scholarly journals Concentration and Detection of Caliciviruses in Water Samples by Reverse Transcription-PCR

2000 ◽  
Vol 66 (10) ◽  
pp. 4383-4388 ◽  
Author(s):  
P. W. Huang ◽  
D. Laborde ◽  
V. R. Land ◽  
D. O. Matson ◽  
A. W. Smith ◽  
...  
Keyword(s):  
Public Health ◽  
Drinking Water ◽  
Surface Waters ◽  
Reverse Transcription ◽  
Water Samples ◽  
Limit Of Detection ◽  
Plaque Assay ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Different Types

ABSTRACT Human caliciviruses (HuCVs) cause waterborne outbreaks of gastroenteritis. Standard indicators of a safe water supply do not adequately predict contamination of water by viruses, including HuCVs. We developed a method to concentrate and detect HuCVs in water samples by using a cultivable primate calicivirus (Pan-1) as a model. Viable Pan-1 was seeded in different types of water and then filtered with a 1MDS filter, eluted with beef extract (BE), and reconcentrated by polyethylene glycol (PEG) precipitation. The viruses in the final samples were tested by plaque assay or by reverse transcription (RT)-PCR following extraction of the RNA with Trizol. Pan-1 was more sensitive to high-pH treatment than poliovirus was; a pH 9.0 BE solution was found to recover 35% more viable Pan-1 than a pH 9.5 BE solution recovered. Pan-1 was recovered from small volumes of deionized, finished, ground, and surface waters at efficiencies of 94, 73, 67, and 64%, respectively, when samples were assayed after elution without further concentration. When larger volumes of water (up to 40 liters) were tested after elution and concentration with PEG, 38, 19, and 14% of the seeded Pan-1 were recovered from finished, ground, and surface waters, respectively. The limit of detection of Pan-1 by RT-PCR was estimated to be 0.75 to 1.5 PFU in 40 liters of finished water. This method may be adapted for monitoring HuCVs in drinking water and other types of water for public health safety.

scholarly journals Detection of mRNA by Reverse Transcription-PCR as an Indicator of Viability in Escherichia coliCells

1998 ◽  
Vol 64 (4) ◽  
pp. 1313-1318 ◽  
Author(s):  
G. E. C. Sheridan ◽  
C. I. Masters ◽  
J. A. Shallcross ◽  
B. M. Mackey
Keyword(s):  
Reverse Transcription ◽  
Room Temperature ◽  
Cellular Viability ◽  
Ethanol Treatment ◽  
Rt Pcr ◽  
Promising Candidate ◽  
Mrna Targets ◽  
Reverse Transcription Pcr ◽  
Different Types ◽  
The Relationship

ABSTRACT The relationship between the detection of mRNA and cellular viability in Escherichia coli was investigated in cells killed by heat or ethanol. Reverse transcription-PCR (RT-PCR) methods were developed for detecting mRNA from rpoH,groEL, and tufA genes. mRNA from all three genes was detected immediately after the cells had been killed by heat or ethanol but gradually disappeared with time when dead cells were held at room temperature. In heat-killed cells, some mRNA targets became undetectable after 2 to 16 h, whereas after ethanol treatment, mRNA was still detected after 16 h. In contrast, 16S rRNA was detected by RT-PCR in all samples containing dead cells and did not disappear during a subsequent incubation of 16 h at room temperature. Of the different types of nucleic acid, mRNA is the most promising candidate for an indicator of viability in bacteria, but its persistence in dead cells depends on the inactivating treatment and subsequent holding conditions.

scholarly journals Sensitive and Rapid Detection of ViableGiardia Cysts and Cryptosporidium parvum Oocysts in Large-Volume Water Samples with Wound Fiberglass Cartridge Filters and Reverse Transcription-PCR

1998 ◽  
Vol 64 (5) ◽  
pp. 1743-1749 ◽  
Author(s):  
Christine Kaucner ◽  
Timothy Stinear
Keyword(s):  
Cryptosporidium Parvum ◽  
Large Volume ◽  
Reverse Transcription ◽  
Water Samples ◽  
Environmental Water ◽  
Significant Advance ◽  
Rt Pcr ◽  
Sensitivity Testing ◽  
Reverse Transcription Pcr ◽  
Primary Sample

ABSTRACT We recently described a reverse transcription-PCR (RT-PCR) for detecting low numbers of viable Cryptosporidium parvumoocysts spiked into clarified environmental water concentrates. We have now modified the assay for direct analysis of primary sample concentrates with simultaneous detection of viable C. parvum oocysts, Giardia cysts, and a novel type of internal positive control (IPC). The IPC was designed to assess both efficiency of mRNA isolation and potential RT-PCR inhibition. Sensitivity testing showed that low numbers of organisms, in the range of a single viable cyst and oocyst, could be detected when spiked into 100-μl packed pellet volumes of concentrates from creek and river water samples. The RT-PCR was compared with an immunofluorescence (IF) assay by analyzing 29 nonspiked environmental water samples. Sample volumes of 20 to 1,500 liters were concentrated with a wound fiberglass cartridge filter. Frequency of detection for viable Giardiacysts increased from 24% by IF microscopy to 69% by RT-PCR. ViableC. parvum oocysts were detected only once by RT-PCR (3%) in contrast to detection of viable Cryptosporidium spp. in four samples by IF microscopy (14%), suggesting thatCryptosporidium species other than C. parvumwere present in the water. This combination of the large-volume sampling method with RT-PCR represents a significant advance in terms of protozoan pathogen monitoring and in the wider application of PCR technology to this field of microbiology.

Enteric viruses in drinking water supplies

2002 ◽  
Vol 2 (3) ◽  
pp. 17-22
Author(s):  
A.P. Wyn-Jones ◽  
J. Watkins ◽  
C. Francis ◽  
M. Laverick ◽  
J. Sellwood
Keyword(s):  
Drinking Water ◽  
Heavy Rainfall ◽  
Liquid Culture ◽  
Plaque Assay ◽  
Enteric Viruses ◽  
Enteric Virus ◽  
The Other ◽  
Raw Water ◽  
Rt Pcr ◽  
Water Supplies

Two rural spring drinking water supplies were studied for their enteric virus levels. In one, serving about 30 dwellings, the water was chlorinated before distribution; in the other, which served a dairy and six dwellings the water was not treated. Samples of treated (40 l) and untreated (20 l) water were taken under normal and heavy rainfall conditions over a six weeks period and concentrated by adsorption/elution and organic flocculation. Infectious enterovirus in concentrates was detected in liquid culture and enumerated by plaque assay, both in BGM cells, and concentrates were also analysed by RT-PCR. Viruses were found in both raw water supplies. Rural supplies need to be analysed for viruses as well as bacterial and protozoan pathogens if the full microbial hazard is to be determined.

scholarly journals Perchlorate Levels in Polish Water Samples of Various Origin

Separations ◽  
2021 ◽  
Vol 8 (4) ◽  
pp. 37
Author(s):  
Przemysław Niziński ◽  
Patrycja Wiśniewska ◽  
Joanna Kończyk ◽  
Rajmund Michalski
Keyword(s):  
Drinking Water ◽  
Water Samples ◽  
Limit Of Detection ◽  
Primary Objective ◽  
Swimming Pool ◽  
Limit Of Quantification ◽  
Reliable Determination ◽  
Pool Water ◽  
Drinking Waters ◽  
Swimming Pool Water

Perchlorate ion (ClO4−) is known as a potent endocrine disruptor and exposure to this compound can result in serious health issues. It has been found in drinking water, swimming pools, and surface water in many countries, however, its occurrence in the environment is still poorly understood. The information on perchlorate contamination of Polish waters is very limited. The primary objective of this study was to assess ClO4− content in bottled, tap, river, and swimming pool water samples from different regions of Poland and provide some data on the presence of perchlorate. We have examined samples of bottled, river, municipal, and swimming pool water using the IC–CD (ion chromatography–conductivity detection) method. Limit of detection and limit of quantification were 0.43 µg/L and 1.42 µg/L, respectively, and they were both above the current health advisory levels in drinking water. The concentration of perchlorate were found to be 3.12 µg/L in one river water sample and from 6.38 to 8.14 µg/L in swimming pool water samples. Importantly, the level of perchlorate was below the limit of detection (LOD) in all bottled water samples. The results have shown that the determined perchlorate contamination in Polish drinking waters seems to be small, nevertheless, further studies are required on surface and river samples. The inexpensive, fast, and sensitive IC–CD method used in this study allowed for a reliable determination of perchlorate in the analyzed samples. To the best of our knowledge, there are no other studies seeking to assess the perchlorate content in Polish waters.

scholarly journals Reverse Transcription-PCR Analysis of Bottled and Natural Mineral Waters for the Presence of Noroviruses

2003 ◽  
Vol 69 (11) ◽  
pp. 6541-6549 ◽  
Author(s):  
Gilbert Thierry Lamothe ◽  
Thierry Putallaz ◽  
Han Joosten ◽  
Joey D. Marugg
Keyword(s):  
Reverse Transcription ◽  
Membrane Filtration ◽  
Limit Of Detection ◽  
Mineral Waters ◽  
Pcr Analysis ◽  
Rna Sequences ◽  
Natural Mineral ◽  
Reverse Transcription Pcr ◽  
Viral Particles ◽  
Nonspecific Amplification

ABSTRACT A seminested reverse transcription-PCR method coupled to membrane filtration was optimized to investigate the presence of norovirus (NV) RNA sequences in bottled and natural mineral waters. The recovery of viral particles by filtration varied between 28 and 45%, while the limit of detection of the overall method ranged from 6 to 95 viral particles. The assay was broadly reactive, as shown by the successful detection of 27 different viral strains representing 12 common genotypes of NVs. A total of 718 bottled and natural mineral water samples were investigated, including 640 samples of finished, spring, and line products (mostly 1 to 1.5 liters), collected from 36 different water brands of various types and from diverse geographic origins over a 2-year period. In addition, 78 samples of larger volume (10 and 400 to 500 liters) and environmental swabs were investigated. From the 1,436 analyses that were performed for the detection of NVs belonging to genogroups I and II, 34 samples (2.44%) were presumptively positive by seminested RT-PCR. However, confirmation by DNA sequence analysis revealed that all presumptive positive results were either due to nonspecific amplification or to cross-contamination. In conclusion, these results do not provide any evidence for the presence of NV genome sequences in bottled waters.

Comet and cup genes in Drosophila spermatogenesis: the first demonstration of post-meiotic transcription

2008 ◽  
Vol 36 (3) ◽  
pp. 540-542 ◽  
Author(s):  
Carine Barreau ◽  
Elizabeth Benson ◽  
Helen White-Cooper
Keyword(s):  
In Situ Hybridization ◽  
Expression Pattern ◽  
Reverse Transcription ◽  
Protein Product ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Single Cyst ◽  
Reverse Transcription Pcr

Post-meiotic transcription is widespread in mammalian spermatogenesis, but is generally believed to be absent from Drosophila spermatogenesis. Genes required during meiosis, in early spermatids or later in spermiogenesis are typically transcribed in primary spermatocytes in Drosophila. Their mRNAs are then stored in the cytoplasm until the protein product is needed. Recently, using in situ hybridization, we identified 17 Drosophila genes, collectively named ‘comets’ and ‘cups’, whose mRNAs are most abundant in, and localize to the distal ends of, elongating spermatids. Using a single-cyst quantitative RT–PCR (reverse transcription–PCR) assay, we confirmed this unusual expression pattern and conclusively demonstrate the existence of post-meiotic transcription in Drosophila spermatids. We found that transcription of comets and cups occurs just before protamines can be detected in spermatid nuclei.

scholarly journals Investigation of Cryptosporidium spp. and Giardia spp. in Lake Eğirdir (Isparta)

2021 ◽  
Author(s):  
Tuğba Sağlam ◽  
Serdar Düşen ◽  
Meral Apaydın Yağcı ◽  
Abdülkadir Yağcı
Keyword(s):  
Public Health ◽  
Drinking Water ◽  
Water Samples ◽  
Average Density ◽  
Agricultural Irrigation ◽  
Control Programs ◽  
Cryptosporidium Spp ◽  
Direct Microscopic Examination ◽  
Different Seasons ◽  
Autumn And Winter

Objective: The aim of this study was to assess both the presence and seasonal variability of Cryptosporidium spp. and Giardia spp. in Eğirdir Lake within the borders of Isparta province, which is used for drinking, agricultural irrigation and recreational purposes. Method: The research was carried out between July 2016 and January 2017 and water samples were taken from five different stations in three different seasons in Lake Eğirdir. After direct microscopic examination of the samples (Native-Lugol method), they were stained with Modified Acid Fast (MAF), and examined under the light microscope for parasites. Results: Cryptosporidium spp and Giardia spp were detected in 15 water samples in summer months, with an average density of 99.2% and 93.3% respectively, in Lake Eğirdir. In addition, both parasites were also detected intensively in autumn and winter Conclusion: The use of Lake Eğirdir for daily needs of people, agriculture andrecreational purposes cause increase in protozoal density. Thus, it is necessary to conduct parasitological studies on Lake Eğirdir, especially during the periods of swimming tourism, to determine the protozoal epidemiology in humans and animals. In addition, it is important to carry out adequate disinfection processes and plan the necessary control programs in terms of public health in the regions where Lake Eğirdir is used as drinking water.

Development of a real-time reverse transcription-PCR assay to detect and quantify group A rotavirus equine-like G3 strains

2021 ◽  
Author(s):  
Eric M. Katz ◽  
Mathew D. Esona ◽  
Rashi Gautam ◽  
Michael D. Bowen
Keyword(s):  
Viral Load ◽  
Real Time ◽  
Reverse Transcription ◽  
Dynamic Range ◽  
Limit Of Detection ◽  
Human Populations ◽  
Pcr Assay ◽  
Group A Rotavirus ◽  
Reverse Transcription Pcr ◽  
Group A

Since 2013, group A rotavirus strains characterized as novel DS-1-like inter-genogroup reassortant ‘equine-like G3’ strains have emerged and spread across five continents among human populations in at least 14 countries. Here we report a novel one-step TaqMan quantitative real-time reverse transcription-PCR assay developed to genotype and quantify the viral load for samples containing rotavirus equine-like G3 strains. Using a universal G forward primer and a newly designed reverse primer and TaqMan probe, we developed and validated an assay with a linear dynamic range of 2.3 × 10 9 – 227 copies per reaction and a limit of detection of 227 copies. The percent positive agreement, percent negative agreement, and precision of our assay were 100.00%, 99.63%, and 100.00%, respectively. This assay can simultaneously detect and quantify the viral load for samples containing DS-1-like inter-genogroup reassortant equine-like G3 strains with high sensitivity and specificity, faster turnaround time, and decreased cost and will be valuable for high-throughput screening of stool samples collected to monitor equine-like G3 strain prevalence and circulation among human populations throughout the world.

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