scholarly journals Real-Time PCR and Sequencing Assays for Rapid Detection and Identification of Avian Schistosomes in Environmental Samples

2015 ◽  
Vol 81 (12) ◽  
pp. 4207-4215 ◽  
Author(s):  
Narayanan Jothikumar ◽  
Bonnie J. Mull ◽  
Sara V. Brant ◽  
Eric S. Loker ◽  
Jeremy Collinson ◽  
...  
Keyword(s):  
Lake Water ◽  
Water Samples ◽  
18S Rdna ◽  
28S Rdna ◽  
Taqman Assay ◽  
Rrna Gene ◽  
Content Type ◽  
Cercarial Dermatitis ◽  
Rdna Sequencing ◽  
Avian Schistosomes

ABSTRACTCercarial dermatitis, also known as swimmer's itch, is an allergenic skin reaction followed by intense itching caused by schistosome cercariae penetrating human skin. Cercarial dermatitis outbreaks occur globally and are frequently associated with freshwater lakes and are occasionally associated with marine or estuarine waters where birds reside year-round or where migratory birds reside. In this study, a broadly reactive TaqMan assay targeting 18S rRNA gene (ribosomal DNA [rDNA]) sequences that was based on a genetically diverse panel of schistosome isolates representing 13 genera and 20 species (the 18S rDNA TaqMan assay) was developed. A PCR assay was also developed to amplify a 28S rDNA region for subsequent sequencing to identify schistosomes. When applied to surface water samples seeded withSchistosoma mansonicercariae, the 18S rDNA TaqMan assay enabled detection at a level of 5S. mansonicercariae in 100 liters of lake water. The 18S rDNA TaqMan and 28S rDNA PCR sequencing assays were also applied to 100-liter water samples collected from lakes in Nebraska and Wisconsin where there were reported dermatitis outbreaks. Avian schistosome DNA was detected in 11 of 34 lake water samples using the TaqMan assay. Further 28S rDNA sequence analysis of positive samples confirmed the presence of avian schistosome DNA and provided a preliminary identification of the avian schistosomes in 10 of the 11 samples. These data indicate that the broadly schistosome-reactive TaqMan assay can be effective for rapid screening of large-volume water samples for detection of avian schistosomes, thereby facilitating timely response actions to mitigate or prevent dermatitis outbreaks. Additionally, samples positive by the 18S rDNA TaqMan assay can be further assayed using the 28S rDNA sequencing assay to both confirm the presence of schistosomes and contribute to their identification.

scholarly journals Comparison of Gull Feces-Specific Assays Targeting the 16S rRNA Genes of Catellicoccus marimammalium and Streptococcus spp.

2012 ◽  
Vol 78 (6) ◽  
pp. 1909-1916 ◽  
Author(s):  
Hodon Ryu ◽  
John F. Griffith ◽  
Izhar U. H. Khan ◽  
Stephen Hill ◽  
Thomas A. Edge ◽  
...  
Keyword(s):  
16S Rrna ◽  
Water Samples ◽  
Environmental Water ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Taqman Assay ◽  
Rrna Gene ◽  
Recreational Waters ◽  
Fecal Samples ◽  
Content Type

ABSTRACTTwo novel gull-specific quantitative PCR (qPCR) assays were developed using 16S rRNA gene sequences from gull fecal clone libraries: a SYBR green assay targetingStreptococcusspp. (gull3) and a hydrolysis TaqMan assay targetingCatellicoccus marimammalium(gull4). The objectives of this study were to compare the host specificity of a previousC. marimammaliumqPCR assay (gull2) with that of the new markers and to examine the presence of the three gull markers in environmental water samples from different geographic locations. Most of the gull fecal samples tested (n= 255) generated positive signals with the gull2 and gull4 assays (i.e., >86%), whereas only 28% were positive with gull3. Low prevalence and abundance of tested gull markers (0.6 to 15%) were observed in fecal samples from six nonavian species (n= 180 fecal samples), whereas the assays cross-reacted to some extent (13 to 31%) with other (nongull) avian fecal samples. The gull3 assay was positive against fecal samples from 11 of 15 avian species, including gull. Of the presumed gull-impacted water samples (n= 349), 86%, 59%, and 91% were positive with the gull2, the gull3, and the gull4 assays, respectively. Approximately 5% of 239 non-gull-impacted water samples were positive with the gull2 and the gull4 assays, whereas 21% were positive witg the gull3 assay. While the relatively high occurrence of gull2 and gull4 markers in waters impacted by gull feces suggests that these assays could be used in environmental monitoring studies, the data also suggest that multiple avian-specific assays will be needed to accurately assess the contribution of different avian sources in recreational waters.

scholarly journals Highly Specific Sewage-DerivedBacteroidesQuantitative PCR Assays Target Sewage-Polluted Waters

2019 ◽  
Vol 85 (6) ◽  
Author(s):  
Shuchen Feng ◽  
Sandra L. McLellan
Keyword(s):  
Population Structure ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Water Samples ◽  
Rrna Gene ◽  
Content Type ◽  
Hypervariable Regions ◽  
Animal Hosts ◽  
Pcr Assays ◽  
The 16S Rrna Gene

ABSTRACTThe identification of sewage contamination in water has primarily relied on the detection of human-associatedBacteroidesusing markers within the V2 region of the 16S rRNA gene. Despite the establishment of multiple assays that target the HF183 cluster (i.e.,Bacteroides dorei) and otherBacteroidesorganisms (e.g.,Bacteroides thetaiotaomicron), the potential for more human-associated markers in this genus has not been explored in depth. We examined theBacteroidespopulation structure in sewage and animal hosts across the V4V5 and V6 hypervariable regions. Using near-full-length cloned sequences, we identified the sequences in the V4V5 and V6 hypervariable regions that are linked to the HF183 marker in the V2 region and found these sequences were present in multiple animals. In addition, the V4V5 and V6 regions contained human fecal marker sequences for organisms that were independent of the HF183 cluster. The most abundantBacteroidesin untreated sewage was not human associated but pipe derived. Two TaqMan quantitative PCR (qPCR) assays targeting the V4V5 and V6 regions of this organism were developed. Validation studies using fecal samples from seven animal hosts (n = 76) and uncontaminated water samples (n = 30) demonstrated the high specificity of the assays for sewage. FreshwaterBacteroideswere also identified in uncontaminated water samples, demonstrating that measures of totalBacteroidesdo not reflect fecal pollution. A comparison of two previously described humanBacteroidesassays (HB and HF183/BacR287) in municipal wastewater influent and sewage-contaminated urban water samples revealed identical results, illustrating the assays target the same organism. The detection of sewage-derivedBacteroidesprovided an independent measure of sewage-impacted waters.IMPORTANCEBacteroidesare major members of the gut microbiota, and host-specific organisms within this genus have been used extensively to gain information on pollution sources. This study provides a broad view of the population structure ofBacteroideswithin sewage to contextualize the well-studied HF183 marker for a human-associatedBacteroides. The study also delineates host-specific sequence patterns across multiple hypervariable regions of the 16S rRNA gene to improve our ability to use sequence data to assess water quality. Here, we demonstrate that regions downstream of the HF183 marker are nonspecific but other potential human-associated markers are present. Furthermore, we show the most abundantBacteroidesin sewage is free living, rather than host associated, and specifically found in sewage. Quantitative PCR assays that target organisms specific to sewer pipes offer measures that are independent of the human microbiome for identifying sewage pollution in water.

scholarly journals Comparison of Filters for Concentrating Microbial Indicators and Pathogens in Lake Water Samples

2012 ◽  
Vol 79 (4) ◽  
pp. 1342-1352 ◽  
Author(s):  
Donna S. Francy ◽  
Erin A. Stelzer ◽  
Amie M. G. Brady ◽  
Carrie Huitger ◽  
Rebecca N. Bushon ◽  
...  
Keyword(s):  
Lake Water ◽  
Water Samples ◽  
Regional Scale ◽  
Cost Effective ◽  
Bacterial Indicators ◽  
Content Type ◽  
Filter Performance ◽  
Dead End ◽  
Field Deployment ◽  
Norovirus Gii

ABSTRACTBacterial indicators are used to indicate increased health risk from pathogens and to make beach closure and advisory decisions; however, beaches are seldom monitored for the pathogens themselves. Studies of sources and types of pathogens at beaches are needed to improve estimates of swimming-associated health risks. It would be advantageous and cost-effective, especially for studies conducted on a regional scale, to use a method that can simultaneously filter and concentrate all classes of pathogens from the large volumes of water needed to detect pathogens. In seven recovery experiments, stock cultures of viruses and protozoa were seeded into 10-liter lake water samples, and concentrations of naturally occurring bacterial indicators were used to determine recoveries. For the five filtration methods tested, the highest median recoveries were as follows: glass wool for adenovirus (4.7%); NanoCeram for enterovirus (14.5%) and MS2 coliphage (84%); continuous-flow centrifugation (CFC) plus Virocap (CFC+ViroCap) forEscherichia coli(68.3%) andCryptosporidium(54%); automatic ultrafiltration (UF) for norovirus GII (2.4%); and dead-end UF forEnterococcus faecalis(80.5%), avian influenza virus (0.02%), andGiardia(57%). In evaluating filter performance in terms of both recovery and variability, the automatic UF resulted in the highest recovery while maintaining low variability for all nine microorganisms. The automatic UF was used to demonstrate that filtration can be scaled up to field deployment and the collection of 200-liter lake water samples.

scholarly journals Development of Quantitative PCR Assays Targeting the 16S rRNA Genes of Enterococcus spp. and Their Application to the Identification of Enterococcus Species in Environmental Samples

2012 ◽  
Vol 79 (1) ◽  
pp. 196-204 ◽  
Author(s):  
Hodon Ryu ◽  
Michael Henson ◽  
Michael Elk ◽  
Carlos Toledo-Hernandez ◽  
John Griffith ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Quantitative Pcr ◽  
Water Samples ◽  
The Other ◽  
Rrna Gene ◽  
Fecal Samples ◽  
Content Type ◽  
Specific Assay ◽  
Enterococcal Species

ABSTRACTThe detection of environmental enterococci has been determined primarily by using culture-based techniques that might exclude some enterococcal species as well as those that are nonculturable. To address this, the relative abundances of enterococci were examined by challenging fecal and water samples against a currently available genus-specific assay (Entero1). To determine the diversity of enterococcal species, 16S rRNA gene-based group-specific quantitative PCR (qPCR) assays were developed and evaluated against eight of the most common environmental enterococcal species. Partial 16S rRNA gene sequences of 439 presumptive environmental enterococcal strains were analyzed to study further the diversity of enterococci and to confirm the specificities of group-specific assays. The group-specific qPCR assays showed relatively high amplification rates with targeted species (>98%), although some assays cross-amplified with nontargeted species (1.3 to 6.5%). The results with the group-specific assays also showed that different enterococcal species co-occurred in most fecal samples. The most abundant enterococci in water and fecal samples wereEnterococcus faecalisandEnterococcus faecium, although we identified more water isolates asEnterococcus casseliflavusthan as any of the other species. The prevalence of the Entero1 marker was in agreement with the combined number of positive signals determined by the group-specific assays in most fecal samples, except in gull feces. On the other hand, the number of group-specific assay signals was lower in all water samples tested, suggesting that other enterococcal species are present in these samples. While the results highlight the value of genus- and group-specific assays for detecting the major enterococcal groups in environmental water samples, additional studies are needed to determine further the diversity, distributions, and relative abundances of all enterococcal species found in water.

scholarly journals Rapid TaqMan-Based Quantification of Chlorophylld-Containing Cyanobacteria in the Genus Acaryochloris

2014 ◽  
Vol 80 (10) ◽  
pp. 3244-3249 ◽  
Author(s):  
Lars Behrendt ◽  
Jeppe L. Nielsen ◽  
Søren J. Sørensen ◽  
Anthony W. D. Larkum ◽  
Jakob R. Winther ◽  
...  
Keyword(s):  
16S Rrna ◽  
Environmental Samples ◽  
Global Distribution ◽  
Coralline Algae ◽  
Taqman Probe ◽  
Gene Copy ◽  
Substantial Contribution ◽  
Taqman Assay ◽  
Rrna Gene ◽  
Content Type

ABSTRACTReports of the chlorophyll (Chl)d-containing cyanobacteriumAcaryochlorishave accumulated since its initial discovery in 1996. The majority of this evidence is based on amplification of the gene coding for the 16S rRNA, and due to the wide geographical distribution of these sequences, a global distribution ofAcaryochlorisspecies was suggested. Here, we present a rapid, reliable, and cost-effective TaqMan-based quantitative PCR (qPCR) assay that was developed for the specific detection ofAcaryochlorisspecies in complex environmental samples. The TaqMan probe showed detection limits of ∼10 16S rRNA gene copy numbers based on standard curves consisting of plasmid inserts. DNA from fiveAcaryochlorisstrains, i.e., MBIC11017, CCMEE5410, HICR111A, CRS, and Awaji-1, exhibited amplification efficiencies of >94% when tested in the TaqMan assay. When used on complex natural communities, the TaqMan assay detected the presence ofAcaryochlorisspecies in four out of eight samples of crustose coralline algae (CCA), collected from temperate and tropical regions. In three out of these TaqMan-positive samples, the presence of Chldwas confirmed via high-performance liquid chromatography (HPLC), and corresponding cell estimates ofAcaryochlorisspecies amounted to 7.6 × 101to 3.0 × 103per mg of CCA. These numbers indicate a substantial contribution of Chld-containing cyanobacteria to primary productivity in endolithic niches. The new TaqMan assay allows quick and easy screening of environmental samples for the presence ofAcaryochlorisspecies and is an important tool to further resolve the global distribution and significance of this unique oxyphototroph.

scholarly journals Phenylobacterium parvum sp. nov., isolated from lake water

2019 ◽  
Vol 69 (4) ◽  
pp. 1169-1172 ◽  
Author(s):  
Chaeyun Baek ◽  
Su-Kyoung Shin ◽  
Hana Yi
Keyword(s):  
Lake Water ◽  
Type Species ◽  
High Ratio ◽  
Rrna Gene ◽  
Enzymatic Reactions ◽  
Aerobic Bacterium ◽  
Phenotypic Properties ◽  
Content Type ◽  
Link Type ◽  
Sequence Similarities

A Gram-stain-negative, rod-shaped and aerobic bacterium, designated HYN0004T, was isolated from lake water. The strain grew at 15–35 °C and pH 7.0–9.0 on R2A. The isoprenoid quinone was Q10 and major polar lipids were phosphatidylglycerol and one unidentified glycolipid. The genome was 2.83 Mb with a DNA G+C content of 69.9 mol%. 16S rRNA gene sequence analyses revealed that HYN0004T represented a member of the genus Phenylobacterium and shared sequence similarities with Phenylobacterium conjunctum (97.8 %), Phenylobacterium koreense (97.5 %), Phenylobacterium aquaticum (97.2 %), and Phenylobacterium heamatophilum (97.0 %). In addition to the low sequence similarities, the phylogenetic tree shapes indicated that HYN0004Trepresents an independent species of this genus. The genomic and phenotypic properties, including small genome size, inability to carry out numerous enzymatic reactions and high ratio of C18 : 1ω6c and/or C18 : 1ω7c in fatty acids, verified the differentiation between HYN0004T and related species. Thus, we propose a novel species of the genus Phenylobacterium , named as Phenylobacterium parvum sp. nov. The type strain is HYN0004T (=KACC 19185T=NBRC 112736T).

scholarly journals Chitinimonas prasina sp. nov., isolated from lake water

2014 ◽  
Vol 64 (Pt_9) ◽  
pp. 3005-3009 ◽  
Author(s):  
Yi Li ◽  
Hong Zhu ◽  
Qiliang Lai ◽  
Xueqian Lei ◽  
Zhangran Chen ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Lake Water ◽  
Type Species ◽  
Gene Sequence ◽  
16S Rrna Gene Sequence ◽  
Rrna Gene ◽  
Rrna Gene Sequence ◽  
Content Type ◽  
Link Type

A Gram-stain-negative, elongated rod-shaped, motile by gliding, green-pigmented, aerobic bacterial strain, designated LY03T, was isolated from lake water in Xiamen, Fujian Province, China. Phylogenetic analysis based on 16S rRNA gene sequencing revealed that the isolate was a member of the genus Chitinimonas , which belongs to the family Burkholderiaceae . Strain LY03T was most closely related to Chitinimonas taiwanensis LMG 22011T (96.02 % 16S rRNA gene sequence similarity), followed by Chitinimonas koreensis KACC 11467T (94.85 %), and the three strains formed a distinct lineage from other strains in the phylogenetic analyses. Optimum conditions for growth were 37 °C, pH 7–9 and without NaCl. The major fatty acids were summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c), C16 : 0 and C10 : 0 3-OH. The DNA G+C content of strain LY03T was 63.6 mol% and the major respiratory quinone was ubiquinone-8 (Q-8). The polar lipids were found to consist of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, unknown polar lipids and unidentified phospholipids. Differential phenotypic properties and phylogenetic distinctiveness distinguished strain LY03T from all other members of the genus Chitinimonas . On the basis of its morphology, physiology, fatty acid composition and 16S rRNA gene sequence data, strain LY03T represents a novel species of the genus Chitinimonas , for which the name Chitinimonas prasina sp. nov. is proposed. The type strain is LY03T ( = MCCC 1F01209T = KCTC 32574T).

scholarly journals Molecular Detection of Campylobacter spp. and Fecal Indicator Bacteria during the Northern Migration of Sandhill Cranes (Grus canadensis) at the Central Platte River

2013 ◽  
Vol 79 (12) ◽  
pp. 3762-3769 ◽  
Author(s):  
Jingrang Lu ◽  
Hodon Ryu ◽  
Jason Vogel ◽  
Jorge Santo Domingo ◽  
Nicholas J. Ashbolt
Keyword(s):  
Water Samples ◽  
Rrna Gene ◽  
Fecal Indicator ◽  
Platte River ◽  
Content Type ◽  
Grus Canadensis ◽  
E Coli ◽  
Sandhill Crane ◽  
Specific Pcr ◽  
Pcr Assays

ABSTRACTThe risk to human health of the annual sandhill crane (Grus canadensis) migration through Nebraska, which is thought to be a major source of fecal pollution of the central Platte River, is unknown. To better understand potential risks, the presence ofCampylobacterspecies and three fecal indicator bacterial groups (Enterococcusspp.,Escherichia coli, andBacteroidetes) was assayed by PCR from crane excreta and water samples collected during their stopover at the Platte River, Nebraska, in 2010. Genus-specific PCR assays and sequence analyses identifiedCampylobacter jejunias the predominantCampylobacterspecies in sandhill crane excreta.Campylobacterspp. were detected in 48% of crane excreta, 24% of water samples, and 11% of sediment samples. The estimated densities ofEnterococcusspp. were highest in excreta samples (mean, 4.6 × 108cell equivalents [CE]/g), while water samples contained higher levels ofBacteroidetes(mean, 5.1 × 105CE/100 ml).Enterococcusspp.,E. coli, andCampylobacterspp. were significantly increased in river water and sediments during the crane migration period, withEnterococcussp. densities (∼3.3 × 105CE/g) 2 to 4 orders of magnitude higher than those ofBacteroidetes(4.9 × 103CE/g),E. coli(2.2 × 103CE/g), andCampylobacterspp. (37 CE/g). Sequencing data for the 16S rRNA gene andCampylobacterspecies-specific PCR assays indicated thatC. jejuniwas the majorCampylobacterspecies present in water, sediments, and crane excreta. Overall, migration appeared to result in a significant, but temporary, change in water quality in spring, when there may be aC. jejunihealth hazard associated with water and crops visited by the migrating birds.

scholarly journals Development and Evaluation of a Quantitative PCR Assay Targeting Sandhill Crane (Grus canadensis) Fecal Pollution

2012 ◽  
Vol 78 (12) ◽  
pp. 4338-4345 ◽  
Author(s):  
Hodon Ryu ◽  
Jingrang Lu ◽  
Jason Vogel ◽  
Michael Elk ◽  
Felipe Chávez-Ramírez ◽  
...  
Keyword(s):  
Host Specificity ◽  
Quantitative Pcr ◽  
Water Samples ◽  
False Negative ◽  
Environmental Water ◽  
Fecal Pollution ◽  
Source Tracking ◽  
Rrna Gene ◽  
Content Type ◽  
Sandhill Crane

ABSTRACTWhile the microbial water quality in the Platte River is seasonally impacted by excreta from migrating cranes, there are no methods available to study crane fecal contamination. Here we characterized microbial populations in crane feces using phylogenetic analysis of 16S rRNA gene fecal clone libraries. Using these sequences, a novel crane quantitative PCR (Crane1) assay was developed, and its applicability as a microbial source tracking (MST) assay was evaluated by determining its host specificity and detection ability in environmental waters. Bacteria from crane excreta were dominated by bacilli and proteobacteria, with a notable paucity of sequences homologous toBacteroidetesandClostridia. The Crane1 marker targeted a dominant clade of unclassifiedLactobacillalessequences closely related toCatellicoccus marimammalium. The host distribution of the Crane1 marker was relatively high, being positive for 69% (66/96) of the crane excreta samples tested. The assay also showed high host specificity, with 95% of the nontarget fecal samples (i.e.,n= 553; 20 different free-range hosts) being negative. Of the presumed crane-impacted water samples (n= 16), 88% were positive for the Crane1 assay, whereas none of the water samples not impacted by cranes were positive (n= 165). Bayesian statistical models of the Crane1 MST marker demonstrated high confidence in detecting true-positive signals and a low probability of false-negative signals from environmental water samples. Altogether, these data suggest that the newly developed marker could be used in environmental monitoring studies to study crane fecal pollution dynamics.

Isolating some fungi strains from diease roots of Dipterocarpus dyeri planted at the nursery in Dong Nai province

2020 ◽  
pp. 187-194
Keyword(s):  
Aspergillus Nidulans ◽  
28S Rdna ◽  
Timber Industry ◽  
Urban Streets ◽  
Rdna Sequencing ◽  
Moist Forest

Dipterocarpus dyeri is a typical plant of tropical evergreen moist forest at Southeast Vietnam. These plants have been planted popularly at parks and urban streets for the shade and it has been commonly materials for timber industry. Multiplication of Dipterocarpus dyeri at nurseries could face to some diseases, such as the withered disease cause serial death. Our study isolated three disease fungi strains from the root areas of the diseased Dipterocarpus dyeri planted Ma Da nursery, Dong Nai province. Result of 28s rDNA sequencing showed these fungi belong to Ophiostoma eucalypticagena, Aspergillus nidulans and Collectotrichum gloeosporioides. This result is base for conducting the following studies to control the withered disease on Dipterocarpus dyeri at the nursery.

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