scholarly journals Significant compartment‐specific impact of different RNA extraction methods and PCR assays on the sensitivity of Hepatitis E virus detection

2021 ◽  
Author(s):  
Patrick Behrendt ◽  
Birgit Bremer ◽  
Daniel Todt ◽  
Eike Steinmann ◽  
Michael Peter Manns ◽  
...  
Keyword(s):  
Hepatitis E Virus ◽  
Rna Extraction ◽  
Virus Detection ◽  
Hepatitis E ◽  
Extraction Methods ◽  
Rna Extraction Methods ◽  
Pcr Assays ◽  
Specific Impact

Rapid Virus Detection Procedure for Molecular Tracing of Shellfish Associated with Disease Outbreaks

2007 ◽  
Vol 70 (4) ◽  
pp. 967-974 ◽  
Author(s):  
ANA MARIA de RODA HUSMAN ◽  
FROUKJE LODDER-VERSCHOOR ◽  
HAROLD H. J. L. van den BERG ◽  
FRANÇOISE S. LE GUYADER ◽  
HILDE van PELT ◽  
...  
Keyword(s):  
Extraction Method ◽  
Rna Extraction ◽  
Virus Detection ◽  
Disease Outbreaks ◽  
Extraction Methods ◽  
Virus Rna ◽  
Pathogenic Viruses ◽  
Rna Extraction Methods ◽  
Digestive Glands ◽  
Outbreak Situation

Detection of pathogenic viruses in oysters implicated in gastroenteritis outbreaks is often hampered by time-consuming, specialist virus extraction methods. Five virus RNA extraction methods were evaluated with respect to performance characteristics and sensitivity on artificially contaminated oyster digestive glands. The two most promising procedures were further evaluated on bioaccumulated and naturally contaminated oysters. The most efficient method was used to trace the source in an outbreak situation. Out of five RNA extraction protocols, PEG precipitation and the RNeasy Kit performed best with norovirus genogroup III–spiked digestive glands. Analyzing 24-h bioaccumulated oysters revealed a slightly better sensitivity with PEG precipitation, but the RNeasy Kit was less prone to concentrate inhibitors. The latter procedure demonstrated the presence of human noroviruses in naturally contaminated oysters and oysters implicated in an outbreak. In this outbreak, in four out of nine individually analyzed digestive glands, norovirus was detected. In one of the oysters and in one of the fecal samples of the clinical cases, identical norovirus strains were detected. A standard and rapid virus extraction method using the RNeasy Kit appeared to be most useful in tracing shellfish as the source in gastroenteritis outbreaks, and to be able to make effective and timely risk management decisions.

Hepatitis E Virus RNA in Commercial Porcine Livers in The Netherlands

2007 ◽  
Vol 70 (12) ◽  
pp. 2889-2895 ◽  
Author(s):  
MARTIJN BOUWKNEGT ◽  
FROUKJE LODDER-VERSCHOOR ◽  
WIM H. M. VAN DER POEL ◽  
SASKIA A. RUTJES ◽  
ANA MARIA DE RODA HUSMAN
Keyword(s):  
The Netherlands ◽  
Hepatitis E Virus ◽  
Blot Hybridization ◽  
Rna Extraction ◽  
Southern Blot Hybridization ◽  
Hepatitis E ◽  
Extraction Methods ◽  
High Dose ◽  
Genotype 3 ◽  
Potential Health

Human hepatitis E virus (HEV) infections by genotype 3 strains in industrialized countries are hypothesized to be caused by pigs. To examine this hypothesis, the potential health risks of transmission routes should be examined. Possible foodborne transmission was studied by quantifying the presence and infectivity of HEV in commercial porcine livers in The Netherlands. A comparison of four tissue disruption and seven RNA extraction methods revealed that mechanical disruption followed by silica-based RNA extraction gave the highest RNA yields and was therefore employed on commercial porcine livers. Four (6.5%) of 62 porcine livers were HEV RNA positive by reverse transcriptase PCR and Southern blot hybridization. Each positive liver was estimated to contain ∼65 PCR-detectable units per g. Sequences were obtained for three of four positive livers and classified as HEV genotype 3. Ninety-three percent similarity to Dutch human HEV sequences and 97% similarity to Dutch swine HEV sequences were observed. To determine whether positive livers contained infectious HEV particles, extracts from livers with known HEV RNA sequences were inoculated intravenously in pigs. Two control pigs were included: one was inoculated with a high dose known to result in infection (104 PCR-detectable units of HEV RNA), and the other was inoculated with a lower concentration of virus that equaled the concentration of PCR-detectable units in commercial livers (∼20 PCR-detectable units). Infection was observed in the high-dose control, but not in other pigs, suggesting a dose-dependent response in pigs. Hence, the implications of HEV RNA in commercial porcine livers in The Netherlands are unknown. However, HEV RNA is present in commercial porcine livers, and sufficient heating of porcine livers before consumption as precautionary measure is recommended.

scholarly journals Comparison of RNA Extraction Methods for Molecular Analysis of Oral Cytology

2016 ◽  
Vol 50 (2) ◽  
pp. 108-115 ◽  
Author(s):  
Alves Mônica Ghislaine Oliveira ◽  
Mario Pérez-Sayáns ◽  
Maria-Elena Padín-Iruegas ◽  
Maria Dolores Reboiras-López ◽  
José Manuel Suarez-Peñaranda ◽  
...  
Keyword(s):  
Molecular Analysis ◽  
Rna Extraction ◽  
Extraction Methods ◽  
Rna Extraction Methods ◽  
Oral Cytology

scholarly journals Optimal RNA Extraction Methods and Development of Synthetic Clones for Seven Strawberry Viruses

2020 ◽  
Vol 26 (3) ◽  
pp. 170-178
Author(s):  
Sun-Jung Kwon ◽  
Ju-Yeon Yoon ◽  
In-Sook Cho ◽  
Bong-Nam Chung
Keyword(s):  
Rna Extraction ◽  
Extraction Methods ◽  
Rna Extraction Methods

scholarly journals Electrical pulse-induced electrochemical biosensor for hepatitis E virus detection

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Ankan Dutta Chowdhury ◽  
Kenshin Takemura ◽  
Tian-Cheng Li ◽  
Tetsuro Suzuki ◽  
Enoch Y. Park
Keyword(s):  
Hepatitis E Virus ◽  
Electrochemical Biosensor ◽  
Virus Detection ◽  
Hepatitis E ◽  
Electrical Pulse

scholarly journals Interlaboratory Validation of a Detection Method for Hepatitis E Virus RNA in Pig Liver

2020 ◽  
Vol 8 (10) ◽  
pp. 1460
Author(s):  
Eva Trojnar ◽  
Matthias Contzen ◽  
Dominik Moor ◽  
Anja Carl ◽  
Sabine Burkhardt ◽  
...  
Keyword(s):  
Hepatitis E Virus ◽  
Detection Method ◽  
Collaborative Study ◽  
False Negative ◽  
Rna Extraction ◽  
Hepatitis E ◽  
Genotype 3 ◽  
Liver Sample ◽  
Pig Liver ◽  
Pork Liver

Background: In the last years, the number of notified hepatitis E cases in humans has continuously increased in Europe. Foodborne infection with the zoonotic hepatitis E virus (HEV) genotype 3 is considered the major cause of this disease. Undercooked liver and raw sausages containing the liver of pigs and wild boar are at high risk of containing HEV. However, so far, no standardized method for the detection of HEV-RNA in pig liver is available. Methods: An international collaborative study on method reproducibility involving 11 laboratories was performed for an HEV-RNA detection method, which consists of steps of sample homogenization, RNA extraction and real-time RT-PCR detection, including a process control. Naturally contaminated pork liver samples containing two different amounts of HEV and a HEV-negative pork liver sample were tested by all laboratories using the method. Results: Valid results were retrieved from 10 laboratories. A specificity of 100% and a sensitivity of 79% were calculated for the method. False negative results were only retrieved from the sample containing very low HEV amounts near the detection limit. Conclusions: The results show that the method is highly specific, sufficiently sensitive and robust for use in different laboratories. The method can, therefore, be applied to routine food control as well as in monitoring studies.

scholarly journals Detection of Viable Cryptosporidium parvum in Soil by Reverse Transcription–Real-Time PCR Targetinghsp70mRNA

2011 ◽  
Vol 77 (18) ◽  
pp. 6476-6485 ◽  
Author(s):  
Zhanbei Liang ◽  
Ann Keeley
Keyword(s):  
Cryptosporidium Parvum ◽  
Reverse Transcription ◽  
Rna Binding ◽  
Direct Detection ◽  
Rna Extraction ◽  
Milk Powder ◽  
Extraction Methods ◽  
Content Type ◽  
Total Rna ◽  
Rna Extraction Methods

ABSTRACTExtraction of high-quality mRNA fromCryptosporidium parvumis a key step in PCR detection of viable oocysts in environmental samples. Current methods for monitoring oocysts are limited to water samples; therefore, the goal of this study was to develop a rapid and sensitive procedure forCryptosporidiumdetection in soil samples. The efficiencies of five RNA extraction methods were compared (mRNA extraction with the Dynabeads mRNA Direct kit after chemical and physical sample treatments, and total RNA extraction methods using the FastRNA Pro Soil-Direct, PowerSoil Total RNA, E.Z.N.A. soil RNA, and Norgen soil RNA purification kits) for the direct detection ofCryptosporidiumwith oocyst-spiked sandy, loamy, and clay soils by using TaqMan reverse transcription-PCR. The study also evaluated the presence of inhibitors by synthesis and incorporation of an internal positive control (IPC) RNA into reverse transcription amplifications, used different facilitators (bovine serum albumin, yeast RNA, salmon DNA, skim milk powder, casein, polyvinylpyrrolidone, sodium hexametaphosphate, andSalmonella entericaserovar Typhi) to mitigate RNA binding on soil components, and applied various treatments (β-mercaptoethanol and bead beating) to inactivate RNase and ensure the complete lysis of oocysts. The results of spiking studies showed thatSalmonellacells most efficiently relieved binding of RNA. With the inclusion ofSalmonelladuring extraction, the most efficient mRNA method was Dynabeads, with a detection limit of 6 × 102oocysts g−1of sandy soil. The most efficient total RNA method was PowerSoil, with detection limits of 1.5 × 102, 1.5 × 103, and 1.5 × 104C. parvumoocysts g−1soil for sandy, loamy, and clay samples, respectively.

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