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This study shows that oligonucleotide sequences are shared between the human genome and primers that have been proposed/used for SARS-CoV-2 detection by polymerase chain reaction (PCR). The high level of sharing (namely, up to 19mer with a maximum number of gaps equal to 2) might bear implications for the diagnostic validity of SARS-CoV-2 detection by PCR.

CRISPR-Cas is an adaptive immunity system of prokaryotes, composed of CRISPR arrays and the associated proteins. The successive addition of spacer sequences in the CRISPR array has made the system a valuable molecular marker, with multiple applications. Due to the high degree of polymorphism of the CRISPR loci, their comparison in bacteria from various sources may provide insights into the evolution and spread of the CRISPR-Cas systems. The aim of this study was to establish a correlation between the enterobacterial CRISPR loci, the sequence of direct repeats (DR), and the number of spacer units, along with the geographical origin and collection source. For this purpose, 3474 genomes containing CRISPR loci from the CRISPRCasdb of Salmonella enterica, Escherichia coli, and Klebsiella pneumoniae were analyzed, and the information regarding the isolates was recorded from the NCBI database. The most prevalent was the I-E CRISPR-Cas system in all three studied taxa. E. coli also presents the I-F type, but in a much lesser percentage. The systems found in K. pneumoniae can be classified into I-E and I-E*. The I-E and I-F systems have two CRISPR loci, while I-E* has only one locus upstream of the Cas cluster. PCR primers have been developed in this study for each CRISPR locus. Distinct clustering was not evident, but statistically significant relationships occurred between the different CRISPR loci and the number of spacer units. For each of the queried taxa, the number of spacers was significantly different (p < 0.01) by origin (Africa, Asia, Australia and Oceania, Europe, North America, and South America) but was not linked to the isolation source type (human, animal, plant, food, or laboratory strains). [ABSTRACT FROM AUTHOR]

Understanding the unique and unexplored microbial environment of xylem sap is starting to be of relevant importance for plant health, as it could include microbes that may protect plants against xylem-limited pathogens, such as Verticillium dahliae and Xylella fastidiosa. In this study, we evaluated the effects that the method for extracting the xylem bacterial communities, the plant age and the PCR primers may have on characterizing the xylem-bacterial-community composition by using an NGS approach. Xylem sap was extracted from xylem vessels by using a Scholander pressure chamber, or by macerating wood shavings that were obtained from xylem tissues by using branches from 10-year-old olive trees, or the entire canopy of 1-year-old olive plantlets. Additionally, we compared four different PCR-primer pairs that target 16S rRNA for their efficacy to avoid the coamplification of mitochondria and chloroplast 16S rRNA, as this represents an important drawback in metabarcoding studies. The highest amplifications in the mitochondria and chloroplast reads were obtained when using xylem woody chips with the PCR1-799F/1062R (76.05%) and PCR3-967F/1391R (99.96%) primer pairs. To the contrary, the PCR2-799F/1115R and PCR4-799F/1193R primer pairs showed the lowest mitochondria 16S rRNA amplification (<27.48%), no chloroplast sequences and the highest numbers of bacterial OTUs identified (i.e., 254 and 266, respectively). Interestingly, only 73 out of 172 and 46 out of 181 genera were shared between the xylem sap and woody chips after amplification with PCR2 or PCR4 primers, respectively, which indicates a strong bias of the bacterial-community description, depending on the primers used. Globally, the most abundant bacterial genera (>60% of reads) included Anoxybacillus, Cutibacterium, Pseudomonas, Spirosoma, Methylobacterium-Methylorubrum and Sphingomonas; however, their relative importance varied, depending on the matrix that was used for the DNA extraction and the primer pairs that were used, with the lowest effect due to plant age. These results will help to optimize the analysis of xylem-inhabiting bacteria, depending on whether whole xylematic tissue or xylem sap is used for the DNA extraction. More importantly, it will help to better understand the driving and modifying factors that shape the olive-xylem-bacterial-community composition. [ABSTRACT FROM AUTHOR]

Simple Summary: Agricultural and environmental sustainability requires effective biosecurity responses that prevent the establishment or spread of exotic insect pests. Understanding where new detections may have come from or if recurrent detections are connected contributes to this. Suitable population genetic markers use relatively rapidly evolving gene regions which render the PCR method species-specific at best. Because resource limitations mean these are pre-emptively developed for the highest risk species, populations of other exotic pests are unable to be characterised at the time. Here we have developed a generic method that is useful across species within the same taxonomic Order, including where there is little or no prior knowledge of their gene sequences. Markers are formed by concomitant sequencing of four gene regions. Sequence concatenation was shown to retrieve higher resolution signatures than standard DNA barcoding. The method is encouragingly universal, as illustrated across species in ten fly and 11 moth superfamilies. Although as-yet untested in a biosecurity situation, this relatively low-tech, off-the-shelf method makes a proactive contribution to the toolbox of quarantine agencies at the time of detection without the need for impromptu species-specific research and development. Biosecurity responses to post-border exotic pest detections are more effective with knowledge of where the species may have originated from or if recurrent detections are connected. Population genetic markers for this are typically species-specific and not available in advance for any but the highest risk species, leaving other less anticipated species difficult to assess at the time. Here, new degenerate PCR primer sets are designed for within the Lepidoptera and Diptera for the 3′ COI, ND3, ND6, and 3′ plus 5′ 16S gene regions. These are shown to be universal at the ordinal level amongst species of 14 and 15 families across 10 and 11 dipteran and lepidopteran superfamilies, respectively. Sequencing the ND3 amplicons as an example of all the loci confirmed detection of population-level variation. This supported finding multiple population haplotypes from the publicly available sequences. Concatenation of the sequences also confirmed that higher population resolution is achieved than for the individual genes. Although as-yet untested in a biosecurity situation, this method is a relatively simple, off-the-shelf means to characterise populations. This makes a proactive contribution to the toolbox of quarantine agencies at the time of detection without the need for unprepared species-specific research and development. [ABSTRACT FROM AUTHOR]

Human milk is the ideal food for infants due to its unique nutritional and immune properties, and more recently human milk has also been recognized as an important source of bacteria for infants. However, a substantial amount of fundamental human milk microbiome information remains unclear, such as the origin, composition and function of the community and its members. There is emerging evidence to suggest that the diversity and composition of the milk microbiome might differ between lactation stages, due to maternal factors and diet, agrarian and urban lifestyles, and geographical location. The evolution of the methods used for studying milk microbiota, transitioning from culture dependent-approaches to include culture-independent approaches, has had an impact on findings and, in particular, primer selection within 16S rRNA gene barcoding studies have led to discrepancies in observed microbiota communities. Here, the current state-of-the-art is reviewed and emerging frontiers essential to improving our understanding of the human milk microbiome are considered.

Recent studies revealed that some intestinal microorganisms anaerobically convert choline to trimethylamine (TMA) by choline TMA-lyase (cutC). TMA is further oxidized to trimethylamine-N-oxide (TMAO), by the liver enzyme flavin-dependent monooxygenase 3 (FMO3). TMA in the serum is correlated with the risk of cardiovascular disease and some other diseases in human. The objective of this study is to study the expression levels of cutC and its activating enzyme (cutD) gene for these microorganisms and their association with TMA production. In this study, we collected 20 TMA producing bacteria strains representing 20 species, and designed primers to evaluate their gene expression levels by reverse transcription quantitative PCR (RT-qPCR). In addition, TMA production was analyzed by UPLC-MS/MS. Results showed that gene expression levels of most individual strains were different when compared with the gene expression level of their glyceraldehyde-3 phosphate dehydrogenase (GAPDH) gene and the TMA production level of gut bacteria may not correlate with their cutC/cutD gene expression levels. Bioinformatic analysis of the CutC protein showed conserved choline binding site residues; cutD showed conserved S-adenosylmethionine (SAM) and two CX2-CX2-CX3 motifs. The present study reports that the TMA production level may not only depend on cutC/cutD gene expression. Other factors may need to be investigated.

Bovine papillomavirus (BPV) types 1 and 2 are recognized as the main cause of equine sarcoids. However, some studies report that up to a quarter of these tumors do not contain detectible BPV1 or BPV2 DNA. The absence of detectible BPV1 or BPV2 in these sarcoids suggests the possible involvement of other papillomavirus types. Currently, five deltapapillomaviruses are recognized to cause mesenchymal neoplasia after cross-species infection. In addition to BPV1 and BPV2, BPV13 has been associated with equine sarcoids in Brazil, BPV14 has been associated with feline sarcoids, and Ovis aries papillomavirus 2 caused a sarcoid-like lesion in a pig. To investigate the cause of equine sarcoids, PCR primers were developed to specifically amplify each of the five different deltapapillomaviruses that have been associated with mesenchymal neoplasia. The specificity of these primers was confirmed using samples of formalin-fixed tissue known to contain each PV type. These primers allow rapid and sensitive detection of deltapapillomavirus DNA in equine sarcoids. As studies have revealed marked regional variability in the cause of equine sarcoids, these primers will be useful to determine the predominant PV type causing sarcoids in a region. Additionally, there is a single report describing mixed infections by BPV1 and BPV2 in equine sarcoids. The specific primer sets are expected to enable more sensitive detection of mixed infections in equine sarcoids. Determining the cause of equine sarcoids is important as vaccines are developed to prevent these common malignant neoplasms. [ABSTRACT FROM AUTHOR]

CRISPR-Cas is an adaptive immunity system of prokaryotes, composed of CRISPR arrays and the associated proteins. The successive addition of spacer sequences in the CRISPR array has made the system a valuable molecular marker, with multiple applications. Due to the high degree of polymorphism of the CRISPR loci, their comparison in bacteria from various sources may provide insights into the evolution and spread of the CRISPR-Cas systems. The aim of this study was to establish a correlation between the enterobacterial CRISPR loci, the sequence of direct repeats (DR), and the number of spacer units, along with the geographical origin and collection source. For this purpose, 3474 genomes containing CRISPR loci from the CRISPRCasdb of Salmonella enterica, Escherichia coli, and Klebsiella pneumoniae were analyzed, and the information regarding the isolates was recorded from the NCBI database. The most prevalent was the I-E CRISPR-Cas system in all three studied taxa. E. coli also presents the I-F type, but in a much lesser percentage. The systems found in K. pneumoniae can be classified into I-E and I-E*. The I-E and I-F systems have two CRISPR loci, while I-E* has only one locus upstream of the Cas cluster. PCR primers have been developed in this study for each CRISPR locus. Distinct clustering was not evident, but statistically significant relationships occurred between the different CRISPR loci and the number of spacer units. For each of the queried taxa, the number of spacers was significantly different (p < 0.01) by origin (Africa, Asia, Australia and Oceania, Europe, North America, and South America) but was not linked to the isolation source type (human, animal, plant, food, or laboratory strains).

[Display omitted] • DNA barcoding is an invaluable tool for fast and accurate taxonomic classification. • Existing DNA barcodes are still insufficient for obtaining well-supported phylogenies. • We present a protocol that produces long amplicons of unlinked loci for spiders. • Amplicons are sequenced at very low cost per specimen with ONT MinION. • Our recovered phylogeny is largely consistent with that of high-cost approaches. Phylogenetic inference has become a standard technique in integrative taxonomy and systematics, as well as in biogeography and ecology. DNA barcodes are often used for phylogenetic inference, despite being strongly limited due to their low number of informative sites. Also, because current DNA barcodes are based on a fraction of a single, fast-evolving gene, they are highly unsuitable for resolving deeper phylogenetic relationships due to saturation. In recent years, methods that analyse hundreds and thousands of loci at once have improved the resolution of the Tree of Life, but these methods require resources, experience and molecular laboratories that most taxonomists do not have. This paper introduces a PCR-based protocol that produces long amplicons of both slow- and fast-evolving unlinked mitochondrial and nuclear gene regions, which can be sequenced by the affordable and portable ONT MinION platform with low infrastructure or funding requirements. As a proof of concept, we inferred a phylogeny of a sample of 63 spider species from 20 families using our proposed protocol. The results were overall consistent with the results from approaches based on hundreds and thousands of loci, while requiring just a fraction of the cost and labour of such approaches, making our protocol accessible to taxonomists worldwide. [ABSTRACT FROM AUTHOR]

This study shows that oligonucleotide sequences are shared between the human genome and primers that have been proposed/used for SARS-CoV-2 detection by polymerase chain reaction (PCR). The high level of sharing (namely, up to 19mer with a maximum number of gaps equal to 2) might bear implications for the diagnostic validity of SARS-CoV-2 detection by PCR. [ABSTRACT FROM AUTHOR]

Understanding the unique and unexplored microbial environment of xylem sap is starting to be of relevant importance for plant health, as it could include microbes that may protect plants against xylem-limited pathogens, such as Verticillium dahliae and Xylella fastidiosa. In this study, we evaluated the effects that the method for extracting the xylem bacterial communities, the plant age and the PCR primers may have on characterizing the xylem-bacterial-community composition by using an NGS approach. Xylem sap was extracted from xylem vessels by using a Scholander pressure chamber, or by macerating wood shavings that were obtained from xylem tissues by using branches from 10-year-old olive trees, or the entire canopy of 1-year-old olive plantlets. Additionally, we compared four different PCR-primer pairs that target 16S rRNA for their efficacy to avoid the coamplification of mitochondria and chloroplast 16S rRNA, as this represents an important drawback in metabarcoding studies. The highest amplifications in the mitochondria and chloroplast reads were obtained when using xylem woody chips with the PCR1-799F/1062R (76.05%) and PCR3-967F/1391R (99.96%) primer pairs. To the contrary, the PCR2-799F/1115R and PCR4-799F/1193R primer pairs showed the lowest mitochondria 16S rRNA amplification (60% of reads) included Anoxybacillus, Cutibacterium, Pseudomonas, Spirosoma, Methylobacterium-Methylorubrum and Sphingomonas; however, their relative importance varied, depending on the matrix that was used for the DNA extraction and the primer pairs that were used, with the lowest effect due to plant age. These results will help to optimize the analysis of xylem-inhabiting bacteria, depending on whether whole xylematic tissue or xylem sap is used for the DNA extraction. More importantly, it will help to better understand the driving and modifying factors that shape the olive-xylem-bacterial-community composition.

Biosecurity responses to post-border exotic pest detections are more effective with knowledge of where the species may have originated from or if recurrent detections are connected. Population genetic markers for this are typically species-specific and not available in advance for any but the highest risk species, leaving other less anticipated species difficult to assess at the time. Here, new degenerate PCR primer sets are designed for within the Lepidoptera and Diptera for the 3′ COI, ND3, ND6, and 3′ plus 5′ 16S gene regions. These are shown to be universal at the ordinal level amongst species of 14 and 15 families across 10 and 11 dipteran and lepidopteran superfamilies, respectively. Sequencing the ND3 amplicons as an example of all the loci confirmed detection of population-level variation. This supported finding multiple population haplotypes from the publicly available sequences. Concatenation of the sequences also confirmed that higher population resolution is achieved than for the individual genes. Although as-yet untested in a biosecurity situation, this method is a relatively simple, off-the-shelf means to characterise populations. This makes a proactive contribution to the toolbox of quarantine agencies at the time of detection without the need for unprepared species-specific research and development.

Bovine papillomavirus (BPV) types 1 and 2 are recognized as the main cause of equine sarcoids. However, some studies report that up to a quarter of these tumors do not contain detectible BPV1 or BPV2 DNA. The absence of detectible BPV1 or BPV2 in these sarcoids suggests the possible involvement of other papillomavirus types. Currently, five deltapapillomaviruses are recognized to cause mesenchymal neoplasia after cross-species infection. In addition to BPV1 and BPV2, BPV13 has been associated with equine sarcoids in Brazil, BPV14 has been associated with feline sarcoids, and Ovis aries papillomavirus 2 caused a sarcoid-like lesion in a pig. To investigate the cause of equine sarcoids, PCR primers were developed to specifically amplify each of the five different deltapapillomaviruses that have been associated with mesenchymal neoplasia. The specificity of these primers was confirmed using samples of formalin-fixed tissue known to contain each PV type. These primers allow rapid and sensitive detection of deltapapillomavirus DNA in equine sarcoids. As studies have revealed marked regional variability in the cause of equine sarcoids, these primers will be useful to determine the predominant PV type causing sarcoids in a region. Additionally, there is a single report describing mixed infections by BPV1 and BPV2 in equine sarcoids. The specific primer sets are expected to enable more sensitive detection of mixed infections in equine sarcoids. Determining the cause of equine sarcoids is important as vaccines are developed to prevent these common malignant neoplasms.

Abstract Dissimilatory nitrate reduction to ammonium (DNRA) is now known to be a more prevalent process in terrestrial ecosystems than previously thought. The key enzyme, a pentaheme cytochrome c nitrite reductase NrfA associated with respiratory nitrite ammonification, is encoded by the nrfA gene in a broad phylogeny of bacteria. The lack of reliable and comprehensive molecular tools to detect diverse nrfA from environmental samples has hampered efforts to meaningfully characterize the genetic potential for DNRA in environmental systems. In this study, modifications were made to optimize the amplification efficiency of previously-designed PCR primers, targeting the diagnostic region of NrfA between the conserved third- and fourth heme binding domains, and to increase coverage to include detection of environmentally relevant Geobacteraceae-like nrfA. Using an alignment of the primers to >270 bacterial nrfA genes affiliated with 18 distinct clades, modifications to the primer sequences improved coverage, minimized amplification artifacts, and yielded the predicted product sizes from reference-, soil-, and groundwater DNA. Illumina sequencing of amplicons showed the successful recovery of nrfA gene fragments from environmental DNA based on alignments of the translated sequences. The new primers developed in this study are more efficient in PCR reactions, although gene targets with high GC content affect efficiency. Furthermore, the primers have a broader spectrum of detection and were validated rigorously for use in detecting nrfA from natural environments. These are suitable for conventional PCR, qPCR, and use in PCR access array technologies that allow multiplex gene amplification for downstream high throughput sequencing platforms. Highlights • New primer sets expand coverage of diverse bacterial nrfA genes involved in the nitrite ammonification step for DNRA. • The coverage of the developed primers now extends to the NrfA clade containing Geobacteraceae. • Primer sets allow efficient PCR amplification of nrfA genes from a wide variety of natural environments. • Primers are suitable for multiple applications including conventional PCR, qPCR, multiplex arrays, and high-throughput sequencing. [ABSTRACT FROM AUTHOR]

In diet metabarcoding analyses, insufficient taxonomic coverage of PCR primer sets generates false negatives that may dramatically distort biodiversity estimates. In this paper, we investigated the taxonomic coverage and complementarity of three cytochrome c oxidase subunit I gene (COI) primer sets based on in silico analyses and we conducted an in vivo evaluation using fecal and spider web samples from different invertivores, environments, and geographic locations. Our results underline the lack of predictability of both the coverage and complementarity of individual primer sets: (a) sharp discrepancies exist observed between in silico and in vivo analyses (to the detriment of in silico analyses); (b) both coverage and complementarity depend greatly on the predator and on the taxonomic level at which preys are considered; (c) primer sets' complementarity is the greatest at fine taxonomic levels (molecular operational taxonomic units [MOTUs] and variants). We then formalized the "one‐locus‐several‐primer‐sets" (OLSP) strategy, that is, the use of several primer sets that target the same locus (here the first part of the COI gene) and the same group of taxa (here invertebrates). The proximal aim of the OLSP strategy is to minimize false negatives by increasing total coverage through multiple primer sets. We illustrate that the OLSP strategy is especially relevant from this perspective since distinct variants within the same MOTUs were not equally detected across all primer sets. Furthermore, the OLSP strategy produces largely overlapping and comparable sequences, which cannot be achieved when targeting different loci. This facilitates the use of haplotypic diversity information contained within metabarcoding datasets, for example, for phylogeography and finer analyses of prey–predator interactions. [ABSTRACT FROM AUTHOR]

Published PCR primers targeting the ammonia monooxygenase gene (amoA) were applied to samples from activated sludge systems operated with low dissolved oxygen (DO) to quantify total and clade-level Nitrospira that perform complete ammonium oxidation (comammox); however, we found these existing primers resulted in significant artifact-associated non-target amplification. This not only overestimated comammox amoA copies but also resulted in numerous false positive detections in the environmental samples tested, as confirmed by gel electrophoresis. Therefore, instead of attempting to quantify comammox diversity, we focused on accurately quantifying the candidate comammox species. We designed specific and sensitive primers targeting 3 candidate species: Candidatus (Ca.) Nitrospira nitrosa, Ca. N. inopinata, and Ca. N. nitrificans. The primers were tested with amoA templates of these candidate species and used to quantify comammox at the species level in low DO activated sludge systems. We found that comammox related to Ca. N. nitrosa were present and abundant in the majority of samples from low DO bioreactors and were not detected in samples from a high DO system. In addition, the greatest abundance of Ca. N. nitrosa was found in bioreactors operated with a long solids retention time. Ca. N. inopinata and Ca. N. nitrificans were only detected sporadically in these samples, indicating a minor role of these comammox in nitrification under low DO conditions. [ABSTRACT FROM AUTHOR]

Background: Q fever is a widespread zoonotic disease caused by Coxiella burnetii. Ticks may act as vectors, and many epidemiological studies aim to assess C. burnetii prevalence in ticks. Because ticks may also be infected with Coxiella-like bacteria, screening tools that differentiate between C. burnetii and Coxiella-like bacteria are essential. Methods: In this study, we screened tick specimens from 10 species (Ornithodoros rostratus, O. peruvianus, O. capensis, Ixodes ricinus, Rhipicephalus annulatus, R. decoloratus, R. geigy, O. sonrai, O. occidentalis, and Amblyomma cajennense) known to harbor specific Coxiella-like bacteria, by using quantitative PCR primers usually considered to be specific for C. burnetii and targeting, respectively, the IS1111, icd, scvA, p1, and GroEL/htpB genes. Results: We found that some Coxiella-like bacteria, belonging to clades A and C, yield positive PCR results when screened with primers initially believed to be C. burnetii-specific. Conclusions: These results suggest that PCR-based surveys that aim to detect C. burnetii in ticks by using currently available methods must be interpreted with caution if the amplified products cannot be sequenced. Future molecular methods that aim at detecting C. burnetii need to take into account the possibility that cross-reactions may exist with Coxiella-like bacteria.

Entamoeba gingivalis normally exists in the human oral cavity, namely in the gums, and brings about some specific diseases. However, it can also trigger some more serious illnesses. Among these are infections of the genital tract, acute osteomyelitis of the mandible and pulmonary abscess. Entamoeba gingivalis identification by light microscopy is difficult, hence polymerase chain reaction (PCR) is used. The contemporary primers for PCR are complement to 18S rRNA. This article informs the reader of the process that was involved in designing new primers for three genes which were thought to be present on the Entamoeba gingivalis genome, but their sequences were unknown. The newly obtained sequences of primers have better properties for identification purposes, compared to these which are currently used.

We isolated and characterized a total of 13 microsatellite loci from Cordulegaster sarracenia (Odonata: Cordulegastridae). Loci were screened in 24 individuals from Louisiana and Texas. Within C. sarracenia, the number of alleles per locus ranged from 0 to 5, and observed and expected heterozygosities ranged from 0.000 to 0.556 and 0.000 to 0.613, respectively. Overall differentiation among study populations was very high (FST = 0.423), suggesting significant geographic population structure with low diversity within populations. Twelve of the 13 primers amplified in C. sayi, C. diastatops, C. maculata, and C. obliqua and polymorphism levels are reported. These new genetic markers will provide tools for addressing a number of population genetic and demographic questions relating to conservation of this rare dragonfly species. [ABSTRACT FROM AUTHOR]

Molecular species identification from biological material collected at field sites has become an established ecological tool. However, extracting and amplifying DNA from degraded field samples, such as prey remains and feces that have been exposed to the elements, remains a challenge and costly. We collected 115 fecal samples of unknown small mammals, resembling fecal droppings of voles and mice (i.e., Cricetidae and Muridae), from a salt marsh in The Netherlands. We modified a previously published protocol into a relatively low-cost method with a PCR success of 95%. We demonstrate that species identification is possible for both Cricetidae and Muridae species using fecal samples of unknown age deposited in the field. For 90 samples, sequences of the variable control region in the mitochondrial genome were obtained and compared to published DNA sequences of small mammals occurring in north European salt marshes. A single sample, probably environmentally contaminated, appeared as Sus scrofa (n = 1). We positively identified house mouse Mus musculus, being the positive control (n = 1), and common vole Microtus arvalis (n = 88). In 81 sequences of 251 nt without ambiguous bases, ten haplotypes were present. These haplotypes, representing the central lineage of the western subspecies M. arvalis arvalis, were separated by 20 mutations from published control region haplotypes of the western European lineages sampled in France. Unlike earlier studies of cytochrome b variation in coastal European populations, we did not find indications of recent purging of genetic variation in our study area. [ABSTRACT FROM AUTHOR]

BACKGROUND The whitefly Bemisia tabaci complex harbours over 40 cryptic species that have been placed in 11 phylogenetically distinct clades based on the molecular characterization of partial mitochondrial DNA COI (mtCOI) gene region. Four cryptic species are currently within the invasive clade, i.e. MED, MEAM1, MEAM2 and IO. Correct identification of these species is a critical step towards implementing reliable measures for plant biosecurity and border protection; however, no standardized B. tabaci -specific primers are currently available which has caused inconsistencies in the species identification processes. RESULTS We report three sets of polymerase chain reaction (PCR) primers developed to amplify the mtCOI region which can be used for genotyping MED, MEAM1 and IO species, and tested these primers on 91 MED, 35 MEAM1 and five IO individuals. PCR and sequencing of amplicons identified a total of 21, six and one haplotypes in MED, MEAM1 and IO respectively, of which six haplotypes were new to the B. tabaci database. CONCLUSION These primer pairs enabled standardization and robust molecular species identification via mtCOI screening of the targeted invasive cryptic species and will improve quarantine decisions. Use of this diagnostic tool could be extended to other species within the complex. © 2017 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Denaturing gradient gel electrophoresis (DGGE) has become a widely used tool to examine microbial community structure. However, when DGGE is applied to evaluate the fungal community of traditional fermentation starters, the choice of hypervariable ribosomal RNA gene regions is still controversial. In the current study, several previously published fungal PCR primer sets were compared and evaluated using PCR-DGGE, with the purpose of screening a suitable primer set to study the fungal community of traditional fermentation starters for Hong Qu glutinous rice wine. Firstly, different primer sets were used to amplify different hypervariable regions from pure fungal cultures. Except NS1/FR1 + and ITS1fGC/ITS4, other primer sets (NL1 +/LS2R, NL3A/NL4GC, FF390/FR1 +, NS1/GCFung, NS3 +/YM951r and ITS1fGC/ITS2r) amplified the target DNA sequences successfully. Secondly, the selected primer sets were further evaluated based on their resolution to distinguish different fungal cultures through DGGE fingerprints. Three primer sets (NL1 +/LS2R, NS1/GCFung and ITS1fGC/ITS2r) were finally selected for investigating the fungal community structure of different traditional fermentation starters for Hong Qu glutinous rice wine. The internal transcribed spacer (ITS) region amplified by ITS1fGC/ITS2r, which is more hypervariable than the 18S rRNA gene and 26S rRNA gene, provides an excellent tool to separate amplification products of different fungal species. Results indicated that PCR-DGGE profile using ITS1fGC/ITS2r showed more abundant fungal species than that using NL1 +/LS2R and NS1/GCFung. Therefore, ITS1fGC/ITS2r is the most suitable primer set for PCR-DGGE analysis of fungal community structure in traditional fermentation starters for Hong Qu glutinous rice wine. DGGE profiles based on ITS1fGC/ITS2r revealed the presence of twenty-four fungal species in traditional fermentation starter. A significant difference of fungal community can be observed directly from DGGE fingerprints and principal component analysis. The statistical analysis results based on the band intensities of fungal DGGE profile showed that Saccharomyces cerevisiae , Saccharomycopsis fibuligera , Rhizopus oryzae , Monascus purpureus and Aspergillus niger were the dominant fungal species. In conclusion, the comparison of several primer sets for fungal PCR-DGGE would be useful to enrich our knowledge of the fungal community structures associated with traditional fermentation starters, which may facilitate the development of better starter cultures for manufacturing Chinese Hong Qu glutinous rice wine. [ABSTRACT FROM AUTHOR]

The high genetic diversity of plants can be a problem when developing molecular methods that require conserved DNA sequences among species. Several chloroplast DNA (cpDNA) regions have been used for the identification of plant DNA from broad taxonomic groups, but many species fail to amplify due to genetic variation at primer-binding sites. Here, we evaluated the conservation degree of four chloroplast DNA (cpDNA) regions commonly used in plant investigations ( atpF-atpH, psbA-trnH, trnL CD and trnL GH). We propose new conserved PCR primers for the study of the most common plant families, designed using consensus sequences obtained from 28 multiple sequences alignments with over 11,000 reference sequences. The new primers were able to amplify all target regions in representative samples from the seven families. The conserved genomic regions and PCR primers can be used in diverse areas of plant research, including DNA barcoding, molecular ecology, metagenomics or phylogeny. [ABSTRACT FROM AUTHOR]

We characterize thirteen polymorphic microsatellite loci isolated from Naja atra genomic libraries, which were enriched for AC-motif microsatellites. The thirteen loci were screened on a group of 48 individuals from two populations, one in Yong’an and the other in Ganzhou. These markers revealed a relatively high degree of genetic diversity (4–12 alleles per locus) and heterozygosity (Ho ranged from 0.213–0.854 and He ranged from 0.301–0.838). Tests for departure from Hardy-Weinberg equilibrium and for linkage disequilibrium were conducted for each of the two populations separately. After sequential Bonferroni correction, none of the 13 loci showed significant departures from Hardy-Weinberg equilibrium. Hierarchical analysis of molecular variance indicated that a small but significant (P < 0.001) proportion (16.0%) of the total variation in the microsatellite DNA data were attributable to differences among populations, indicating geographical structuring and restricted gene flow. It could be attributable to the Wuyi mountains in the area having a sufficiently isolating effect to significantly reduce gene flow. Our microsatellite data also showed a low Nm (1.31) value in the two populations from mainland China. Thus, the Yong’an and Ganzhou populations could be treated as distinct evolutionarily significant units (ESUs). The high level of polymorphism revealed by these microsatellite markers will be useful for the study of gene flow, population structure and evolutionary history of N. atra.

Dye-decolorizing peroxidases (DyPs) were classified as a new family of heme peroxidase in 2007. Produced by various bacteria, they are the first bacterial enzymes known able to degrade lignin and dyes, for which their application in waste treatment and pretreatment of lignocellulosic biomass could be envisaged. In this work, a PCR primer pair was created and tested that enabled the detection and quantification of a wide range of bacterial genes of P class DyP in complex matrices. In addition, a phylogenetic tree was built with all available sequences of DyP genes available, offering a first overview of their presence in the bacteria kingdom. [ABSTRACT FROM AUTHOR]

Waterborne viruses are increasingly being considered in risk assessment schemes. In general, virus detection by culture methods is time consuming. In contrast, detection by quantitative polymerase chain reaction (qPCR) is more rapid and therefore, more suitable for monitoring. At present, qPCR lacks the essential ability for discriminating between infectious and non-infectious viruses, thus limiting its applicability for monitoring disinfection processes. In this study, a method was developed to quantify UV inactivation by long amplicon (LA)-qPCR. Bacteriophage phiX174 was used as a surrogate for human pathogenic viruses. A qPCR protocol was developed with new sets of primers, resulting in amplicon lengths of 108, 250, 456, 568, 955, 1063, 1544, and 1764 nucleotides. The log reduction of gene copies increased with increasing amplicon length. Additional treatment with the intercalating dye, PMA, had no effect, indicating that the bacteriophage capsids were not damaged by low pressure UV irradiation. A qPCR of nearly the complete genome (approx. 5000 nucleotides) showed similar results to the plaque assay. The log reduction in qPCR correlates with [specific amplicon length x UV dose]. The normalized DNA effect constant can be applied to calculate phiX174 inactivation based on qPCR detection. [ABSTRACT FROM AUTHOR]

The polymorphisms of soil fungal rDNA Internal Transcribed Spacer regions were studied in Korean pine forests of various ages (10-100-year-old trees) by means of cloned libraries, and analyzed to determine the effects of the trees' developmental stage on soil fungal community structure. The obtained Shannon diversity index (H) and richness (S) indicated that the diversity of the soil fungal community increased significantly with the development of Korean pines ( P < 0.05). In addition, cluster analysis (UPGMA) showed that the soil fungal community variety associated with differently aged Korean pines was higher than 50 %. The soil fungal community diversity correlated significantly with the N content and C/N ratio of the soil ( P < 0.05). The results of this study indicate that the age of in Korean pine can affect soil fungal community by altering soil properties, which in turn could affect the nutrient cycling in the forest ecosystem. [ABSTRACT FROM AUTHOR]

Mitochondrial genomes (mtDNAs or mitogenomes) of seed plants are characterized by a notoriously unstable organization on account of which available so-called universal or consensus primers may fail to fulfil their foreseen function - amplification of various mtDNA regions in a broad range of plant taxa. Thus, the primers developed for groups assumed to have similar organization of their mitogenomes, such as families, may facilitate a broader usage of more variable non-coding portions of these genomes in group members. Using in silico PCR method and six available complete mitogenomes of Fabaceae, it has been demonstrated that only three out of 36 published universal primer and three Medicago sativa-specific primer pairs that amplify various mtDNA regions are suitable for six representatives of the Fabaceae family upon minor modifications, and develop 21 Fabaceae-specific primer pairs for amplification of all 14 cis-splicing introns in genes of NADH subunits (nad genes) which represent the most commonly used noncoding mtDNA regions in various studies in plants. Using the same method and six available complete mitogenomes of representatives of related families Cucurbitaceae, Euphorbiaceae and Rosaceae and a model plant, Arabidopsis thaliana, it has further been demonstrated that applicability of newly developed primer pairs for amplification of nad introns in more or less related taxa was dependent not only on species evolutionary distances but also on their genome sizes. A reported set of 24 primer pairs is a valuable resource which may facilitate a broader usage of mtDNA variability in future studies at both intra- and inter-specific levels in Fabaceae, which is the third largest family of flowering plants rarely studied at the mtDNA level, and in other more or less related taxa. [ABSTRACT FROM AUTHOR]

Fungi have developed a wide assortment of enzymes to break down pectin, a prevalent polymer in plant cell walls that is important in plant defense and structure. One enzyme family used to degrade pectin is the glycosyl hydrolase family 28 (GH28). In this study we developed primers for the amplification of GH28 coding genes from a database of 293 GH28 sequences from 40 fungal genomes. The primers were used to successfully amplify GH28 pectinases from all Ascomycota cultures tested, but only three out of seven Basidiomycota cultures. In addition, we further tested the primers in PCRs on metagenomic DNA extracted from senesced tree leaves from different forest ecosystems, followed by cloning and sequencing. Taxonomic specificity for Ascomycota GH28 genes was tested by comparing GH28 composition in leaves to internal transcribed spacer (ITS) amplicon composition using pyrosequencing. All sequences obtained from GH28 primers were classified as Ascomycota; in contrast, ITS sequences indicated that fungal communities were up to 39% Basidiomycetes. Analysis of leaf samples indicated that both forest stand and ecosystem type were important in structuring fungal communities. However, site played the prominent role in explaining GH28 composition, whereas ecosystem type was more important for ITS composition, indicating possible genetic drift between populations of fungi. Overall, these primers will have utility in understanding relationships between fungal community composition and ecosystem processes, as well as detection of potentially pathogenic Ascomycetes. [ABSTRACT FROM AUTHOR]

Despite the advent of next-generation sequencing, the polymerase chain reaction (PCR) and Sanger sequencing remain useful tools for molecular identification and systematics. To date, molecular systematics of scale insects has been constrained by the paucity of loci that researchers have been able to amplify with available PCR primers. Due to the rapid molecular evolution of scale insects, "universal" primers, and even primers developed for their sister taxon the Aphidoidea, typically fail. We used transcriptome data for two diaspidids, Acutaspis umbonifera (Newstead) and Chrysomphalus aonidum (Linnaeus), together with a published aphid genome, to design novel PCR primer sets for scale insects. Our primers amplify fragments of eight single-copy genes: ATP-dependent RNA helicase (DHX8), translation initiation factor 5 (IF5X1), DNA replication licensing factor (Mcm2), double-strand break repair protein (MRE11A), serine/threonineprotein phosphatase (PPP1CB), DNA-directed RNA polymerase II (RNApII), ribonucleoside-diphosphate reductase (RRM1), signal recognition particle receptor (SRPα), neuronal PAS domain-containing protein 4 (NPAS4), and cleft lip and palate transmembrane protein 1 (TP1). Here we report the results of tests of amplification success and phylogenetic utility of these primer sets across the Diaspididae and nine other families of Coccomorpha. [ABSTRACT FROM AUTHOR]

Freshwater biodiversity is under pressure from the detrimental effects of climate change, habitat degradation, biological invasion, and overfishing. Environmental DNA (eDNA) obtained directly from environmental samples can be used to evaluate the distribution of aquatic species. We developed a new set of universal PCR primers (16 S 200) for eDNA metabarcoding from mitochondrial DNA sequences. Using the new 16 S 200 primers, we detected the presence of 27 fish species distributed across four families in Lake Gehu, in southeastern China. eDNA metabarcoding and live capture methods identified 16 species in common; however, the two methods identified nine and 12 unique species, respectively. Data from eDNA metabarcoding were more consistent with results from gill net capture methods than from ground cage capture. The present study supports the effectiveness of eDNA for rapidly assessing the species composition of fish communities.This is the first attempt to designed new sets of primers for eDNA-based metabarcoding analysis of freshwater fish in China. Despite pitfalls and limitations, eDNA metabarcoding is a promising approach for assessing fish communities in freshwater lakes. Current applications of eDNA are widespread, but the new technology requires further refinement. The eDNA metabarcoding is an efficient and cost-effective method that can be used in conjunction with traditional survey methods for analyzing fish communities. [ABSTRACT FROM AUTHOR]

Background: A priority for conservation is the identification of endemic populations. We developed microsatellite markers for common raven (Corvus corax), a bird species with a Holarctic distribution, to identify and assess endemic populations in Alaska. Results: From a total of 50 microsatellite loci, we isolated and characterized 15 loci. Eight of these loci were polymorphic and readily scoreable. Eighteen to 20 common ravens from Fairbanks, Alaska were genotyped showing the following variability: 3-8 alleles per locus, 0.25-0.80 observed heterozygosity (Ho), and 0.30-0.80 expected heterozygosity (He). All loci were in Hardy-Weinberg equilibrium and linkage equilibrium and many loci amplified and were polymorphic in related taxa. Conclusions: These loci will be used to identify endemic populations of common raven and assess their genetic diversity and connectivity. [ABSTRACT FROM AUTHOR]

16S rDNA sequencing is an established technique for molecular identification and phylogenetic studies of bacteria. In most of the previous studies, single set of primers (16S forward and 16S reverse) was unlikely to amplify and sequence full fragment of 16S gene and the product size remained shorter. Thus, an improved method was adopted to sequence the full-length 16S rDNA fragment employing an additional set of internal primers (Henk 16S and Hoff 16S) along with external primers (Loff 16SF and Loff 16SR) in a single run to enhance the precision in bacterial identification by getting a sequence length of over 1300 bp. Moreover, we presented another way to tackle the missing sequences and some faulty readings in the raw sequence data that appear to be another impediment causing imprecise identification of bacteria. Therefore, peaks cross-matching was done while editing the raw sequence data prior to alignment through NCBI BLAST. Finally, CAP3 sequence assembly online program was employed for precise assembling of the sequence data obtained from each of the four primers to obtain a single contiguous sequence for greater precision in bacterial identification. This approach will certainly help taxonomists to describe novel bacterial species. [ABSTRACT FROM AUTHOR]

Recent metagenomic analyses of Yellowstone National Park (YNP) thermal spring communities suggested the presence of minor archaeal populations that simultaneous PCR-based assays using traditional 'universal' 16S rRNA gene primers failed to detect. Here we use metagenomics to identify PCR primers effective at detecting elusive members of the Archaea, assess their efficacy, and describe the diverse and novel archaeal community from a circum-neutral thermal spring from the Bechler region of YNP. We determined that a less commonly used PCR primer, Arch349F, captured more diversity in this spring than the widely used A21F primer. A search of the PCR primers against the RDP 16S rRNA gene database indicated that Arch349F also captured the largest percentage of Archaea, including 41 % more than A21F. Pyrosequencing using the Arch349F primer recovered all of the phylotypes present in the clone-based portion of the study and the metagenome of this spring in addition to several other populations of Archaea, some of which are phylogenetically novel. In contrast to the lack of amplification with traditional 16S rRNA gene primers, our comprehensive analyses suggested a diverse archaeal community in the Bechler spring, with implications for recently discovered groups such as the Geoarchaeota and other undescribed archaeal groups. [ABSTRACT FROM AUTHOR]

We isolated and characterized a total of 33 microsatellite loci from the Patagonian sprat Sprattus fuegensis, a recent exploited marine resource with a conservation status unknowing. Loci were screened in 24 individuals from the inshore waters of the Aysén Fjord, Chile. The number of alleles per locus ranged from 7 to 24, observed heterozygosity ranged from 0.217 to 0.875, and the probability of identity values ranged from 0.006 to 0.133. These new loci will provide tools for examining population genetic structure, estimating effective population size and provide information to fisheries management and conservation. [ABSTRACT FROM AUTHOR]

We isolated and characterized a total of 22 microsatellite loci for the leafcutter ant, Acromyrmex lundii. The loci were screened for 24 individuals from southern Brazil and Uruguay. The number of alleles per locus ranged from 5 to 20, the observed heterozygosity ranged from 0.417 to 0.917, and the probability of identity values ranged from 0.011 to 0.38. These genetic markers will be useful for understanding the population and conservation biology of the leafcutter ant A. lundii and closely related species, and will provide novel insights into the evolutionary biology of social parasitism and leafcutter ant mating systems. [ABSTRACT FROM AUTHOR]

We isolated and characterized a total of 31 microsatellite loci from the mole salamander, Ambystoma talpoideum. Loci were screened in 20 individuals from a single location in Aiken, South Carolina. The number of alleles per locus ranged from 3 to 11, observed heterozygosity ranged from 0.000 to 0.700, and the probability of identity values ranged from 0.031 to 0.400. These new loci will provide tools for examining the genetic diversity, structure, mating system, and adult morph determination of A. talpoideum. [ABSTRACT FROM AUTHOR]

The Saccharomycetales or 'true yeasts' consist of more than 800 described species, including many of scientific, medical and commercial importance. Considerable progress has been made in determining the phylogenetic relationships of these species, largely based on r DNA sequences, but many nodes for early-diverging lineages cannot be resolved with r DNA alone. r DNA is also not ideal for delineating recently diverged species. From published full-genome sequence data, we have identified 14 regions of protein-coding genes that can be PCR-amplified in a large proportion of a diverse collection of 25 yeast species using degenerate primers. Phylogenetic analysis of the sequences thus obtained reveals a well-resolved phylogeny of the Saccharomycetales with many branches having high bootstrap support. Analysis of published sequences from the Saccharomyces paradoxus species complex shows that these protein-coding gene fragments are also informative about genealogical relationships amongst closely related strains. Our set of protein-coding gene fragments is therefore suitable for analysing both ancient and recent evolutionary relationships amongst yeasts. [ABSTRACT FROM AUTHOR]