Detection of enteroviruses in marine waters by direct RT-PCR and cell culture

1995 ◽  
Vol 31 (5-6) ◽  
pp. 323-328 ◽  
Author(s):  
K. A. Reynolds ◽  
C. P. Gerba ◽  
I. L. Pepper
Keyword(s):  
Cell Culture ◽  
Polymerase Chain Reaction Amplification ◽  
Cost Effective ◽  
The United States ◽  
Water Runoff ◽  
Rt Pcr ◽  
Marine Waters ◽  
Storm Water Runoff ◽  
Routine Monitoring ◽  
Reaction Amplification

Sewage outfalls and storm water runoff introduces pathogenic human enteric viruses into marine coastal waters, which may pose a potential public health risk. Although members of the enterovirus group have been suggested as possible indicators of sewage pollution in marine waters, the lack of rapid, sensitive and cost effective methods have prevented routine monitoring in the United States. This study compared traditional cell culture and direct RT-PCR (reverse transcriptase-polymerase chain reaction) amplification for detection of an enterovirus. Poliovirus could be recovered from 100 L of artificial seawater with an average efficiency of 77%, using adsorption and elution from electronegative filters. Viruses were eluted from the filters with 1.5% beef extract for viruses (BEV) adjusted to pH 9.5 and reconcentrated by organic flocculation to a volume of 30 mL. Substances which interfered with detection by RT-PCR were removed by treatment of the concentrates with sephadex and chelex resins. Direct RT-PCR could detect 2.5 and 0.025 PFU (plaque forming units) for single (25 cycles) and double PCR (2 × 25 cycles) in 10 μL of pure culture poliovirus samples, respectively. These methods are currently being applied to assess the occurrence of enteroviruses at marine bathing beaches influenced by sewage discharges.

Polymerase chain reaction amplification and typing of rotavirus in environmental samples

1995 ◽  
Vol 31 (5-6) ◽  
pp. 371-374 ◽  
Author(s):  
R. Gajardo ◽  
R. M. Pintó ◽  
A. Bosch
Keyword(s):  
Polymerase Chain Reaction ◽  
Environmental Samples ◽  
Polymerase Chain Reaction Amplification ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Group A ◽  
Reaction Amplification ◽  
Stool Specimens ◽  
Polymerase Chain

A reverse transcription polymerase chain reaction (RT-PCR) assay is described that has been developed for the detection and serotyping of group A rotavirus in stool specimens and concentrated and non-concentrated sewage specimens.

scholarly journals Monitoring the abundance of plastic debris in the marine environment

2009 ◽  
Vol 364 (1526) ◽  
pp. 1999-2012 ◽  
Author(s):  
Peter G. Ryan ◽  
Charles J. Moore ◽  
Jan A. van Franeker ◽  
Coleen L. Moloney
Keyword(s):  
Statistical Power ◽  
Standing Stock ◽  
Cost Effective ◽  
Water Runoff ◽  
Temporal Heterogeneity ◽  
Plastic Debris ◽  
Storm Water Runoff ◽  
Beach Dynamics ◽  
Spatial And Temporal Heterogeneity ◽  
Population Sizes

Plastic debris has significant environmental and economic impacts in marine systems. Monitoring is crucial to assess the efficacy of measures implemented to reduce the abundance of plastic debris, but it is complicated by large spatial and temporal heterogeneity in the amounts of plastic debris and by our limited understanding of the pathways followed by plastic debris and its long-term fate. To date, most monitoring has focused on beach surveys of stranded plastics and other litter. Infrequent surveys of the standing stock of litter on beaches provide crude estimates of debris types and abundance, but are biased by differential removal of litter items by beachcombing, cleanups and beach dynamics. Monitoring the accumulation of stranded debris provides an index of debris trends in adjacent waters, but is costly to undertake. At-sea sampling requires large sample sizes for statistical power to detect changes in abundance, given the high spatial and temporal heterogeneity. Another approach is to monitor the impacts of plastics. Seabirds and other marine organisms that accumulate plastics in their stomachs offer a cost-effective way to monitor the abundance and composition of small plastic litter. Changes in entanglement rates are harder to interpret, as they are sensitive to changes in population sizes of affected species. Monitoring waste disposal on ships and plastic debris levels in rivers and storm-water runoff is useful because it identifies the main sources of plastic debris entering the sea and can direct mitigation efforts. Different monitoring approaches are required to answer different questions, but attempts should be made to standardize approaches internationally.

Field-Scale Demonstration of Electrocoagulation and Enhanced Media Filtration for Treatment of Shipyard Storm Water

2001 ◽  
Vol 17 (04) ◽  
pp. 191-201
Author(s):  
Maria E. Pulido ◽  
Enrique J. La Motta ◽  
Reddy M. Nandipati ◽  
Juan C. Josse
Keyword(s):  
Treatment Process ◽  
Effective Means ◽  
Cost Effective ◽  
Storm Water ◽  
Water Runoff ◽  
Waste Streams ◽  
Storm Water Runoff ◽  
Effective Technology ◽  
Solid Residuals ◽  
Ship Ballast Water

The use of electrocoagulation and enhanced media filtration was evaluated for the treatment of storm water runoff from shipyards. A 15-gallon per minute (gpm) fieldscale unit was operated at Litton Avondale Industries Shipyard in New Orleans, Louisiana. The study indicated that electrocoagulation and enhanced media filtration is a cost-effective technology for treatment of shipyard storm water runoff. This technology is simple to operate and has the potential to provide a cost-effective means to treat various process wastewaters generated in shipyards. The study also indicates a need for further research on the management of solid residuals generated in the treatment process. Further research is also needed to assess the effectiveness of this technology for the treatment of other related waste streams such as ship ballast water and bilge water.

scholarly journals Mutation Associated with Orange Fruit Color Increases Concentrations of β-Carotene in a Sweet Pepper Variety (Capsicum annuum L.)

Foods ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1225
Author(s):  
Nasya Tomlekova ◽  
Velichka Spasova-Apostolova ◽  
Ivelin Pantchev ◽  
Fatma Sarsu
Keyword(s):  
Polymerase Chain Reaction Amplification ◽  
Cost Effective ◽  
Sweet Pepper ◽  
Breeding Programs ◽  
Health Related ◽  
Reaction Amplification ◽  
Induced Mutagenesis ◽  
Orange Fruit ◽  
Β Carotene

Pepper is the second most important vegetable crop in Bulgarian agriculture and has become the subject of extensive breeding programs that frequently employ induced mutagenesis. The success of breeding programs can be enhanced by the efficient and integral application of different biochemical and molecular methods to characterize specific mutant alleles. On the other hand, identifying new cost-effective methods is important under a limited-resources environment. In this paper we compare the levels of five health-related carotenoid compounds of fruits (α-carotene, β-carotene, lutein, β-cryptoxanthin, zeaxanthin) between a mutant variety Oranzheva kapia (possessing high ß-carotene concentration) and a corresponding initial pepper variety Pazardzhishka kapia 794. Both varieties are intended for fresh consumption. Pepper is a major natural source of β-carotene. It was observed that fruit at both commercial and botanical maturity from mutant variety had greater α-carotene and β-carotene concentrations to the initial variety (7.49 and 1.94 times higher, respectively) meaning that the mutant was superior in fruit quality to the initial genotype. Two hydroxylase enzymes, converting α- and β-carotene to lutein and zeaxanthin, respectively, are known to exist in pepper and are encoded by two genes on chromosomes 3 and 6-CrtZchr03 and CrtZchr06. The molecular characterization of the mutant variety through locus-specific Polymerase chain reaction amplification, gene cloning and sequencing as well as expression was performed. Our results suggest that the increased ß-carotene accumulation in the mutant variety Oranzheva kapia results from a biosynthetic pathway breakdown due to deletion of CrtZchr03 gene.

scholarly journals The ABL-BCR fusion gene is expressed in chronic myeloid leukemia

Blood ◽  
1993 ◽  
Vol 81 (1) ◽  
pp. 158-165 ◽  
Author(s):  
JV Melo ◽  
DE Gordon ◽  
NC Cross ◽  
JM Goldman
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Fusion Gene ◽  
Polymerase Chain Reaction Amplification ◽  
Direct Sequencing ◽  
Rt Pcr ◽  
Reading Frame ◽  
Chromosome 9Q ◽  
Reaction Amplification ◽  
Polymerase Chain

Abstract Although the BCR-ABL hybrid gene on chromosome 22q-plays a pivotal role in the pathogenesis of chronic myeloid leukemia (CML), little is known of the reciprocal chimeric gene, ABL-BCR, formed on chromosome 9q+. By reverse transcription/polymerase chain reaction amplification (RT/PCR) we have detected ABL-BCR mRNA in cells from 31 of 44 BCR-ABL positive CML patients and 3 of 5 CML cell lines. Of the 34 positive samples, 31 had classical t(9;22) (q34;q11) translocations; in 3 samples there was no Philadelphia (Ph) and/or 9q+ chromosomes. ABL-BCR expression consisted of ABL(Ib)-BCR mRNA in 26 patients and of both ABL(Ib)-BCR and ABL(Ia)-BCR mRNA species in 6 patients. The ABL-BCR transcripts encoded one or, more rarely, both of the two potential junctions, designated ABL-b3 and ABL-b4, which differed in size by 75 bp. In 2 patients, the BCR exon b3 was not present in either the BCR-ABL or the corresponding ABL-BCR transcript, whereas in 5 patients exon b3 was present in both transcripts. Direct sequencing of PCR fragments representing the full-length coding sequence of ABL-BCR cDNAs type Ib- b3, Ia-b3, Ib-b4, and Ia-b4 showed an open reading frame predicted to encode fusion proteins of 370 to 414 amino-acids. If an ABL-BCR gene product is produced in CML cells, it may be relevant as a mechanism for deregulating the GTPase activating protein (GAP) function of BCR.

scholarly journals The ABL-BCR fusion gene is expressed in chronic myeloid leukemia

Blood ◽  
1993 ◽  
Vol 81 (1) ◽  
pp. 158-165 ◽  
Author(s):  
JV Melo ◽  
DE Gordon ◽  
NC Cross ◽  
JM Goldman
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Fusion Gene ◽  
Polymerase Chain Reaction Amplification ◽  
Direct Sequencing ◽  
Rt Pcr ◽  
Reading Frame ◽  
Chromosome 9Q ◽  
Reaction Amplification ◽  
Polymerase Chain

Although the BCR-ABL hybrid gene on chromosome 22q-plays a pivotal role in the pathogenesis of chronic myeloid leukemia (CML), little is known of the reciprocal chimeric gene, ABL-BCR, formed on chromosome 9q+. By reverse transcription/polymerase chain reaction amplification (RT/PCR) we have detected ABL-BCR mRNA in cells from 31 of 44 BCR-ABL positive CML patients and 3 of 5 CML cell lines. Of the 34 positive samples, 31 had classical t(9;22) (q34;q11) translocations; in 3 samples there was no Philadelphia (Ph) and/or 9q+ chromosomes. ABL-BCR expression consisted of ABL(Ib)-BCR mRNA in 26 patients and of both ABL(Ib)-BCR and ABL(Ia)-BCR mRNA species in 6 patients. The ABL-BCR transcripts encoded one or, more rarely, both of the two potential junctions, designated ABL-b3 and ABL-b4, which differed in size by 75 bp. In 2 patients, the BCR exon b3 was not present in either the BCR-ABL or the corresponding ABL-BCR transcript, whereas in 5 patients exon b3 was present in both transcripts. Direct sequencing of PCR fragments representing the full-length coding sequence of ABL-BCR cDNAs type Ib- b3, Ia-b3, Ib-b4, and Ia-b4 showed an open reading frame predicted to encode fusion proteins of 370 to 414 amino-acids. If an ABL-BCR gene product is produced in CML cells, it may be relevant as a mechanism for deregulating the GTPase activating protein (GAP) function of BCR.

Incidence of enteroviruses in Mamala Bay, Hawaii using cell culture and direct polymerase chain reaction methodologies

1998 ◽  
Vol 44 (6) ◽  
pp. 598-604 ◽  
Author(s):  
Kelly A Reynolds ◽  
Kimberly Roll ◽  
Roger S Fujioka ◽  
Charles P Gerba ◽  
Ian L Pepper
Keyword(s):  
Cell Culture ◽  
Polymerase Chain Reaction ◽  
Water Samples ◽  
Health Hazard ◽  
Effective Means ◽  
Detection Sensitivity ◽  
Rt Pcr ◽  
Marine Waters ◽  
Chain Reaction ◽  
Polymerase Chain

The consequence of point and nonpoint pollution sources, discharged into marine waters, on public recreational beaches in Mamala Bay, Hawaii was evaluated using virus cell culture and direct reverse transcriptase - polymerase chain reaction (RT-PCR). Twelve sites, nine marine, two freshwater (one stream and one canal), and one sewage, were assessed either quarterly or monthly for 1 year to detect the presence of human enteric viruses. Water samples were concentrated from initial volumes of 400 L to final volumes of 30 mL using Filterite electronegative cartridge filters and a modified beef extract elution procedure. Cell culture was applied using the Buffalo Green Monkey kidney cell line to analyze samples for enteroviruses. Positive samples were also evaluated by RT-PCR, using enterovirus-specific primers. Levels of RT-PCR inhibition varied with each concentrated sample. Resin column purification increased PCR detection sensitivity by at least one order of magnitude in a variety of sewage outfall and recreational marine water samples but not in the freshwater canal samples. Using cell culture, viable enteroviruses were found in 50 and 17% of all outfall and canal samples, respectively. Samples were positive at beaches 8% of the time. These data illustrate the potential public health hazard associated with recreational waters. Using direct PCR, viruses were detected at the outfall but were not found in any beach or canal samples, in part, owing to substances that inhibit PCR. Therefore, conventional cell culture is the most effective means of detecting low levels of infectious enteroviruses in environmental waters, whereas direct RT-PCR is rendered less effective by inhibitory compounds and low equivalent reaction volumes.Key words: enteroviruses, RT-PCR, cell culture, marine waters.

scholarly journals The Need of Non-traditional Techniques to Screen for the Virus

2020 ◽  
Vol 8 (T1) ◽  
pp. 1-2
Author(s):  
Davide Borroni ◽  
Kunal Gadhvi
Keyword(s):  
Early Stage ◽  
False Negative ◽  
Cost Effective ◽  
The United States ◽  
Viral Metagenomics ◽  
Present Moment ◽  
Rt Pcr ◽  
Viral Genomes ◽  
Reverse Transcription Pcr ◽  
Positive Rate

BACKGROUND: At the present moment, the etiological diagnosis of SARS-CoV-2 is based on the polymerase chain reaction (PCR). False negative cases are increasingly reported in several studies using reverse transcription-PCR (RT-PCR). For example, the positive rate of RT-PCR for throat swabs was reported to be about 60% in early stage of COVID-19. AIM: We aimed to present metagenomic next-generation sequencing (mNGS) as a potential tool to detect pathogens. METHODS: In the recent year, mNGS is shown the potential to detect pathogens without the need of hypothesis guided approach and is proven to be highly effective. RESULTS: A recent prospective study in the United States compared the diagnostic performance of routine diagnostic tests with mNGS and showed that mNGS detected a bacteria or virus in the CSF of 13 of 58 patients presenting with meningoencephalitis who were negative for or not assessed with routine diagnostic test including PCR. NGS also has the advantage to cover entire viral genomes. CONCLUSION: As viral metagenomics has significantly improved in recent years and become more cost effective, we think that a change in the approach toward a shot-gun metagenomic testing should be explored and could potentially aid the diagnosis of COVID-19 cases and the management of this pandemic.

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