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In the present study, we combined morphological and phylogenetic methods to characterize Myxobolus pseudonobilis n. sp. infecting Hypophthalmichthys molitrix Valenciennes, 1844 from Chongqing, China. The morphology and molecular characteristics of M. pseudonobilis n. sp. were distinct from those of other previously described Myxobolus species. Mature myxospores were ovoid in frontal view with spore dimensions of 10.0 ± 0.4 (9.3-10.9) μm in length and 8.5 ± 0.2 (7.9-9.0) μm in width. Two polar capsules occupying approximately half of the myxospore length were unequal in size. The larger polar capsule containing 6 to 7 filament coils measured 5.2 ± 0.3 (4.5-5.8) μm in length and 3.6 ± 0.2 (3.2-3.9) μm in width, while the smaller capsule with 4 to 5 filament coils measured 3.9 ± 0.3 (3.0-4.4) μm in length and 2.5 ± 0.3 (2.1-3.6) μm in width. The comparison of molecular characteristics demonstrated similarities and genetic distances of 18S rDNA sequences of 95.19% - 98.20% and 1.82% - 5.46%, respectively, between M. pseudonobilis n. sp. and its morphologically similar species, and secondary structures were also distinctly different. Moreover, phylogenetic analysis showed that M. pseudonobilis n. sp. was clustered with other myxobolids possessing spores with a blunt anterior end and branched independently. In addition, the morphology of myxosporeans as an important indicator was discussed., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Acanthocephalans of the order Echinorhynchida are one of the most diverse groups in their phylum, with approximately 470 species classified into 11 families that largely consist of parasites of freshwater, brackish and marine fishes and, sporadically, reptiles and amphibians distributed worldwide. Previous phylogenies inferred with molecular data have supported the paraphyly or polyphyly of some families, suggesting that most of them have been diagnosed based on unique combinations of characters, rather than shared derivative features. We expand the taxonomic sampling of several genera such as Acanthocephalus, Echinorhynchus and Pseudoacanthocephalus of Echinorhynchidae from diverse biogeographical zones in the Americas, Europe and Asia with the aim of testing the monophyly of the family by using two molecular markers. Sequences from small (SSU) and large (LSU) subunits of ribosomal DNA were obtained for six species representing the genera Acanthocephalus and Echinorhynchus from the Neotropical, Nearctic, Palearctic and Oriental regions. These sequences were aligned with other sequences available in the GenBank dataset from Echinorhynchidae. Phylogenetic trees inferred with the combined (SSU + LSU) and the individual data sets consistently placed the genera Acanthocephalus, Pseudoacanthocephalus and Echinorhynchus into three independent lineages. Two families, Paracanthocephalidae Golvan, 1960, and Pseudoacanthocephalidae Petrochenko, 1956, were resurrected to accommodate the genera Acanthocephalus and Pseudoacanthocephalus, respectively. The species of the genus Acanthocephalus from the Nearctic, Palearctic and Oriental biogeographic regions formed a clade that was well supported. However, Acanthocephalus amini from the Neotropical region was nested inside Arhythmacanthidae. Therefore, the genus Calakmulrhynchus was created to accommodate A. amini and resolve the paraphyly of Acanthocephalus. Finally, the diagnoses of the families Echinorhynchidae and Arhythmacanthidae were amended. The molecular phylogenies should be used as a taxonomic framework to find shared derived characters (synapomorphies) and build a more robust classification scheme that reflects the evolutionary history of the acanthocephalans., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Nephridiophagids are unicellular eukaryotes that parasitize the Malpighian tubules of numerous insects. Their life cycle comprises multinucleate vegetative plasmodia that divide into oligonucleate and uninucleate cells, and sporogonial plasmodia that form uninucleate spores. Nephridiophagids are poor in morphological characteristics, and although they have been tentatively identified as early-branching fungi based on the SSU rRNA gene sequences of three species, their exact position within the fungal tree of live remained unclear. In this study, we describe two new species of nephridiophagids (Nephridiophaga postici and Nephridiophaga javanicae) from cockroaches. Using long-read sequencing of the nearly complete rDNA operon of numerous further species obtained from cockroaches and earwigs to improve the resolution of the phylogenetic analysis, we found a robust affiliation of nephridiophagids with the Chytridiomycota-a group of zoosporic fungi that comprises parasites of diverse host taxa, such as microphytes, plants, and amphibians. The presence of the same nephridiophagid species in two only distantly related cockroaches indicates that their host specificity is not as strict as generally assumed.

Leishmaniasis is a worldwide neglected disease, encompassing asymptomatic infections and different clinical forms, such as American Tegumentary Leishmaniasis (ATL) which is part of the complex of diseases caused by protozoan parasites from Leishmania genus, transmitted by sand fly vectors. As a neglected disease, much effort is still needed in treatment and diagnosis. Currently, ATL diagnosis is mainly made by parasite detection by microscopy. The sensitivity of the method varies, and factors such as collection procedures interfere. Molecular approaches, specially based on Real Time PCR (qPCR) technique, has been widely used to detect Leishmania infection and to quantify parasite load, once it is a simple, rapid and sensitive methodology, capable to detect low parasite concentrations and less prone to variability. Although many studies have been already published addressing the use of this technique, an improvement on these methodologies, including an analytical validation, standardization and data association is demanded. Moreover, a proper validation by the assay by the use of clinical samples is still required. In this sense, the purpose of the present work is to compare the performance of qPCR using two commonly used targets (18S rDNA and HSP70) with an internal control (RNAse P) in multiplex reactions. Additionally, we validated reactions by assaying 88 samples from patients presenting different clinical forms of leishmaniasis (cutaneous, mucosal, recent and old lesions), representing the diversity found in Brazil's Amazon Region. Following the methodology proposed herein, the results indicate the use of both qPCR assays, 18S rDNA and HSP70, to achieve a very good net sensitivity (98.5%) and specificity (100%), performing simultaneous or sequential testing, respectively. With this approach, our main goal is to conclude the first step of a further multicenter study to propose the standardization of detection and quantification of Leishmania., Competing Interests: The authors have declared that no competing interests exist.

Sculpin fishes belonging to the family Cottidae represent a large and complex group, inhabiting a wide range of freshwater, brackish-water, and marine environments. Numerous studies based on analysis of their morphology and genetic makeup frequently provided controversial results. In the present work, we sequenced complete mitochondrial (mt) genomes and fragments of nuclear ribosomal DNA (rDNA) of the fourhorn sculpin Myoxocephalus quadricornis and some related cottids to increase the power of phylogenetic and taxonomic analyses of this complex fish group. A comparison of the My. quadricornis mt genomes obtained by us with other complete mt genomes available in GenBank has revealed a surprisingly low divergence (3.06 ± 0.12%) with Megalocottus platycephalus and, at the same time, a significantly higher divergence (7.89 ± 0.16%) with the species of the genus Myoxocephalus . Correspondingly, phylogenetic analyses have shown that My. quadricornis is clustered with Me. platycephalus but not with the Myoxocephalus species. Completely consistent patterns of divergence and tree topologies have been obtained based on nuclear rDNA. Thus, the multi-gene data in the present work indicates obvious contradictions in the relationships between the Myoxocephalus and Megalocottus species studied. An extensive phylogenetic analysis has provided evidence for a closer affinity of My. quadricornis with the species of the genus Megalocottus than with the species of the genus Myoxocephalus . A recombination analysis, along with the additional GenBank data, excludes introgression and/or incorrect taxonomic identification as the possible causative factors responsible for the observed closer affinity between the two species from different genera. The above facts necessitate realignment of the genera Myoxocephalus and Megalocottus . The genetic data supports the two recognized genera, Myoxocephalus and Megalocottus , but suggests changing their compositions through transferring My. quadricornis to the genus Megalocottus . The results of the present study resolve the relationships within a complex group of sculpin fishes and show a promising approach to phylogenetic systematics (as a key organizing principle in biodiversity research) for a better understanding of the taxonomy and evolution of fishes and for supplying relevant information to address various fish biodiversity conservation and management issues.

This paper, we introduced Sub-terahertz (Sub-THz) technology to identify nematode DNA sequence. First, data mining technology and restriction enzyme digestion were used to cut out two corresponding sequences, each containing about 100 base pairs that could represent the characteristic fragments of Bursaphelenchus xylophilus (Bx) and Bursaphelenchus mucronatus (Bm) rDNA in internal transcribed spacer 1 (ITS1) region. Then, vibration spectra of the two enzyme-cut sequences were measured by Fourier transform infrared spectroscopy (FTIR). Meanwhile, the spectrum was analyzed by molecular dynamics method. It was found that the calculated and experimental spectra of the two enzyme-cut sequences were consistent, although the differences of the sequences could not be well reflected in the spectra. The vibration modes corresponding to diverse absorption peaks in the spectra were quite different, which were closely related to the internal bases sequencing. This can be used as an indicator for identifying Bx and Bm DNA. Moreover, the normal mode analysis (NMA) method was first adopted for spectral attribution analysis of DNA long sequences. Finally, the vibration spectra of shorter sequences predicted by second-order Markov chains and Monte Carlo method were studied. To some extent, the predicted short sequences can represent the complete sequence as the initial calculation structure., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Repeated quantitative measurement of bacterial DNA on whole blood has been shown to be a promising method for monitoring bloodstream infection (BSI) with selected bacterial species. To enable broad use of this method, we developed a quantitative droplet digital PCR (ddPCR) method for 16S rDNA. It was validated with species-specific ddPCRs for Staphylococcus aureus (nuc), Streptococcus pneumoniae (lytA), and Escherichia coli (uidA) on spiked whole blood samples and on repeated whole blood samples (days 0, 1-2, 3-4, 6-8, and 13-15) from 83 patients with BSI with these pathogens. In these patients, 16S rDNA and species-specific DNA were detected in 60% and 61%, respectively, at least at one time-point. The highest positivity rates were seen in S. aureus BSI, where 92% of the patients were 16S rDNA-positive and 85% nuc-positive. Quantitative 16S rDNA and species-specific DNA showed strong correlations in spiked samples (r = 0.98; p < 0.0001) and clinical samples (r = 0.84; p < 0.0001). Positivity for 16S rDNA was rapidly cleared in patients with S. pneumoniae and E. coli BSI, but more slowly and sometimes persisted, in those with S. aureus BSI. The initial 16S rDNA load was higher in BSI patients with sepsis (Sepsis-3 definition) than without sepsis (median 2.38 vs. 0 lg10 copies/mL; p = 0.031) and in non-survivors than in survivors (median 2.83 vs. 0 lg10 copies/mL; p = 0.006). 16S rDNA ddPCR appears to be a promising method for bacterial DNA monitoring during BSI. The clinical value of such monitoring should be further studied., Competing Interests: The authors have declared that no competing interests exist.

Household contacts (HHC) of leprosy patients exhibit high-risk of developing leprosy and contact tracing is helpful for early diagnosis. From 2011 to 2018,2,437 HHC were examined in a clinic in Rio de Janeiro, Brazil and 16S qPCR was used for diagnosis and monitoring of contacts. Fifty-four HHCs were clinically diagnosed with leprosy at intake. Another 25 exhibited leprosy-like skin lesions at intake, 8 of which were confirmed as having leprosy (50% of which were qPCR positive) and 17 of which were diagnosed with other skin diseases (6% qPCR positive). In skin biopsies, qPCR presented a sensitivity of 0.50 and specificity of 0.94. Furthermore, 955 healthy HHCs were followed-up for at least 3 years and skin scrapings were collected from earlobes for qPCR detection. Positive qPCR indicated a non-significant relative risk of 2.52 of developing the disease. During follow-up, those who progressed towards leprosy exhibited 20% qPCR positivity, compared to 9% of those who remained healthy. Disease-free survival rates indicated that age had a significant impact on disease progression, where patients over 60 had a greater chance of developing leprosy [HR = 32.4 (3.6-290.3)]. Contact tracing combined with qPCR may assist in early diagnosis and age is a risk factor for leprosy progression.

The ciliate genus Spirostomum comprises eight morphospecies, inhabiting diverse aquatic environments worldwide, where they can be used as water quality indicators. Although Spirostomum species are relatively easily identified using morphological methods, the previous nuclear rDNA-based phylogenies indicated several conflicts in morphospecies delineation. Moreover, the single locus phylogenies and previous analytical approaches could not unambiguously resolve phylogenetic relationships among Spirostomum morphospecies. Here, we attempt to investigate species boundaries and evolutionary history of Spirostomum taxa, using 166 new sequences from multiple populations employing one mitochondrial locus (CO1 gene) and two nuclear loci (rRNA operon and alpha-tubulin gene). In accordance with previous studies, relationships among the eight Spirostomum morphospecies were poorly supported statistically in individual gene trees. To overcome this problem, we utilised for the first time in ciliates the Bayesian coalescent approach, which accounts for ancestral polymorphisms, incomplete lineage sorting, and recombination. This strategy enabled us to robustly resolve deep relationships between Spirostomum species and to support the hypothesis that taxa with compact macronucleus and taxa with moniliform macronucleus each form a distinct lineage. Bayesian coalescent-based delimitation analyses strongly statistically supported the traditional morphospecies concept but also indicated that there are two S. minus-like cryptic species and S. teres is non-monophyletic. Spirostomum teres was very likely defined by a set of ancestral features of lineages that also gave rise to S. yagiui and S. dharwarensis. However, molecular data from type populations of the morphospecies S. minus and S. teres are required to unambiguously resolve the taxonomic problems.

Extracellular DNA (exDNA) pool in aquatic environments is a valuable source for biomonitoring and bioassessment. However, degradation under particular environmental conditions can hamper exDNA detectability over time. In this study, we analyzed how different biotic and abiotic factors affect the degradation rate of extracellular environmental DNA using 16S rDNA sequences extracted from the sediment of a eutrophic lake and Anabaena variabilis cultured in the laboratory. We exposed the extracted exDNA to different levels of temperature, light, pH, and bacterial activity, and quantitatively analyzed the concentration of exDNA during 4 days. The solution containing bacteria for microbial activity treatment was obtained from the lake sediment using four consecutive steps of filtration; two mesh filters (100 μm and 60 μm mesh) and two glass fiber filters (2.7 μm and 1.2 μm pore-sized). We found that temperature individually and in combination with bacterial abundance had significant positive effects on the degradation of exDNA. The highest degradation rate was observed in samples exposed to high microbial activity, where exDNA was completely degraded within 1 day at a rate of 3.27 day -1 . Light intensity and pH had no significant effects on degradation rate of exDNA. Our results indicate that degradation of exDNA in freshwater ecosystems is driven by the combination of both biotic and abiotic factors and it may occur very fast under particular conditions.

Tuberculosis caused by Mycobacterium tuberculosis (M. tuberculosis) remains one of the most serious infectious diseases all over the world. The key to reduce the spread and mortality rate of tuberculosis is to develop faster and more sensitive approaches for detection of M. tuberculosis. However, current detection methods can not meet the requirements of rapid clinical M. tuberculosis detection in terms of detection time. Herein, a new 16S rDNA multichannel series piezoelectric quartz crystal (MSPQC) sensor based on Exonuclease III (Exo III)-aided target recycling has been developed for rapid detection of M. tuberculosis. The specific 16S rDNA fragment of M. tuberculosis was used as biomarker, DNA capture probes complementary to the biomarker were designed and modified on the surface of AuNPs. The Exo III which could recognise hybrid duplexes and selectively digest DNA capture probe was used to assist digestion cycle by digesting DNA capture probe and releasing the intact target fragment. After all DNA probes loading on the surface of AuNPs were removed, the surface of AuNPs was exposed and conductive connection was formed between the nanogap network electrode by self-catalytic growth of exposed AuNPs in the glucose and HAuCl 4 solution. This resulted in sensitive response of M. tuberculosis sensor and M. tuberculosis was detected by recording this response. The limit of detection (LOD) of the method was 20 CFU/mL and the detection time was less than 3 h. It was expected to be widely used in detection methods of M. tuberculosis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Torymus sinensis Kamijo (Hymenoptera: Torymidae) is an alien parasitoid that is used in many areas of the world for biological control the Asian chestnut gall wasp, Dryocosmus kuriphilus Yasumatsu (Hymenoptera: Cynipidae). In Italy, this parasitoid was imported from Japan in 2003 and subsequently multiplied and released throughout the country. In this study, a phylogenetic investigation was carried out on insects from three different sites in northern Tuscany (Italy). Moreover, the possible hybridization between T. sinensis and some native Torymus species was evaluated. The conserved region 18S rRNA gene and the hypervariable ITS2 (Internal Transcribed Spacer 2) region of the ribosomal cistrone were selected as molecular markers. Sequencing the amplified products, after cloning, ruled out any hybridization between T. sinensis and the native Torymus species, and also confirmed the presence of two haplotypes for the Tuscan population of T. sinensis both for the region of the 18S rRNA gene as well as for the ITS2 region. These results confirm that the environmental impact of the alien parasitoid T. sinensis in the study site is acceptable, although an extensive and repeated monitoring would be desirable., (© The Author(s) 2019. Published by Oxford University Press on behalf of Entomological Society of America.)

In the presented work identification of microorganisms isolated from various types of honeys was performed. Martix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and 16S rDNA sequencing were applied to study environmental bacteria strains.With both approches, problematic spore-forming Bacillus spp, but also Staphylococcus spp., Lysinibacillus spp., Micrococcus spp. and Brevibacillus spp were identified. However, application of spectrometric technique allows for an unambiguous distinction between species/species groups e.g.B. subtilis or B. cereus groups. MALDI TOF MS and 16S rDNA sequencing allow for construction of phyloproteomic and phylogenetic trees of identified bacterial species. Furthermore, the correlation beetween physicochemical properties, geographical and botanical origin and the presence bacterial species in honey samples were investigated., Competing Interests: The authors have read the journal's policy and the authors of this manuscript have the following competing interests: MK is a paid employee of the Bruker Daltonik GmbH company. Bruker Daltonik GmbH company was not an additional funder of the study nor had influence on the study design or interpretation. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

This study evaluated the feasibility of using a silicon carbide (SiC) anaerobic ceramic membrane bioreactor (AnCMBR) to co-manage domestic wastewater (DWW) and food waste recycling wastewater (FRW). A pilot-scale SiC-AnCMBR was put into operation for 140 days under two different organic loading rates (OLRs): 5 kg COD m -3  d -1 (OLR 5) and 3 kg COD m -3  d -1 (OLR 3). The organic removal efficiency was 93.5 ± 3.7% over the operational period. Methane production increased significantly after sludge re-seeding at OLR 3. rDNA and rRNA microbial results showed that the active archaeal community was affected by sludge re-seeding, whereas the active bacterial community was not, indicating that a shift in the active archaeal community was responsible for the increased methane production. Our results thus suggest that SiC-AnCMBRs are a promising option for co-managing DWW and FRW., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Premise of the Study: Polyploidy has been long recognized as an important force in plant evolution. Previous studies had suggested widespread occurrence of polyploidy and the allopolyploid origin of several species in the diverse neotropical genus Lachemilla (Rosaceae). Nonetheless, this evidence has relied mostly on patterns of cytonuclear discordance, and direct evidence from nuclear allelic markers is still needed., Methods: Here we used PCR target enrichment in combination with high throughput sequencing to obtain multiple copies of the nuclear ribosomal (nr) DNA cistron and 45 regions of the plastid genome (cpDNA) from 219 accessions representing 48 species of Lachemilla and to explore the allopolyploid origin of species in this group., Key Results: We were able to identify multiple nrDNA ribotypes and establish clear evidence of allopolyploidy in 33 species of Lachemilla, showing that this condition is common and widespread in the genus. Additionally, we found evidence for three autopolyploid species. We also established multiple, independent origins of several allopolyploid species. Finally, based solely on the cpDNA phylogeny, we identified that the monotypic genus Farinopsis is the sister group of Lachemilla and allied genera within subtribe Fragariinae., Conclusions: Our study demonstrates the utility of the nuclear ribosomal DNA cistron to detect allopolyploidy when concerted evolution of this region is not complete. Additionally, with a robust chloroplast phylogeny in place, the direction of hybridization events can be established, and multiple, independent origins of allopolyploid species can be identified., (© 2019 Botanical Society of America.)

Objectives: This study genotyped oral isolates of Candida albicans and C. dubliniensis by analyzing 25S rDNA transposable intron and evaluated their virulence attributes in oral candidiasis., Design: C. albicans and C. dubliniensis were isolated from oral cavity of normal carriers (n = 100) and oral candidiasis patients (n = 100), genotyped by PCR, and virulence properties, namely, secreted phospholipase and proteinase activities (using an agar plate method) and binding to buccal epithelial cells, were determined. In addition, antifungal sensitivity was assayed for all Candida isolates., Results: C. albicans genotypes A, B, C and D (C. dubliniensis) were identified. Genotype B was the most prevalent in both healthy and candidiasis groups and had highest buccal epithelial cell binding ability but lowest secreted phospholipase activity. Genotype C was the third most prevalent, with higher frequency in patients than normal carriers. Genotype A, the second most prevalent, was equally found in both groups. There were no significant differences in secreted proteinase activity among the three C. albicans genotypes. C. dubliniensis, the least prevalent, was more frequent in healthy carriers and demonstrated minimal levels of the virulence properties. When all Candida isolates were compared based on groups of subjects, only secreted phospholipase activity was significantly higher in isolates from candidiasis patients. All Candida isolates were susceptible to clotrimazole, fluconazole, miconazole and nystatin., Conclusions: Genotyping based on the 25S rDNA transposable intron region provided a simple method allowing studies of the pathogenicity of each genotype., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Previous studies for enumerating fungal communities on pine needles relied entirely on phenotypic diversity (microscopy) or identification based on DNA sequence data from those taxa recovered via cultural studies. To bypass limitations of the culturing methods and provide a more realistic diversity estimate, we employed and assessed a PCR-DGGE based method coupled with rDNA phylogenetic sequence analyses to characterize fungal taxa associated with pine needles. Fresh (living) and decayed needles from three hosts of the Pinaceae (Keteleeria fortunei, Pinus elliottii and P. massoniana) were examined. Morphological studies reveal that the most abundant species associated with decayed needles were Cladosporium cladosporioides and an unidentified Trichoderma species followed by Gliocephalotrichum sp., Gliocladium sp., Lophodermium pinastri, Paecilomyces varioti, Phaeostalagmus cyclosporus and a Phoma sp, which are commonly occurring fungi. Community genomic data from freshly collected and decayed pine needles recovered 40 operational taxonomic units, which appear to be mostly undetected members of the natural fungal consortium. Sequence analyses revealed a number of phylotypes or "species" that were not recovered using traditional morphological and cultural approaches previously used. Phylogenetic data from partial 18S rDNA sequence data reveal that most phylotypes represent potential novel phylogenetic fungal lineages with affinities to the Dothideomycetes, Leotiomycetes, Lecanoromycetes and Sordariomycetes and were not identical to previously known endophytes or saprobes. Although the major ecological roles of these phylotypes in pine needles are still enigmatic, this study provides new insights in hidden fungal diversity that mycologists are possibly ignoring given the discrepancies associated with available methods. To what extent do previously recovered identified species (either as saprobes or endophytes) from morphological or culturing studies act as pioneer decomposers or constitute an integral part of endophytic community warrants further investigation.

Sequencing and classification of microbial taxa within forensically relevant biological fluids has the potential for applications in the forensic science and biomedical fields. The quantity of bacterial DNA from human samples is currently estimated based on quantity of total DNA isolated. This method can miscalculate bacterial DNA quantity due to the mixed nature of the sample, and consequently library preparation is often unreliable. We developed an assay that can accurately and specifically quantify bacterial DNA within a mixed sample for reliable 16S ribosomal DNA (16S rDNA) library preparation and high throughput sequencing (HTS). A qPCR method was optimized using universal 16S rDNA primers, and a commercially available bacterial community DNA standard was used to develop a precise standard curve. Following qPCR optimization, 16S rDNA libraries from saliva, vaginal and menstrual secretions, urine, and fecal matter were amplified and evaluated at various DNA concentrations; successful HTS data were generated with as low as 20 pg of bacterial DNA. Changes in bacterial DNA quantity did not impact observed relative abundances of major bacterial taxa, but relative abundance changes of minor taxa were observed. Accurate quantification of microbial DNA resulted in consistent, successful library preparations for HTS analysis., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objectives: To identify the drop-off location of victims in drowning cases, and confirm whether it is a fatal drowning or the victim is thrown into the water after death by detecting part of 5.8S sequence and second internal transcribed spacer （ITS2） （5.8S+ITS2） of diatom rDNA in water and organs., Methods: Two cases identified by diatom examination, which received by Nanjing Municipal Public Security Bureau Forensic Center, were taken as the research objects. The difference of the population structure of algae in water and human tissue was analysed by length polymorphism of 5.8S+ITS2 marker., Results: In case 1, similar species of diatom were detected from victim's lung and liver tissues and the water sample. Two kinds of DNA fragments with length of 330 bp and 376 bp were detected from victim's lung tissue and the water sample using 5.8S+ITS2 marker, which could confirm the victim was drowning before death. In case 2, there was no diatom found in victim's lung and liver tissues. Only one kind of DNA fragment with length of 331 bp and low relative fluorescence unit （RFU） was obtained from victim's lung tissue using 5.8S+ITS2 marker, thus the victim was thrown into the water after death., Conclusions: The experimental results of the two cases in present study are consistent with the actual facts and the result of the diatom microscopic examination. The difference of population structure of specific microorganism in water and human tissue can be detected by 5.8S+ITS2 marker, which can help to identify the drop-off location of victims in drowning cases, and confirm whether it is a fatal drowning or the victim is thrown into the water after death., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Editorial Department of Journal of Forensic Medicine.)

Background: Candida albicans, Candida glabrata, and Candida parapsilosis are three prevalent causes of candidiasis, worldwide. These species are considered as nine medically important complex species. Limited knowledge about these newly recognized species prompted us to develop a one-step, multiplex PCR to detect and identify them in clinical settings., Methods: Primers targeting Hyphal Wall Protein I gene for the C. albicans, C. dubliniensis, C. africana, Intergenic Spacer for the C. glabrata, C. nivariensis, C. bracarensis, and Intein and ITS rDNA for the C. parapsilosis, C. orthopsilosis, and C. metapsilosis were designed. Using 168 CBS reference strains and 280 clinical isolates, the specificity and reproducibility of the developed assay were evaluated., Results: Our developed assay successfully identified and distinguished all the nine species. No cross-reaction with closely- and distantly-related yeast species, Aspergillus species and human DNA was observed, resulting in 100% specificity. The ambiguous results obtained by MALDI-TOF for C. albicans and C. africana were corrected by our 9-plex PCR assay. This assay identified all the cryptic complex species from two test sets from Iran and China, correctly., Conclusions: Our developed multiplex assay is accurate, specific, cost/time-saving, and works without the tedious DNA extraction steps. It could be integrated into routine clinical laboratories and as a reliable identification tool and has the potential to be implemented into epidemiological studies to broaden the limited knowledge of cryptic species complexes.

Objective: The genetic data related to Turkish ticks is limited. Therefore, this study was aimed to perform of the molecular characterization based on 16S rDNA gene region of some ixodid tick species in Turkey., Methods: The molecular characterizations of partial 16S rDNA gene region belonging to 17 individual ixodid tick species in Turkey have been made and their phylogenetic relationships based on the relevant gene region have been analysed., Results: The 16S rDNA phylogenetic relationships between the isolates obtained from this study and the homolog isolates have been revealed. The 16S rDNA data belonging to Ixodes laguri has been obtained for the first time and its phylogenetic location has been demonstrated. Original genetic data related to Haemaphysalis inermis, Ha. parva and Ha. sulcata, which have a limited number of genetic information, has been obtained. Besides, the new genetic data belonging to Dermacentor marginatus, D. reticulatus, Hyalomma anatolicum, H. detritum, Ha. inermis, Ha. parva, Ha. punctata, Ha. sulcata and I. laguri has been recorded for the first time from Turkey., Conclusion: The molecular phylogenetic characterization based on 16S rDNA gene region of 17 ixodid tick species in Turkey has been performed. The obtained data has made a significant contribution to the genetic knowledge of the mentioned ticks species.

The rising incidence of invasive fungal infections and the expanding spectrum of fungal pathogens makes early and accurate identification of the causative pathogen a daunting task. Diagnostics using molecular markers enable rapid identification of fungi, offer new insights into infectious disease dynamics, and open new possibilities for infectious disease control and prevention. We performed a retrospective study using clinical specimens (N = 233) from patients with suspected fungal infection previously subjected to culture and/or internal transcribed spacer (ITS) PCR. We used these specimens to evaluate a high-throughput screening method for fungal detection using automated DNA extraction (QIASymphony), fungal ribosomal small subunit (18S) rDNA RT-PCR and amplicon sequencing. Fungal sequences were compared with sequences from the curated, commercially available SmartGene IDNS database for pathogen identification. Concordance between 18S rDNA RT-PCR and culture results was 91%, and congruence between 18S rDNA RT-PCR and ITS PCR results was 94%. In addition, 18S rDNA RT-PCR and Sanger sequencing detected fungal pathogens in culture negative (N = 13) and ITS PCR negative specimens (N = 12) from patients with a clinically confirmed fungal infection. Our results support the use of the 18S rDNA RT-PCR diagnostic workflow for rapid and accurate identification of fungal pathogens in clinical specimens.

Colistin sulfate is widely used in both human and veterinary medicine. However, its effect on the microbial ecologyis unknown. In this study, we determined the effect of colistin sulfate on the diversity of soil microorganisms by amplified rDNA restriction analysis (ARDRA) and high-throughput sequencing.ARDRAshowed that the diversity of DNA from soil microorganisms was reduced after soil was treated with colistin sulfate, with the most dramatic reductionobserved after 35days of treatment. High-throughput sequencing showed that the Chao1 and abundance-based coverage estimators (ACE) were reduced in the soils treated with colistin sulfate for 35 dayscompared to those treated with colistin sulfate for 7days. Furthermore, Chao1 and ACE tended to be lower when higher concentration of colistin sulfate was used, suggesting that the microbial abundance is reduced by colistin sulfate in a dose-dependent manner. Shannon index showed that the diversity of soil microorganism was reduced upon treatment with colistin sulfate compared to the untreated control group. Following 7days of treatment, Bacillus, Clostridiumand Sphingomonas were sensitive to all the concentration of colistin sulfate used in this study. Following 35days of treatment, the abundance of Choroplast, Haliangium, Pseudomonas, Lactococcus, and Clostridium was significantly decreased. Our results demonstrated that colistin sulfate especially at high concentration (≥5mg/kg) could alter the population structure of microorganisms and consequently the microbial community function in soil., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Ribosomal (r)RNA and rDNA have been golden molecular markers in microbial ecology. However, it remains poorly understood how ribotype copy number (CN)-based characteristics are linked with diversity, abundance, and activity of protist populations and communities observed at organismal levels. Here, we applied a single-cell approach to quantify ribotype CNs in two ciliate species reared at different temperatures. We found that in actively growing cells, the per-cell rDNA and rRNA CNs scaled with cell volume (CV) to 0.44 and 0.58 powers, respectively. The modeled rDNA and rRNA concentrations thus appear to be much higher in smaller than in larger cells. The observed rRNA:rDNA ratio scaled with CV 0.14 . The maximum growth rate could be well predicted by a combination of per-cell ribotype CN and temperature. Our empirical data and modeling on single-cell ribotype scaling are in agreement with both the metabolic theory of ecology and the growth rate hypothesis, providing a quantitative framework for linking cellular rDNA and rRNA CNs with body size, growth (activity), and biomass stoichiometry. This study also demonstrates that the expression rate of rRNA genes is constrained by cell size, and favors biomass rather than abundance-based interpretation of quantitative ribotype data in population and community ecology of protists., (© 2017 The Authors. Journal of Eukaryotic Microbiology published by Wiley Periodicals, Inc. on behalf of International Society of Protistologists.)

The Gambierdiscus genus is a group of benthic dinoflagellates commonly associated with ciguatera fish poisoning (CFP), which is generally found in tropical or sub-tropical regions around the world. Morphologically similar species within the genus can vary in toxicity; however, species identifications are difficult or sometimes impossible using light microscopy. DNA sequencing of ribosomal RNA genes (rDNA) is thus often used to identify and describe Gambierdiscus species and ribotypes, but the expense and time can be prohibitive for routine culture screening and/or large-scale monitoring programs. This study describes a restriction fragment length polymorphism (RFLP) typing method based on analysis of the large subunit rDNA that can successfully identify at least nine of the described Gambierdiscus species and two Fukuyoa species. The software programs DNAMAN 6.0 and Restriction Enzyme Picker were used to identify a set of restriction enzymes (SpeI, HpyCH4IV, and TaqαI) capable of distinguishing most of the known Gambierdiscus species for which DNA sequences were available. This assay was tested using in silico analysis and cultured isolates, and species identifications of isolates assigned by RFLP typing were confirmed by DNA sequencing. To verify the assay and assess intra-specific heterogeneity in RFLP patterns, identifications of 63 Gambierdiscus isolates comprising ten Gambierdiscus species, one ribotype, and two Fukuyoa species were confirmed using RFLP typing, and this method was subsequently employed in the routine identification of isolates collected from the Caribbean Sea. The RFLP assay presented here reduces the time and cost associated with morphological identification via scanning electron microscopy and/or DNA sequencing, and provides a phylogenetically sensitive method for routine Gambierdiscus species assignment., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Harmful algal blooms (HABs) are global threats to marine ecosystems, fisheries, and human health. Therefore, developing effective and accurate methods for identifying causative algae and monitoring seawater quality is urgent. However, traditional, microscopy-based methods are complex, inaccurate, and time-consuming. Here, we present a novel method for effective and sensitive detection of Chattonella marina using hyper-branched rolling circle amplification (HRCA) and HRCA-based strip test (HBST). The large subunit (LSU) ribosomal DNA (rDNA) D1-D2 region of C. marina was firstly sequenced to design a species-specific padlock probe (PLP). The HRCA reaction with two amplification primers and further HBST for C. marina was established. The optimized reaction conditions for HRCA were PLP concentration, 20 pM; ligation temperature, 65 °C; ligation time, 60 min; amplification temperature, 61 °C; and amplification time, 60 min. The developed HBST detection procedure involved HRCA reaction, test strip preparation, hybridization, coloration, and judgment of hybridization by the naked eye. Specificity and sensitivity of the established methods were validated. Moreover, the results showed that the established detection methods were specific and sensitive to C. marina. The detection limits of HRCA and HBST assays were 10 copies and 1 copy μL -1 of plasmid with LSU rDNA of C. marina, which are of two and three respective magnitude orders higher than conventional PCR. Finally, the protocols were applied to the simulated field samples and the results showed that the developed HBST assay had higher detection sensitivity than HRCA and PCR. In conclusion, the methods presented in this study are promising for sensitive, intuitive, and specific detection of C. marina in field monitoring natural samples and may provide a good detection model for other harmful algae in the future.

Plant-based food ingredients such as garlic, Chinese leek, Chinese onion, green onion and onion are widely used in many cuisines around the world. However, these ingredients known as the "five forbidden vegetables" (FFVs) are not allowed in some vegetarian diets. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for the detection of FFVs using five respective LAMP primer sets. The specific primers targeted the ITS1-5.8S-ITS2 nuclear ribosomal DNA sequence regions among the five vegetables. The results demonstrated that the identification of FFVs using the newly developed LAMP assay is more sensitive than the traditional PCR method. Using pepper, basil, parsley, chili and ginger as references, established LAMP primer sets showed high specificity for the identification of the FFV species. Moreover, when FFVs were mixed with other plant ingredients at different ratios (100:0, 50:50, 20:80, 10:90, 5:95, 2:98, and 1:99), no cross-reactivity was evident using LAMP. Finally, genomic DNAs extracted from boiled and steamed FFVs in processed foods were used as templates; the performance of the LAMP reaction was not influenced using validated LAMP primers. Not only can FFV ingredients be identified but commercial foods containing FFVs can also be authenticated. This LAMP method will be useful for the authentication of FFVs in practical food markets in the future.

Chromatin immunoprecipitation (ChIP) is a valuable technique for localizing proteins of interest to specific genomic sites and determining the relative abundance of these proteins at these sites. The ChIP method entails chemical cross-linking of proteins to genomic DNA, isolation of protein-DNA conjugates, and purification of DNA from conjugates. Real-time polymerase chain reactions are used to identify and quantify isolated genomic sequences. Here we describe how to localize yeast proteins to gene sequences residing within the nucleolus, i.e., ribosomal DNA (rDNA).

If the in situ growth rate of filamentous bacteria in activated sludge can be quantified, researchers can more accurately assess the effect of operating conditions on the growth of filaments and improve the mathematical modeling of filamentous bulking. We developed a method to quantify the in situ specific growth rate of Sphaerotilus natans (a model filament) in activated sludge using the species-specific 16S rRNA:rDNA ratio. Primers targeting the 16S rRNA of S. natans were designed, and real-time PCR and RT-PCR were used to quantify DNA and RNA levels of S. natans, respectively. A positive linear relationship was found between the rRNA:rDNA ratio (from 440 to 4500) and the specific growth rate of S. natans (from 0.036 to 0.172 h -1 ) using chemostat experiments. The in situ growth rates of S. natans in activated sludge samples from three water reclamation facilities were quantified, illustrating how the approach can be applied in a complex environment such as activated sludge., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The detection of plankton DNA is one of the important methods for the diagnosis of drowning from postmortem tissues. This study investigated the quantities of picoplankton (Cyanobacteria) DNA in the lung, liver, kidney tissues and blood of drowned and non-drowned rabbits, and the sensitivity of detection of picoplankton DNA by polymerase chain reaction (PCR) detect for the diagnosis of death from drowning. For this purpose, the DNA of the 16S ribosomal RNA gene of picoplankton was quantitatively assayed from the tissues of drowned and non-drowned rabbits immersed in water after death. Each of the liver, kidney and lung tissues and blood were obtained from drowned and non-drowned rabbits. Picoplankton DNA in the tissues was extracted using the DNeasy® Blood & Tissue kit to determine the yield of picoplankton DNA from each tissue. TaqMan real-time PCR was performed for quantitative analysis of picoplankton DNA. Target DNA was detected in the liver, kidney and lung samples obtained from the drowned rabbits, while no picoplankton DNA was detected in the non-drowned rabbit tissues (except in lung samples). The results verified that direct PCR for the detection of picoplankton DNA is useful for the diagnosis of drowning. Although we observed seasonal changes in the quantity of picoplankton in river water, we were able to detect DNA from various organs of drowned bodies during the season when picoplankton were not the most abundant., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

We previously reported the characterization 5S ribosomal DNA (rDNA) clusters in the common sea urchin Paracentrotus lividus and demonstrated the presence of DNA methylation-dependent silencing of embryo specific 5S rDNA cluster in adult tissue. In this work, we show genetic and epigenetic characterization of 18S-26S rDNA clusters in this specie. The results indicate the presence of three different 18S-26S rDNA clusters with different Non-Transcribed Spacer (NTS) regions that have different chromosomal localizations. Moreover, we show that the two largest clusters are hyper-methylated in the promoter-containing NTS regions in adult tissues, as in the 5S rDNA. These findings demonstrate an analogous epigenetic regulation in small and large rDNA clusters and support the logical synchronism in building ribosomes. In fact, all the ribosomal RNA genes must be synchronously and equally transcribed to perform their unique final product., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Ultrafast-metagenomic sequence classification using exact alignments (Kraken) is a novel approach to classify 16S rDNA sequences. The classifier is based on mapping short sequences to the lowest ancestor and performing alignments to form subtrees with specific weights in each taxon node. This study aimed to evaluate the classification performance of Kraken with long 16S rDNA random environmental sequences produced by cloning and then Sanger sequenced. A total of 480 clones were isolated and expanded, and 264 of these clones formed contigs (1352 ± 153 bp). The same sequences were analyzed using the Ribosomal Database Project (RDP) classifier. Deeper classification performance was achieved by Kraken than by the RDP: 73% of the contigs were classified up to the species or variety levels, whereas 67% of these contigs were classified no further than the genus level by the RDP. The results also demonstrated that unassembled sequences analyzed by Kraken provide similar or inclusively deeper information. Moreover, sequences that did not form contigs, which are usually discarded by other programs, provided meaningful information when analyzed by Kraken. Finally, it appears that the assembly step for Sanger sequences can be eliminated when using Kraken. Kraken cumulates the information of both sequence senses, providing additional elements for the classification. In conclusion, the results demonstrate that Kraken is an excellent choice for use in the taxonomic assignment of sequences obtained by Sanger sequencing or based on third generation sequencing, of which the main goal is to generate larger sequences., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Trematode cercariae are commonly found in many freshwater gastropods. These cercariae can serve to identify the occurrence of such trematodes as Centrocestus formosanus, Haplorchis taichui, Haplorchoides sp, and Stellantchasmus falcatus, which are important parasites in Chiang Mai Province, Thailand. As the species of these cercariae cannot be identified accurately based on morphology, this study employed sequencing of a fragment of 28S ribosomal DNA and phylogenetic analysis to identify the trematode cercariae found in freshwater gastropods in Chiang Mai Province. Eight types of trematode cercariae were identified, namely, distome cercaria (grouped with Philophthalmus spp clade), echinostome cercaria (grouped with Echinostoma spp clade), furcocercous cercaria (grouped with Posthodiplostomum sp/Alaria taxideae/Hysteromorpha triloba clade), monostome cercaria (grouped with Catatropis indicus clade), parapleurolophocercous cercaria (grouped with Haplorchoides sp clade), pleurolophocercous cercaria (grouped with Centrocestusformosanus clade), transversotrema cercaria (grouped with Transversotrema spp clade), and xiphidiocercaria (grouped with Prosthodendrium spp clade). These results provide important information that can be used for identifying these parasites in epidemiological surveys.

The bacterial communities present in smokeless tobacco (ST) products have not previously reported. In this study, we used Next Generation Sequencing to study the bacteria present in U.S.-made dry snuff, moist snuff and Sudanese toombak. Sample diversity and taxonomic abundances were investigated in these products. A total of 33 bacterial families from four phyla, Actinobacteria, Firmicutes, Proteobacteria and Bacteroidetes, were identified. U.S.-produced dry snuff products contained a diverse distribution of all four phyla. Moist snuff products were dominated by Firmicutes. Toombak samples contained mainly Actinobacteria and Firmicutes (Aerococcaceae, Enterococcaceae, and Staphylococcaceae). The program PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) was used to impute the prevalence of genes encoding selected bacterial toxins, antibiotic resistance genes and other pro-inflammatory molecules. PICRUSt also predicted the presence of specific nitrate reductase genes, whose products can contribute to the formation of carcinogenic nitrosamines. Characterization of microbial community abundances and their associated genomes gives us an indication of the presence or absence of pathways of interest and can be used as a foundation for further investigation into the unique microbiological and chemical environments of smokeless tobacco products.

Though the use of metagenomic methods to sample below-ground fungal communities is common, the use of similar methods to sample plants from their underground structures is not. In this study we use high throughput sequencing of the ribulose-bisphosphate carboxylase large subunit (rbcL) plastid marker to study the plant community as well as the internal transcribed spacer and large subunit ribosomal DNA (rDNA) markers to investigate the fungal community from two wetland sites. Observed community richness and composition varied by marker. The two rDNA markers detected complementary sets of fungal taxa and total fungal composition clustered according to primer rather than by site. The composition of the most abundant plants, however, clustered according to sites as expected. We suggest that future studies consider using multiple genetic markers, ideally generated from different primer sets, to detect a more taxonomically diverse suite of taxa compared with what can be detected by any single marker alone. Conclusions drawn from the presence of even the most frequently observed taxa should be made with caution without corroborating lines of evidence.

One of the most important challenges in agriculture is to determine the effectiveness and environmental impact of certain farming practices. The aim of present study was to determine and compare the taxonomic composition of the microbiomes established in soil following long-term exposure (14 years) to a conventional and organic farming systems (CFS and OFS accordingly). Soil from unclared forest next to the fields was used as a control. The analysis was based on RT-PCR and pyrosequencing of 16S rRNA genes of bacteria and archaea. The number of bacteria was significantly lower in CFS than in OFS and woodland. The highest amount of archaea was detected in woodland, whereas the amounts in CFS and OFS were lower and similar. The most common phyla in the soil microbial communities analyzed were Proteobacteria (57.9%), Acidobacteria (16.1%), Actinobacteria (7.9%), Verrucomicrobia (2.0%), Bacteroidetes (2.7%) and Firmicutes (4.8%). Woodland soil differed from croplands in the taxonomic composition of microbial phyla. Croplands were enriched with Proteobacteria (mainly the genus Pseudomonas), while Acidobacteria were detected almost exclusively in woodland soil. The most pronounced differences between the CFS and OFS microbiomes were found within the genus Pseudomonas, which significantly (p<0,05) increased its number in CFS soil compared to OFS. Other differences in microbiomes of cropping systems concerned minor taxa. A higher relative abundance of bacteria belonging to the families Oxalobacteriaceae, Koribacteriaceae, Nakamurellaceae and genera Ralstonia, Paenibacillus and Pedobacter was found in CFS as compared with OFS. On the other hand, microbiomes of OFS were enriched with proteobacteria of the family Comamonadaceae (genera Hylemonella) and Hyphomicrobiaceae, actinobacteria from the family Micrococcaceae, and bacteria of the genera Geobacter, Methylotenera, Rhizobium (mainly Rhizobium leguminosarum) and Clostridium. Thus, the fields under OFS and CFS did not differ greatly for the composition of the microbiome. These results, which were also confirmed by cluster analysis, indicated that microbial communities in the field soil do not necessarily differ largely between conventional and organic farming systems.

Background: Although high-throughput sequencing, such as Illumina-based technologies (e.g. MiSeq), has revolutionized microbial ecology, adaptation of amplicon sequencing for environmental microbial community analysis is challenging due to the problem of low base diversity., Results: A new phasing amplicon sequencing approach (PAS) was developed by shifting sequencing phases among different community samples from both directions via adding various numbers of bases (0-7) as spacers to both forward and reverse primers. Our results first indicated that the PAS method substantially ameliorated the problem of unbalanced base composition. Second, the PAS method substantially improved the sequence read base quality (an average of 10 % higher of bases above Q30). Third, the PAS method effectively increased raw sequence throughput (~15 % more raw reads). In addition, the PAS method significantly increased effective reads (9-47 %) and the effective read sequence length (16-96 more bases) after quality trim at Q30 with window 5. In addition, the PAS method reduced half of the sequencing errors (0.54-1.1 % less). Finally, two-step PCR amplification of the PAS method effectively ameliorated the amplification biases introduced by the long barcoded PCR primers., Conclusion: The developed strategy is robust for 16S rRNA gene amplicon sequencing. In addition, a similar strategy could also be used for sequencing other genes important to ecosystem functional processes.

Black yeast-like fungi and relatives as agents of cerebral phaeohyphomycosis are often encountered in human fatal brain abscesses and lead to almost 100% mortality despite the application of antifungal and surgical therapy. We report to our knowledge the first case of brain infection due to Rhinocladiella mackenziei in a 54-year-old immunocompetent male in Iran where R. mackenziei has not been reported previously. The initial diagnosis was brain fungal infection because of pigmented, irregular, branched, septated hyphae based on histopathological staining. The patient was treated with intravenous amphotericin B deoxycholate (0.5mg/kg/day) combined with oral itraconazole (200mg twice daily), nevertheless, his neurological function deteriorated rapidly and ultimately the patient died due to respiratory failure later two weeks. R. mackenziei was identified based on the sequencing of internal transcribed spacer (ITS rDNA region) (KJ140287). Therefore, considerable attention for this life-threatening infection is highly recommended., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

An efficient loop-mediated isothermal amplification procedure (LAMP) for the detection of "Candidatus Liberibacter solanacearum" (Lso), the bacterial causal agent of potato zebra chip (ZC) disease, is described in this chapter. Similar to the polymerase chain reaction (PCR), the LAMP employs a bacterial polymerase to amplify specific DNA sequences. However, the method differs from conventional PCR in that it uses six primers specific to the target region to generate a loop structure and autocycling strand displacement rather than thermocycling for sequence amplification. Moreover, unlike PCR that requires agarose gel electrophoresis for resolution, the positive LAMP results can be visualized directly as a precipitate within the reaction tubes. The 16S rDNA gene of "Ca. Liberibacter solanacearum" was used as the target for the design of the six LAMP primers. The LAMP technique is a reliable, rapid, and cost-effective method of detecting the "Ca. Liberibacter solanacearum" pathogen in the potato/tomato psyllid, Bactericera cockerelli, and in field-grown potato plants and tubers.

Probiotic microorganisms are those which exert a positive exect on the growth of the host, when administered as a dietary mixture in an adequate amount. They form the best alternative to the use of antibiotics for controlling enteric diseases in poultry farm animals, especially in the light of the gruesome problems of development of antibiotic resistance in enteric pathogens and the contamination of poultry products with antibiotics. 16S rDNA sequencing which has gained wide popularity amongst microbiologists for the molecular characterization and identification of newly discovered isolates provides accurate identification of isolates down to the level of sub-species (strain). It's most important advantage over the traditional biochemical characterization methods are that it can provide an accurate identification of strains with atypical phenotypic characters as well. The following work is an application of 16S rRNA gene sequencing approach to identify a novel, alkaline protease producing bacteria, from poultry farm waste. The sample was collected from a local poultry farm in the Guntur district, Andhra Pradesh, India. Subsequently the sample was serially diluted and the aliquots were incubated for a suitable time period following which the suspected colony was subjected to 16S rDNA sequencing. The results showed the isolate to be a novel, high alkaline protease producing bacteria, which was named Bacillus firmus isolate EMBS023, after characterization the sequence of isolate was deposited in GenBank with accession number JN990980.

Background and Objective: Acute exacerbations of chronic obstructive pulmonary disease (AE-COPD) are leading causes of mortality in hospital intensive care units. We sought to determine whether dental plaque biofilms might harbor pathogenic bacteria that can eventually cause lung infections in patients with severe AE-COPD., Material and Methods: Paired samples of subgingival plaque biofilm and tracheal aspirate were collected from 53 patients with severe AE-COPD. Total bacterial DNA was extracted from each sample individually for polymerase chain reaction amplification and/or generation of bacterial 16S rDNA sequences and cDNA libraries. We used a metagenomic approach, based on bacterial 16S rDNA sequences, to compare the distribution of species present in dental plaque and lung., Results: Analysis of 1060 sequences (20 clones per patient) revealed a wide range of aerobic, anaerobic, pathogenic, opportunistic, novel and uncultivable bacterial species. Species indistinguishable between the paired subgingival plaque and tracheal aspirate samples (97-100% similarity in 16S rDNA sequence) were dental plaque pathogens (Aggregatibacter actinomycetemcomitans, Capnocytophaga sputigena, Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola) and lung pathogens (Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa and Streptococcus pneumoniae). Real-time polymerase chain reaction of 16S rDNA indicated lower levels of Pseudomonas aeruginosa and Porphyromonas gingivalis colonizing the dental plaques compared with the paired tracheal aspirate samples., Conclusion: These results support the hypothesis that dental bacteria may contribute to the pathology of severe AE-COPD., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Paracoccidioides brasiliensis (P. brasiliensis) is a thermo-dimorphic fungus that causes paracoccidioidomycosis. Brazil epidemiological data shows that endemic areas are the subtropical regions, especially where agricultural activities predominate such as the Southeast, South, and Midwest. There are several tests to diagnose paracoccidioidomycosis, but they have many limitations such as low sensitivity, high cost, and a cross-reacting problem. In this work, gold nanoprobes were used to identify P. brasiliensis as an alternative diagnostic technique, which is easier to apply, costs less, and has great potential for application. The specific Ribosomal sequence of P. brasiliensis DNA was amplified and used to design the nanoprobes using a thiol-modified oligonucleotide. The results of positive and negative tests were done by UV-visible and Fourier Transform Infrared (FT-IR) measurements. The deconvolution of FT-IR sample spectra showed differences in the vibrational modes from the hydrogen bridge NHN and NHO bands that form the double helix DNA for samples matching the DNA sequence of nanoprobes that could be used to classify the samples., (Copyright © 2013 Elsevier B.V. All rights reserved.)

High-throughput sequencing technology, coupled with the use of conserved marker genes, has allowed for the understanding of communities of microbes (both culturable and unculturable) as well as their phylogenetic placement. The recent explosion of sequencing data prompted the development of software that could process the vast amount of data generated and phylogenetically differentiate groups of samples. Host-associated microbial studies have revealed that microbes are highly varied between individuals and fluctuate within an individual. Large-scale studies are being undertaken that include collection of extensive environmental data to help uncover the forces that shape microbial communities.

Aiming to examine whether the genetic background of the crude drugs derived from four Yunnanese Swertia plants and their chemical constituent profiles correlate, we analyzed the nucleotide sequences of their nuclear ribosomal DNA regions including ITS1, 5.8S ribosomal RNA gene, and ITS2, together with those of Japanese S. japonica and S. pseudochinensis from Hebei Province. The result that two of the Yunnanese Swertia plants, S. binchuanensis and S. punicea, were genetically similar may explain their similarity in chemical constituent profiles. On the other hand, in spite of differences in chemical profile, S. decora and S. pseudochinensis were genetically close. The other Yunnanese Swertia plants, S. delavayi, and S. japonica, stood at intermediate positions between these two genetically similar pairs. The result suggests that although genetic background would have an influence, environmental factors, e.g., soil and weather conditions, might be critical for their production of secondary metabolites.

Deliberate or accidental contamination of food, feed, and water supplies poses a threat to human health worldwide. A rapid and sensitive detection technique that could replace the current labor-intensive and time-consuming culture-based methods is highly desirable. In addition to species-specific assays, such as PCR, there is a need for generic methods to screen for unknown pathogenic microorganisms in samples. This work presents a metagenomics-based direct-sequencing approach for detecting unknown microorganisms, using Bacillus cereus (as a model organism for B. anthracis) in bottled water as an example. Total DNA extraction and 16S rDNA gene sequencing were used in combination with principle component analysis and multicurve resolution to study detection level and possibility for identification. Results showed a detection level of 10(5) to 10(6) CFU/L. Using this method, it was possible to separate 2 B. cereus strains by the principal component plot, despite the close sequence resemblance. A linear correlation between the artificial contamination level and the relative amount of the Bacillus artificial contaminant in the metagenome was observed, and a relative amount value above 0.5 confirmed the presence of Bacillus. The analysis also revealed that background flora in the bottled water varied between the different water types that were included in the study. This method has the potential to be adapted to other biological matrices and bacterial pathogens for fast screening of unknown bacterial threats in outbreak situations.

Extracorporeal membrane oxygenation (ECMO) represents a temporary life-saving therapy for respiratory or circulatory failure, but infections during ECMO support are a life-threatening complication. Surface-related infections of ECMO are mentioned, but rarely described in the literature. A universal rDNA polymerase chain reaction (PCR) test was used to investigate the potential microbiological colonization of membrane oxygenators (MOs) in 20 patients undergoing ECMO. The overall patient-based positivity by PCR was 45%. Gram-positive bacteria (71%) represented the most abundant microorganisms on MO surfaces, followed by Gram-negative bacteria (22%) and fungi (7%). The most frequently detected causative pathogens were staphylococci (58%). Bacterial mixed infections represented 56% of all infections. In four PCR-positive cases, the pathogens detected on the MO surfaces were also found by blood culture or by culture of specimens obtained from the infectious focus. In conclusion, hollow fiber membranes of MOs can be colonized by microorganisms and appear to be a potential source of bacterial and fungal infections in ECMO patients. These infections may pose an increased risk for clinical worsening. As a consequence, persistent septic complications have to be discussed as an indication for MO exchange. The initial results suggest that the applied PCR assay is a valuable tool to investigate MOs.

Conventional nested PCR is a very sensitive and specific method for the diagnosis of visceral leishmaniasis. However, this type of PCR is notorious for contamination problems related to the processing of the product between the first and the second PCR steps. In order to have a PCR method that is just as efficient but without the risk of contamination, we attempted the optimization of a single-tube nested PCR (STNPCR) method. During the first and the second PCR steps, we used the small subunit of ribosomal RNA (ssu rRNA) and the ribosomal internal transcribed spacer (ITS) as targets, respectively. The performances of STNPCR and nested PCR in detecting the DNA of Leishmania chagasi were compared. In the case of STNPCR, the inner primers were immobilized on the interior of the tube cap by means of adsorption microtubes and then were solubilized before the second reaction. This procedure eliminated the need to open the microtube, which could have led to false-positive results through cross-contamination. The detection limit for the purified L. chagasi DNA was 1 fg by using nested PCR and 10 fg by using STNPCR. We also tested the specificity of the system against other parasites, and observed that Trypanosoma cruzi DNA was amplified with a detection limit of up to 1 pg. This study not only presents a promising tool for the diagnosis of visceral leishmaniasis, but also provides a new tool for the diagnosis of Chagas disease, either in mono-infection by T. cruzi or in co-infection with Leishmania spp., (Copyright © 2013 Elsevier Inc. All rights reserved.)