling pathway, up-regulates the expression of NAD(P)H quinone oxidoreductase 1 (NQO1) and other genes and it could inhibit apoptosis. In contrast, the melatonin receptor blockade down-regulates the expression of Nrf2 pathway-related genes, but significantly up-regulates the expression of apoptotic genes. It was indicated that MLT activates the Nrf2 pathway through the melatonin receptor and alleviates H2O2-induced oxidative stress and apoptosis in antler MSCs. This study provides a theoretical basis for further studying the oxidative stress and antioxidant process of antler MSCs and, thereby, increasing antler yields.

DNA barcodes and microsatellites: How they complement for species identification in the complex genus Tamarix (Tamaricaceae)

AbstractDNA barcoding allows the identification of an organism by comparing the sequence of selected DNA regions (barcodes) with a previously compiled database, and it can be useful for taxonomic identification of species in complex genera, such as Tamarix. Many species of this genus show convergent morphology, which leads to frequent errors in their identification. Highly variable genetic markers, such as microsatellites or short sequence repeats (SSR), could be used to differentiate species where DNA barcodes fail. Here, we tested the ability of both, 5 different marker regions (rbcL, matK, ITS, trnH‐psbA, and ycf1), and 14 microsatellites, to properly identify Tamarix species, especially those from the Mediterranean Basin, and compared the pros and cons of the different analytical methods for species identification. DNA barcoding allows the genetic identification of certain species in Tamarix. The two‐locus barcodes matK + ITS and ITS + ycf1 were the best‐performing combinations, allowing up to 69% and 70%, respectively, correct identification. However, DNA barcoding failed in phylogenetically close groups, such as many Mediterranean species. The use of SSR can aid the identification of species, and the combination of both types of data (DNA barcoding and SSR) improved the success. The combination of data was especially relevant in detecting the presence of hybridization processes, which are common in the genus. However, caution must be exercised when choosing the clustering methods for the SSR data since different methods can lead to very different results.

Identification of tuna species in raw and processed products using DNA mini-barcoding of the mitochondrial control region

Accurate species identification methods are needed to combat tuna fraud, improve tuna stock regulation, and mitigate health risks associated with mislabeled tuna products. The objective of this study was to conduct a market survey of raw and processed tuna products using a DNA mini-barcoding system based on the mitochondrial control region (CR). A total of 80 samples of raw, dried, and canned tuna products were collected at the retail level for CR mini-barcoding analysis. The samples underwent DNA extraction, polymerase chain reaction (PCR), and DNA sequencing of the 236-bp CR mini-barcode. The resulting sequences were searched against GenBank using the nucleotide Basic Local Alignment Search Tool (BLAST) to determine the species. The study achieved species identification for 100% of the raw samples, 95% of the dried samples, and 50% of the canned samples, for an overall success rate of 86% (69/80 samples). Mislabeling occurred in 11 of the identified samples (16%), including 8 products marketed as raw, dried, or canned yellowfin tuna, 2 samples marketed as dried or canned skipjack tuna, and 1 raw fillet sold as bluefin tuna. Overall, the DNA mini-barcoding system proved to be a promising method in identifying tuna species in both raw and processed samples. However, testing with a secondary marker is required in some cases to resolve instances of possible species introgression. Future research should explore optimization of this method for improved identification of canned tuna samples. • A total of 80 samples of raw, dried, and canned tuna products were tested. • Samples underwent DNA mini-barcoding of the mitochondrial control region. • PCR amplification was successful for 100% of samples. • Species identification was achieved for 86% of samples. • Mislabeling occurred in 16% of the identified samples.

A fast multi-source information fusion strategy based on deep learning for species identification of boletes

Wild mushroom market is an important economic source of Yunnan province in China, and its wild mushroom resources are also valuable wealth in the world. This work will put forward a method of species identification and optimize the method in order to maintain the market order and protect the economic benefits of wild mushrooms. Here we establish deep learning (DL) models based on the two-dimensional correlation spectroscopy (2DCOS) images of near-infrared spectroscopy from boletes, and optimize the identification effect of the model. The results show that synchronous 2DCOS is the best method to establish DL model, and when the learning rate was 0.01, the epochs were 40, using stipes and caps data, the identification effect would be further improved. This method retains the complete information of the samples and can provide a fast and noninvasive method for identifying boletes species for market regulators.

SSR Loci Analysis in Transcriptome and Molecular Marker Development in Polygonatum sibiricum.

To study the SSR loci information and develop molecular markers, a total of 435,858 unigenes in transcriptome of Polygonatum sibiricum were used to explore SSR. The distribution frequency of SSR and the basic characteristics of repeat motifs were analyzed using MISA software, and SSR primers were designed by Primer 3.0 software and then validated by PCR. Moreover, the gene function analysis of SSR Unigene was obtained by Blast. The results showed that 112,728 SSR loci were found in the transcriptome of Polygonatum sibiricum, which distributed in 435,858 unigenes with a distribution frequency of 25.86%. Mo-nucleotide and Di-nucleotide repeat were the main types, accounted for 83.83% of all SSRs. The repeat motifs of A/T and AC/GT were the predominant repeat types of Mo-nucleotide and Di-nucleotide, respectively. A total of 113,305 pairs of SSR primers with the potential to produce polymorphism were designed for maker development. One hundred and fifty-four of the 500 randomly selected primers not only produced fragments with expected molecular size but also had high polymorphism, which could accurately separate the tested varieties. The gene function of unigenes containing SSR was mostly related to the molecular function of Polygonatum sibiricum. The SSR markers in transcriptome of Polygonatum sibiricum show rich type, strong specificity, and high potential of polymorphism, which will benefit the candidate gene mining and marker-assisted breeding. The developed markers can also provide technical methods for molecular identification of intraspecific species of Polygonatum Mill. and maker-assisted breeding of superior varieties of Polygonatum Mill.

Benefits and limitations of a new genome-based PCR-RFLP genotyping assay (GB-RFLP): A SNP-based detection method for identification of species in extremely young adaptive radiations.

High‐throughput DNA sequencing technologies make it possible now to sequence entire genomes relatively easily. Complete genomic information obtained by whole‐genome resequencing (WGS) can aid in identifying and delineating species even if they are extremely young, cryptic, or morphologically difficult to discern and closely related. Yet, for taxonomic or conservation biology purposes, WGS can remain cost‐prohibitive, too time‐consuming, and often constitute a “data overkill.” Rapid and reliable identification of species (and populations) that is also cost‐effective is made possible by species‐specific markers that can be discovered by WGS. Based on WGS data, we designed a PCR restriction fragment length polymorphism (PCR‐RFLP) assay for 19 Neotropical Midas cichlid populations (Amphilophus cf. citrinellus), that includes all 13 described species of this species complex. Our work illustrates that identification of species and populations (i.e., fish from different lakes) can be greatly improved by designing genetic markers using available “high resolution” genomic information. Yet, our work also shows that even in the best‐case scenario, when whole‐genome resequencing information is available, unequivocal assignments remain challenging when species or populations diverged very recently, or gene flow persists. In summary, we provide a comprehensive workflow on how to design RFPL markers based on genome resequencing data, how to test and evaluate their reliability, and discuss the benefits and pitfalls of our approach.

Optimization of cerebrospinal fluid microbial DNA metagenomic sequencing diagnostics

Infection in the central nervous system is a severe condition associated with high morbidity and mortality. Despite ample testing, the majority of encephalitis and meningitis cases remain undiagnosed. Metagenomic sequencing of cerebrospinal fluid has emerged as an unbiased approach to identify rare microbes and novel pathogens. However, several major hurdles remain, including establishment of individual limits of detection, removal of false positives and implementation of universal controls. Twenty-one cerebrospinal fluid samples, in which a known pathogen had been positively identified by available clinical techniques, were subjected to metagenomic DNA sequencing. Fourteen samples contained minute levels of Epstein-Barr virus. The detection threshold for each sample was calculated by using the total leukocyte content in the sample and environmental contaminants found in the bioinformatic classifiers. Virus sequences were detected in all ten samples, in which more than one read was expected according to the calculations. Conversely, no viral reads were detected in seven out of eight samples, in which less than one read was expected according to the calculations. False positive pathogens of computational or environmental origin were readily identified, by using a commonly available cell control. For bacteria, additional filters including a comparison between classifiers removed the remaining false positives and alleviated pathogen identification. Here we show a generalizable method for identification of pathogen species using DNA metagenomic sequencing. The choice of bioinformatic method mainly affected the efficiency of pathogen identification, but not the sensitivity of detection. Identification of pathogens requires multiple filtering steps including read distribution, sequence diversity and complementary verification of pathogen reads.

Rapid and accurate identification of pathogenic bacteria at the single-cell level using laser tweezers Raman spectroscopy and deep learning.

We report a new method for the rapid identification of pathogenic bacterial species at the single-cell level that combines laser tweezers Raman spectroscopy (LTRS) with deep learning (DL). LTRS can accurately measure single-cell Raman spectra (scRS) without destroying and labeling cells. Based on the scRS data, DL rapidly and accurately identifies pathogenic bacteria. We measured scRS of 15 species bacteria using homemade LTRS. For each species, approximately, 160 cells from three different patients were measured, one patient's data were used as test set, and the rest after being augmented was used as training set. A residual network (ResNet) model, trained on the augmented training set, achieved an accuracy of 94.53% on the test set. Moreover, we applied gradient-weighted class activation mapping to visualize the proposed model. Finally, we demonstrated the advantages of ResNet over traditional machine-learning algorithms.

Chloroplast Genome Evolution and Species Identification of Styrax (Styracaceae).

The genus Styrax L. consists of approximately 130 species distributed in the Americas, eastern Asia, and the Mediterranean region. The phylogeny and evolutionary history of this genus are not clear. Knowledge of the phylogenetic relationships and the method for species identification will be critical for the evolution of this genus. In this study, we sequenced the chloroplast genome of 17 Styrax samples and added 17 additional chloroplast genome sequences from GenBank. The data were used to investigate chloroplast genome evolution, infer phylogenetic relationships, and access the species identification rate within Styrax. The Styrax chloroplast genome contains typical quadripartite structures, ranging from 157,641 bp to 159,333 bp. The chloroplast genome contains 114 unique genes. The P distance among the Styrax species ranged from 0.0003 to 0.00611. Seventeen small inversions and SSR sites were discovered in the Styrax chloroplast genome. By comparing with the chloroplast genome sequences, six mutation hotspots were identified, and the markers ycf1b and trnT-trnL were identified as the best Styrax-specific DNA barcodes. The specific barcodes and superbarcode exhibited higher discriminatory power than universal barcodes. Chloroplast phylogenomic results improved the resolution of the phylogenetic relationships of Styrax compared to previous analyses.

Comparative study of DNA extraction to initiate harmonized protocol for a simple method of species identification: Fresh and canned Tuna case study

ABSTRACT Seven different methods of DNA extraction were performed and compared in order to have the better DNA recovery in terms of quality and quantity. A PCR assay was realised based on the Cytb mitochondrial gene to identify 53 commercial food products of wide range diversity (Scombridae Thunnus species) commercialised in the Tunisian market, and we checked whether these products were correctly labelled under European and national legislations. Phylogenetic analyses on Cytb mitochondrial gene were used to study the relationships among the considered species. Tuna species was easily differentiated and confirmed by direct sequencing. The results showed a high deficiency in labelling: 96% of canned tuna did not specify the species, although the name of the species had to be mentioned. Partial least squares regression (PLS) showed relatively high values (45%) in all analysed data, suggesting substantial difference between the extraction procedures and matrices with SDS/PCI method being the most convenient.

One Day in Denmark: Nationwide point-prevalence survey of human bacterial isolates and comparison of classical and whole-genome sequence-based species identification methods.

ObjectivesImplementing whole-genome sequencing (WGS) technologies in clinical microbiology laboratories can increase the amount and quality of information available for healthcare practitioners. In this study, we analysed the applicability of this method and determined the distribution of bacterial species processed in clinical settings in Denmark.MethodsWe performed a point-prevalence study of all bacterial isolates (n = 2,009) processed and reported in the Clinical Microbiology Laboratories in Denmark in one day in January 2018. We compared species identification as performed by classical methods (MALDI-TOF) and by bioinformatics analysis (KmerFinder and rMLST) of WGS (Illumina NextSeq) data. We compared the national point-prevalence of bacterial isolates observed in clinical settings with the research attention given to those same genera in scientific literature.ResultsThe most prevalent bacterium was Escherichia coli isolated from urine (n = 646), followed by Staphylococcus spp. from skin or soft tissues (n = 197). The distribution of bacterial species throughout the country was not homogeneous. We observed concordance of species identification for all methods in 95.7% (n = 1,919) of isolates, furthermore obtaining concordance for 99.7% (n = 1,999) at genus level. The number of scientific publications in the country did not correlate with the number of bacterial isolates of each genera analysed in this study.ConclusionsWGS technologies have the potential to be applied in clinical settings for routine diagnostics purposes. This study also showed that bioinformatics databases should be continuously improved and results from local point-prevalence surveys should not be applied at national levels without previously determining possible regional variations.

Rapid identification of Amanita citrinoannulata poisoning using colorimetric and real-time fluorescence and loop-mediated isothermal amplification (LAMP) based on the nuclear ITS region

Health concerns and financial losses caused by mushroom poisoning have been reported worldwide. Amanita citrinoannulata, a poisonous mushroom commonly found in China, results in a toxic reaction in humans after mistaken ingestion. To reduce the mistaken ingestion of poisonous mushrooms and to improve clinical diagnosis of mushroom poisoning, a rapid mushroom species identification method is required. Such identification methods could be advantageous in the identification of other poisonous mushroom species. This study developed two rapid and sensitive methods for the detection of A. citrinoannulata utilizing colorimetric and real-time loop-mediated isothermal amplification (LAMP) technology and specifically designed primers for the internal transcribed spacer (ITS) genes of A. citrinoannulata. The methods demonstrated high sensitivity as 0.2 ng of A. citrinoannulata DNA could be detected, with no cross-reaction with 41 non-target mushroom species. The entire detection process could be completed within 40 min without requiring complex instruments and can be observed by the naked eye. Therefore, these novel methods can be used for the identification of fresh and cooked mushroom samples and vomit samples, which contain only 1% A. citrinoannulata. Furthermore, these methods facilitate the detection of mushroom poisoning, and thus, have potential to reduce the number of mushroom poisoning-related deaths worldwide.