scholarly journals Quantitative PCR assay for the detection of Aedes vigilax in mosquito trap collections containing large numbers of morphologically similar species and phylogenetic analysis of specimens collected in Victoria, Australia

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Peter T. Mee ◽  
Shani Wong ◽  
Karen Brown ◽  
Stacey E. Lynch
Keyword(s):  
Phylogenetic Analysis ◽  
Quantitative Pcr ◽  
Coastal Areas ◽  
Qpcr Assay ◽  
Detection Sensitivity ◽  
Similar Species ◽  
Pest Species ◽  
Mosquito Trap ◽  
Aedes Vigilax ◽  
Large Numbers

Abstract Background Aedes vigilax is one of the most significant arbovirus vector and pest species in Australia’s coastal regions. Occurring in multiple countries, this mosquito species occurs as a species complex which has been separated into three clades with two detected in Australia. Until recently, Ae. vigilax has largely been absent from Victoria, only occasionally caught over the years, with no reported detections from 2010 to 2016. Complicating the detection of Ae. vigilax is the shared sympatric distribution to the morphologically similar Ae. camptorhynchus, which can exceed 10,000 mosquitoes in a single trap night in Victoria. Currently, there are no molecular assays available for the detection of Ae. vigilax. We aim to develop a quantitative PCR (qPCR) for the detection of Ae. vigilax, with the specificity and sensitivity of this assay assessed as well as a method to process whole mosquito traps. Methods Trapping was performed during the 2017–2020 mosquito season in Victoria in two coastal areas across these 3 consecutive years. A qPCR assay was designed to allow rapid identification of Ae. vigilax as well as a whole mosquito trap homogenizing and processing methodology. Phylogenetic analysis was performed to determine which clade Ae. vigilax from Victoria was closest to. Results Aedes vigilax was successfully detected each year across two coastal areas of Victoria, confirming the presence of this species. The qPCR assay was proven to be sensitive and specific to Ae. vigilax, with trap sizes up to 1000 mosquitoes showing no inhibition in detection sensitivity. Phylogenetic analysis revealed that Ae. vigilax from Victoria is associated with clade III, showing high sequence similarity to those previously collected in New South Wales, Queensland and Western Australia. Conclusions Aedes vigilax is a significant vector species that shares an overlapping distribution to the morphologically similar Ae. camptorhynchus, making detection difficult. Here, we have outlined the implementation of a specific and sensitive molecular screening assay coupled with a method to process samples for detection of Ae. vigilax in collections with large numbers of non-target species. Graphical abstract

Quantitative PCR Using a Nanofluidic Platform to Quantify Bcr-Abl mRNA in Patients Who Are Negative by Routine Quantitative PCR Testing.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3300-3300 ◽  
Author(s):  
Vivian G. Oehler ◽  
Bret Helton ◽  
Thea Kalebic ◽  
Jerald P. Radich
Keyword(s):  
Quantitative Pcr ◽  
Cell Lines ◽  
Qpcr Assay ◽  
Detection Sensitivity ◽  
Molecular Remission ◽  
Normal Peripheral Blood ◽  
Negative Cell ◽  
Over Time ◽  
Qualitative Pcr

Abstract Abstract 3300 Poster Board III-188 Imatinib mesylate (IM) has dramatically changed the management of chronic phase (CP) chronic myeloid leukemia (CML), with more than 80% of treated patients alive and free from transformation after 8 years of therapy. Long-term studies suggest that molecular response appears to increase in some patients over time and a significant minority of patients eventually obtain a “molecular remission”, where quantitative PCR (QPCR) for the bcr-abl transcript is negative. Two IM cessation trials have been conducted in a total of 90 patients who were consistently negative by QPCR. In each trial ∼40-50% of patients had molecular relapse after IM was stopped. Thus, despite the fact that IM, as well as dasatinib and nilotinib, do not appear to target CML stem cells, it appears that some patients may be able to stop IM therapy in the short-term, if not the long-term. These data suggest that different “kinetic” profiles exist: in some patients bcr-abl continues to decline, whereas in others it may plateau or even increase. In order to study this phenomenon, a more sensitive method of molecular detection is required. We utilized a nanofluidic platform (Fluidigm Corporation, South San Francisco, CA) to detect and quantify rare copies of bcr-abl mRNA. In this assay, detection sensitivity is augmented by sample partitioning prior to PCR, such that multiple QPCR reactions are performed simultaneously in hundreds of replicates. Initial bcr-abl copy number is calculated using the Poisson distribution. The assay was first optimized to reproducibly detect a single copy of bcr-abl using plasmids and bcr-abl positive cell lines in a background of bcr-abl negative cell lines. We then examined longitudinal samples (4-5 samples per patient) from 8 CP CML patients enrolled on the Novartis-sponsored TOPS trial who were in a molecular remission by QPCR. Among 33 patient samples, 7 samples were positive by QPCR and 26 samples were negative by QPCR. The Fluidigm nanofluidic QPCR assay was positive in all 7 samples positive by QPCR and was positive in 11 of 26 quantitative negative PCR samples (42%). Eleven of the 33 samples were also negative by a sensitive nested qualitative PCR assay performed in duplicate. Bcr-abl was detected using the nanofluidic QPCR assay in 1 of 11 of these samples (9%). Bcr-abl was not detected in 3 normal peripheral blood samples or in 3 bcr-abl negative cell lines. In one patient nanofluidic QPCR over time identified increasing bcr-abl 6 months before molecular relapse was evident by routine QPCR (Figure 1a,b). In 5 patients bcr-abl decreased or was stable over time (Figure 1b). In two patients persistently negative by both quantitative and nested qualitative PCR no bcr-abl was detected over time. Conclusion The nanofluidic QPCR assay can detect bcr-abl in ∼40% of cases negative by routine QPCR and can detect increasing bcr-abl copies many months before it is evident by routine QPCR testing. Interestingly, this detection frequency is similar to the relapse rate in molecular remission patients after IM is discontinued. This observation suggests the assay may be valuable in monitoring for molecular relapse, potentially in ongoing IM cessation trials. Disclosures Radich: Novartis: Consultancy, Honoraria, Research Funding.

scholarly journals Distribution of Bacillus thuringiensis subsp. israelensis in Soil of a Swiss Wetland Reserve after 22 Years of Mosquito Control

2011 ◽  
Vol 77 (11) ◽  
pp. 3663-3668 ◽  
Author(s):  
Valeria Guidi ◽  
Nicola Patocchi ◽  
Peter Lüthy ◽  
Mauro Tonolla
Keyword(s):  
Spatial Distribution ◽  
Bacillus Thuringiensis ◽  
Confidence Interval ◽  
Quantitative Pcr ◽  
Mosquito Control ◽  
Soil Samples ◽  
Sampling Point ◽  
Content Type ◽  
Large Numbers ◽  
Continuous Accumulation

ABSTRACTRecurrent treatments withBacillus thuringiensissubsp.israelensisare required to control the floodwater mosquitoAedes vexansthat breeds in large numbers in the wetlands of the Bolle di Magadino Reserve in Canton Ticino, Switzerland. Interventions have been carried out since 1988. In the present study, the spatial distribution of restingB. thuringiensissubsp.israelensisspores in the soil was measured. TheB. thuringiensissubsp.israelensisconcentration was determined in soil samples collected along six transects covering different elevations within the periodically flooded zones. A total of 258 samples were processed and analyzed by quantitative PCR that targeted an identical fragment of 159 bp for theB. thuringiensissubsp.israelensis cry4Aaandcry4Bagenes.B. thuringiensissubsp.israelensisspores were found to persist in soils of the wetland reserve at concentrations of up to 6.8 log per gram of soil. Continuous accumulation due to regular treatments could be excluded, as the decrease in spores amounted to 95.8% (95% confidence interval, 93.9 to 97.7%). The distribution of spores was correlated to the number ofB. thuringiensissubsp.israelensistreatments, the elevation of the sampling point, and the duration of the flooding periods. The number ofB. thuringiensissubsp.israelensistreatments was the major factor influencing the distribution of spores in the different topographic zones (P< 0.0001). These findings indicated thatB. thuringiensissubsp.israelensisspores are rather immobile after their introduction into the environment.

scholarly journals Accurate, Fast and Cost-Effective Diagnostic Test for Monosomy 1p36 Using Real-Time Quantitative PCR

2014 ◽  
Vol 2014 ◽  
pp. 1-8
Author(s):  
Pricila da Silva Cunha ◽  
Heloisa B. Pena ◽  
Carla Sustek D’Angelo ◽  
Celia P. Koiffmann ◽  
Jill A. Rosenfeld ◽  
...  
Keyword(s):  
Real Time ◽  
Diagnostic Test ◽  
Quantitative Pcr ◽  
Target Genes ◽  
Cost Effective ◽  
Qpcr Assay ◽  
Deletion Syndrome ◽  
Comparative Genomic ◽  
Real Time Quantitative Pcr ◽  
Subtelomeric Deletion

Monosomy 1p36 is considered the most common subtelomeric deletion syndrome in humans and it accounts for 0.5–0.7% of all the cases of idiopathic intellectual disability. The molecular diagnosis is often made by microarray-based comparative genomic hybridization (aCGH), which has the drawback of being a high-cost technique. However, patients with classic monosomy 1p36 share some typical clinical characteristics that, together with its common prevalence, justify the development of a less expensive, targeted diagnostic method. In this study, we developed a simple, rapid, and inexpensive real-time quantitative PCR (qPCR) assay for targeted diagnosis of monosomy 1p36, easily accessible for low-budget laboratories in developing countries. For this, we have chosen two target genes which are deleted in the majority of patients with monosomy 1p36:PRKCZandSKI. In total, 39 patients previously diagnosed with monosomy 1p36 by aCGH, fluorescentin situhybridization (FISH), and/or multiplex ligation-dependent probe amplification (MLPA) all tested positive on our qPCR assay. By simultaneously using these two genes we have been able to detect 1p36 deletions with 100% sensitivity and 100% specificity. We conclude that qPCR ofPRKCZandSKIis a fast and accurate diagnostic test for monosomy 1p36, costing less than 10 US dollars in reagent costs.

scholarly journals Development of an Internal Control for Evaluation and Standardization of a Quantitative PCR Assay for Detection of Helicobacter pylori in Drinking Water

2007 ◽  
Vol 73 (22) ◽  
pp. 7380-7387 ◽  
Author(s):  
Keya Sen ◽  
Nancy A. Schable ◽  
Dennis J. Lye
Keyword(s):  
Helicobacter Pylori ◽  
Drinking Water ◽  
Quantitative Pcr ◽  
Water Samples ◽  
Sodium Thiosulfate ◽  
Qpcr Assay ◽  
E Coli ◽  
H Pylori ◽  
Labeled Probe ◽  
Treated Drinking Water

ABSTRACT Due to metabolic and morphological changes that can prevent Helicobacter pylori cells in water from growing on conventional media, an H. pylori-specific TaqMan quantitative PCR (qPCR) assay was developed that uses a 6-carboxyfluorescein-labeled probe (A. E. McDaniels, L. Wymer, C. Rankin, and R. Haugland, Water Res. 39:4808-4816, 2005). However, proper internal controls are needed to provide an accurate estimate of low numbers of H. pylori in drinking water. In this study, the 135-bp amplicon described by McDaniels et al. was modified at the probe binding region, using PCR mutagenesis. The fragment was incorporated into a single-copy plasmid to serve as a PCR-positive control and cloned into Escherichia coli to serve as a matrix spike. It was shown to have a detection limit of five copies, using a VIC dye-labeled probe. A DNA extraction kit was optimized that allowed sampling of an entire liter of water. Water samples spiked with the recombinant E. coli cells were shown to behave like H. pylori cells in the qPCR assay. The recombinant E. coli cells were optimized to be used at 10 cells/liter of water, where they were shown not to compete with 5 to 3,000 cells of H. pylori in a duplex qPCR assay. Four treated drinking water samples spiked with H. pylori (100 cells) demonstrated similar cycle threshold values if the chlorine disinfectant was first neutralized by sodium thiosulfate.

Protubera beijingensis sp. nov. (Protophallaceae, Phallales) from China

Phytotaxa ◽  
2018 ◽  
Vol 348 (2) ◽  
pp. 133
Author(s):  
GUOJIE LI ◽  
DEJIANG DENG ◽  
JINKANG WEI ◽  
CHULONG ZHANG ◽  
RUILIN ZHAO ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
New Species ◽  
North China ◽  
Morphological Description ◽  
Similar Species ◽  
Multigene Phylogenetic Analysis ◽  
Globally Distributed ◽  
A New Species

The genus Protubera includes gasteroid species. Its members are globally distributed in tropical, subtropical, and temperate areas, and presently, six species are recognized. In this paper, Protubera beijingensis from North China is described as a new species. Its morphological description and illustration are provided in detail and compared with morphologically similar species. A multigene phylogenetic analysis based on nLSU, atp6, and rpb2 sequences of the genus Protubera also identifies this organism as a new species within Protubera.

scholarly journals Automated multiplex nucleic acid tests for rapid detection of SARS-CoV-2, influenza A and B infection with direct reverse-transcription quantitative PCR (dirRT-qPCR) assay in a centrifugal microfluidic platform

RSC Advances ◽  
2020 ◽  
Vol 10 (56) ◽  
pp. 34088-34098
Author(s):  
Minghui Ji ◽  
Yun Xia ◽  
Jacky Fong-Chuen Loo ◽  
Lang Li ◽  
Ho-Pui Ho ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Quantitative Pcr ◽  
Reverse Transcription ◽  
Rapid Detection ◽  
Influenza A ◽  
Qpcr Assay ◽  
Multiplex Detection ◽  
Microfluidic Platform ◽  
Reverse Transcription Quantitative Pcr ◽  
Nucleic Acid Tests

Development of a microfluidic disc-direct reverse-transcription quantitative PCR platform to perform automated multiplex nucleic acid tests for rapid multiplex detection of disease infection.

scholarly journals Novel Molecular Synapomorphies Demarcate Different Main Groups/Subgroups of Plasmodium and Piroplasmida Species Clarifying Their Evolutionary Relationships

Genes ◽  
2019 ◽  
Vol 10 (7) ◽  
pp. 490 ◽  
Author(s):  
Sharma ◽  
Gupta
Keyword(s):  
Phylogenetic Analysis ◽  
Phylogenetic Trees ◽  
Plasmodium Species ◽  
Protein Sequences ◽  
Evolutionary Relationships ◽  
Major Life ◽  
Comparative Analyses ◽  
Large Numbers ◽  
Conserved Signature Indels ◽  
Relationship Of

The class Hematozoa encompasses several clinically important genera, including Plasmodium, whose members cause the major life-threating disease malaria. Hence, a good understanding of the interrelationships of organisms from this class and reliable means for distinguishing them are of much importance. This study reports comprehensive phylogenetic and comparative analyses on protein sequences on the genomes of 28 hematozoa species to understand their interrelationships. In addition to phylogenetic trees based on two large datasets of protein sequences, detailed comparative analyses were carried out on the genomes of hematozoa species to identify novel molecular synapomorphies consisting of conserved signature indels (CSIs) in protein sequences. These studies have identified 79 CSIs that are exclusively present in specific groups of Hematozoa/Plasmodium species, also supported by phylogenetic analysis, providing reliable means for the identification of these species groups and understanding their interrelationships. Of these CSIs, six CSIs are specifically shared by all hematozoa species, two CSIs serve to distinguish members of the order Piroplasmida, five CSIs are uniquely found in all Piroplasmida species except B. microti and two CSIs are specific for the genus Theileria. Additionally, we also describe 23 CSIs that are exclusively present in all genome-sequenced Plasmodium species and two, nine, ten and eight CSIs which are specific for members of the Plasmodium subgenera Haemamoeba, Laverania, Vinckeia and Plasmodium (excluding P. ovale and P. malariae), respectively. Additionally, our work has identified several CSIs that support species relationships which are not evident from phylogenetic analysis. Of these CSIs, one CSI supports the ancestral nature of the avian-Plasmodium species in comparison to the mammalian-infecting groups of Plasmodium species, four CSIs strongly support a specific relationship of species between the subgenera Plasmodium and Vinckeia and three CSIs each that reliably group P. malariae with members of the subgenus Plasmodium and P. ovale within the subgenus Vinckeia, respectively. These results provide a reliable framework for understanding the evolutionary relationships among the Plasmodium/Piroplasmida species. Further, in view of the exclusivity of the described molecular markers for the indicated groups of hematozoa species, particularly large numbers of unique characteristics that are specific for all Plasmodium species, they provide important molecular tools for biochemical/genetic studies and for developing novel diagnostics and therapeutics for these organisms.

scholarly journals Development and Application of a Quantitative PCR Detection Method to Quantify Venturia oleaginea in Asymptomatic Olive (Olea europaea) Leaves

2020 ◽  
Vol 110 (3) ◽  
pp. 547-555
Author(s):  
Silvia Scibetta ◽  
Giovanni E. Agosteo ◽  
Ahmed Abdelfattah ◽  
Maria G. Li Destri Nicosia ◽  
Santa O. Cacciola ◽  
...  
Keyword(s):  
Quantitative Pcr ◽  
High Throughput Sequencing ◽  
Current Knowledge ◽  
Leaf Growth ◽  
Its Region ◽  
Qpcr Assay ◽  
Detection Methods ◽  
Effective Control ◽  
Its2 Region

Olive leaf spot (OLS), caused by Venturia oleaginea, is one of the most common and serious diseases of olive trees in the Mediterranean region. Understanding the pathogen life cycle is important for the development of effective control strategies. Current knowledge is incomplete owing to a lack of effective detection methods. It is extremely difficult to culture V. oleaginea in vitro, so primers were designed to amplify and sequence the internal transcribed spacer ITS1-5.8S-ITS2 region of the fungus directly from infected olive leaves. Sanger sequencing indicated a unique ITS region present in the European strains screened, confirming the appropriateness of the target region for developing a quantitative PCR (qPCR) assay. Furthermore, high-throughput sequencing of the same region excluded the presence of other Venturia species in the olive phyllosphere. The qPCR assay proved very specific and sensitive, enabling the detection of approximately 26 copies of target DNA. The analysis of symptomless leaves during early stages of the epidemic from the end of winter through spring revealed a similar quantity of pathogen DNA regardless of the leaf growth stage. In contrast, the pathogen titer changed significantly during the season. Data indicated that leaf infections start earlier than expected over the season and very young leaves are as susceptible as adult leaves. These findings have important practical implications and suggest the need for improved scheduling of fungicide treatments. The qPCR assay represents a valuable tool providing quantitative results and enables detection of V. oleaginea in all olive organs, including those in which OLS cannot be studied using previously available methods.

scholarly journals Tzeananiaceae, a new pleosporalean family associated with Ophiocordyceps macroacicularis fruiting bodies in Taiwan

MycoKeys ◽  
2018 ◽  
Vol 37 ◽  
pp. 1-17 ◽  
Author(s):  
Hiran A. Ariyawansa ◽  
Alan J.L. Phillips ◽  
Wei-Yu Chuang ◽  
Ichen Tsai
Keyword(s):  
Phylogenetic Analysis ◽  
Morphological Variation ◽  
Fruiting Body ◽  
New Record ◽  
Fungal Strain ◽  
Monotypic Genus ◽  
Fruiting Bodies ◽  
Large Numbers ◽  
Thin Walled ◽  
Six Genes

The order Pleosporales comprises a miscellaneous group of fungi and is considered to be the largest order of the class Dothideomycetes. The circumscription of Pleosporales has undergone numerous changes in recent years due to the addition of large numbers of families reported from various habitats and with a large amount of morphological variation. Many asexual genera have been reported in Pleosporales and can be either hyphomycetes or coelomycetes. Phoma-like taxa are common and have been shown to be polyphyletic within the order and allied with several sexual genera. During the exploration of biodiversity of pleosporalean fungi in Taiwan, a fungal strain was isolated from mycelium growing on the fruiting body of an Ophiocordyceps species. Fruiting structures that developed on PDA were morphologically similar to Phoma and its relatives in having pycnidial conidiomata with hyaline conidia. The fungus is characterised by holoblastic, cylindrical, aseptate conidiogenous cells and cylindrical, hyaline, aseptate, guttulated, thin-walled conidia. Phylogenetic analysis based on six genes, ITS, LSU, rpb2, SSU, tef1 and tub2, produced a phylogenetic tree with the newly generated sequences grouping in a distinct clade separate from all of the known families. Therefore, a new pleosporalean family Tzeananiaceae is established to accommodate the monotypic genus Tzeanania and the species T.taiwanensis in Pleosporales, Dothideomycetes. The Ophiocordyceps species was identified as O.macroacicularis and this is a new record in Taiwan.

scholarly journals Development of a quantitative PCR assay for detection of redside shiner (Richardsonius balteatus) from environmental DNA

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Marshal S. Hoy ◽  
Carl O. Ostberg
Keyword(s):  
Quantitative Pcr ◽  
Water Samples ◽  
Effective Means ◽  
Environmental Dna ◽  
Sympatric Species ◽  
Negative Control ◽  
Control Site ◽  
Qpcr Assay ◽  
Redside Shiner ◽  
Richardsonius Balteatus

Abstract Objective A quantitative PCR (qPCR) assay for the detection of redside shiner (Richardsonius balteatus) environmental DNA (eDNA) was designed as a side product of a larger project aimed at using eDNA to determine the presence and geographic extent of native and non-native fishes in the reservoirs and associated tributaries above the three mainstem dams (Ross, Diablo, Gorge) on the Skagit River, Washington, USA. The eDNA survey results can be used to help guide additional sampling efforts that include traditional sampling methods, such as electrofishing and netting. Results The redside shiner qPCR assay (RSSCOI_540-601) was validated by testing for sensitivity using redside shiner genomic DNA from three different populations and by testing for specificity against 30 potentially sympatric species. No non-target amplification was observed in our validation tests. We then evaluated the assay on field-collected water samples where there are known populations of redside shiner and a negative control site where the target species is known to be absent. The field-collected water samples tested positive at the redside shiner sites and tested negative at the negative control site. The assay could provide resource managers with an effective means for surveying and monitoring redside shiner populations.

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