Distribution of Norwalk Virus within Shellfish Following Bioaccumulation and Subsequent Depuration by Detection Using RT-PCR

Consumption of raw bivalve mollusks contaminated with pathogens from human feces continues to present a human health risk. The purpose of this study was to monitor the uptake, localization, and removal of Norwalk virus (NV) in shellfish (oyster and clam) tissues by analyzing virus distribution in selected dissected tissues. Live shellfish were allowed to bioaccumulate different input titers of NV for time periods from 4 to 24 h. In some experiments, depuration by shellfish that bioaccumulated NV and Escherichia coli bacteria was allowed to proceed for 24 or 48 hours. Dissected stomach (St), digestive diverticula (DD), adductor muscle (AM), and hemolymph cells (HC) tissues were assayed for NV by the reverse transcription polymerase chain reaction (RT-PCR) method. An intemal RNA standard control was added to the RT-PCR to identify the presence of inhibitors to RT-PCR. NV titers in DD tissues before and after depuration were estimated using quantitative RT-PCR end-point dilution. NV was found in the alimentary tract (DD or St) at all concentrations of input virus, but was present more frequently after exposure to higher levels of virus. NV was detected in AM and HC only following exposure to higher levels of virus. In experiments where depuration by oysters was continued for 48 h, depuration of bacteria was efficient (95% reduction of bacteria), but minimal (7%) reduction of NV titers from DD tissues was detected. These findings indicate that NV can localize both within and outside the alimentary tract of shellfish, and NV is poorly depurated using conditions favorable for E. coli depuration.

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A SYBR green, real-time RT-PCR method to detect and quantitate Norwalk virus in stools

Journal of Virological Methods ◽

10.1016/j.jviromet.2003.10.011 ◽

2004 ◽

Vol 116 (1) ◽

pp. 63-70 ◽

Cited By ~ 66

Author(s):

Gary P. Richards ◽

Michael A. Watson ◽

David H. Kingsley

Keyword(s):

Real Time ◽

Sybr Green ◽

Norwalk Virus ◽

Rt Pcr ◽

Pcr Method

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Evaluation of multiplex SYBR green real-time PCR assay for detection of pathogenic Escherichia coli

International Journal of Engineering & Technology ◽

10.14419/ijet.v9i2.30389 ◽

2020 ◽

Vol 9 (2) ◽

pp. 448

Author(s):

Ema Komalasari ◽

Winiati P. Rahayu ◽

Siti Nurjanah

Keyword(s):

Escherichia Coli ◽

Real Time ◽

Real Time Pcr ◽

Sybr Green ◽

Rt Pcr ◽

E Coli ◽

Melting Curves ◽

Pathogenic Escherichia Coli ◽

Wide Range ◽

Pcr Method

Pathogenic Escherichia coli (E. coli) has been implicated in a wide range of disease causing infections. It is essential to generate a method for detecting and differentiating each pathotype of E. coli which is more quickly and efficiently by using less reagent. This study aimed to evaluate a SYBR Green multiplex real-time PCR method for detecting four types of pathogenic E. coli. Two of multiplex real-time PCR system, 6-plex and 3-plex, were set to detect six different virulence factors from ETEC, EPEC, EHEC, and EIEC and evaluate the melting curves and specificity compared to simplex method. The results showed that 3-plex rt-PCR method gave more reliable melting curves than 6-plex. The 3-plex rt-PCR also provided similar melting value (Tm) to simplex system. The results of this specificity assay supported the selection of 3-plex rt-PCR conditions for detection of pathogenic E. coli.

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DETECTION AND SEQUENCING GENES IRON, IUTA, AND HLYF IN AVIAN PATHOGENIC ESCHERICIA COLI

Buletin Veteriner Udayana ◽

10.24843/bulvet.2019.v11.i02.p16 ◽

2019 ◽

pp. 229

Author(s):

Nyoman Anandiya Ramaditya ◽

I Nengah Kerta Besung ◽

I Gusti Ngurah Kade Mahardika

Keyword(s):

The Other ◽

Marker Genes ◽

Chain Reaction ◽

Gene Markers ◽

E Coli ◽

The World ◽

Pcr Method ◽

Polymerase Chain ◽

Escherichia Coli Bacteria ◽

Pathogenic Gene

Research has been carried out to detect pathogenic gene markers of Avian Pathogenic Eschericia coli (APEC) iroN, iutA, and hlyF in Escherichia coli bacteria isolated from organs of sick chickens in Bali, and to determine phylogenetic relationships between those marker genes in Bali and in the other countries in the world. Six isolates of E. coli bacteria with codes E2, E3, E7, E8, E9, and E10 were used in this study. The isolates were isolated from domestic chicken in 2018. All genes were detected using the Polymerase Chain Reaction (PCR) method. The genes of iroN, iutA, and hlyF could be detected from all isolates. Well readable sequence of iroN, hlyF, and iutA was 659 bp, 518 bp, and 250 bp, respectively. All three genes were homogenous. Phylogical analysis shows that all pathogenic markers share same cluster with the pathogenic E. coli from all countries in the world.

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Environmental Effect of HV Towers at the Cerebellum and Cognitive Impairments in the Monkey

Basic and Clinical Neuroscience Journal ◽

10.32598/bcn.2021.1340.5 ◽

2021 ◽

pp. 1-24

Author(s):

Hamed Aliyari ◽

◽

Hedayat Sahraei ◽

Mohammad Bagher Menhaj ◽

Masoomeh Kazemi ◽

...

Keyword(s):

High Voltage ◽

Rhesus Monkeys ◽

Visual Learning ◽

Rt Pcr ◽

Electrical Field ◽

Cerebral Mri ◽

Before And After ◽

Pcr Method ◽

Cerebellar Anatomy ◽

Male Rhesus

Today, living or working of people in the vicinity and even under the high-voltage lines is a pernicious environmental hazard to humans. The male rhesus monkey is used to investigate the effects of fields produced by high-voltage towers. In this study, the function and level of effect in rhesus monkeys' brain have been investigated in cerebellum's cognitive, biological and structural perspective. Two monkeys have been used, one as a control and the second under test. The monkey under test was subjected to a simulated HV electrical field of 3 kV/m for 4 hours a day for a one month. Behavioral tests were performed using a device designed and built for this purpose. Concentration analysis of adrenocorticotropic hormones (ACTH) and inspection of glucocorticoid receptor gene's (GR) expression were performed by the RT-PCR method. Changes in cerebellar anatomy with MRI images were examined. All tests were performed before and after the test period and were compared with the control monkey. Cognitive tests showed a significant reduction for the monkey that was exposed to a high-voltage electrical field in the first week after field imposition compared with the same time before. Also, the expression of the GR gene was decreased and the concentration of ACTH hormone in plasma was increased. Surveying the level of cerebral MRI images did not show any difference, but hemorrhage was evident in a part of the cerebellum. The results of cognitive, biological and MRI tests in the tested monkey showed a decrease in the visual learning and memory indices.

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Development of a real-time RT-PCR method for the detection of Citrus tristeza virus (CTV) and its implication in studying virus distribution in planta

3 Biotech ◽

10.1007/s13205-021-02976-5 ◽

2021 ◽

Vol 11 (10) ◽

Author(s):

Sunil B. Kokane ◽

Pragati Misra ◽

Amol D. Kokane ◽

Mrugendra G. Gubyad ◽

Ashish J. Warghane ◽

...

Keyword(s):

Real Time ◽

Citrus Tristeza Virus ◽

In Planta ◽

Rt Pcr ◽

Pcr Method ◽

Tristeza Virus ◽

Virus Distribution ◽

Citrus Tristeza

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Sewage impact on shellfish microbial contamination

Water Science & Technology ◽

10.2166/wst.2004.0035 ◽

2004 ◽

Vol 50 (1) ◽

pp. 117-124 ◽

Cited By ~ 25

Author(s):

M. Pommepuy ◽

F. Dumas ◽

M.P. Caprais ◽

P. Camus ◽

C. Le Mennec ◽

...

Keyword(s):

Microbial Contamination ◽

Sewage Treatment ◽

Human Origin ◽

Rt Pcr ◽

Model Calculations ◽

Microbial Water Quality ◽

Sewage Treatment Plants ◽

E Coli ◽

Pcr Method ◽

The Impact

Coastal areas are frequently contaminated by microorganisms of human origin, due to high population density and low seawater renewal. To evaluate the impact of wastewater input on shellfish quality, a study was conducted in Brittany (France) over a period of 20 months. A hydrodynamic model was used to simulate wastewater impact on microbial water quality. To validate the model, wastewater from the three main sewage treatment plants and shellfish from three sites were sampled monthly. Bacterial indicators (E. coli), F-RNA phages were searched for by culture and noroviruses by RT-PCR and hybridisation. These microorganisms were detected in the three effluents and clams, with no marked seasonal variation. The microbial concentrations in the two oyster beds, distant from the effluent outfall, were low, and only three of the samples were positive for norovirus. For simulation, the winter wastewater inputs of E. coli and phages were calculated and an estimation for norovirus flux was made from the epidemic situation in the population. The microbial behaviour was included in the model by a decay-rate factor. Results from the model calculations were found to be very similar to E. coli and phage concentrations observed in shellfish. For noroviruses, the model indicated that shellfish distant from the wastewater input were under the detection limit of the RT-PCR method. This study demonstrated the use of modelisation to interpret norovirus contamination in various areas.

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