Detection of enteroviruses and hepatitis a virus RNA in cow milk by RT-PCR (original) (raw)
Related papers
International Journal of Food Microbiology, 2008
Enteric viruses are important agents of foodborne disease. Unfortunately, robust, quantitative methods for sampling and analysis of enteric and other viruses in processed or complex foods are not well-established. As a result, epidemiologically determined etiologies or pathogen sources in foodborne outbreaks are rarely confirmed by virological analysis. In this study, an acid-adsorption elution concentration (AEC) method previously used to monitor virus occurrence and investigate enteric virus outbreaks in shellfish was adapted for examination of processed food items, namely tomato sauce and blended strawberries. Hepatitis A virus (HAV), poliovirus, and coliphage MS2 (MS2) were seeded in 10 or 30 g samples of tomato sauce or blended strawberries, recovered by AEC, and quantified by cell culture infectivity assay. In addition, nested reverse transcription-polymerase chain reaction (RT-PCR) and TaqMan RT-PCR assays were used to detect HAV RNA. Viruses were efficiently adsorbed to foods as an initial concentration step, with infectious HAV and MS2 adsorption of 67% and 93%, respectively, to tomato sauce, and 89% and 99%, respectively, to blended strawberries. Forty-three to 65% of HAV and poliovirus were subsequently eluted and recovered from tomato sauce using 0.5 M threonine, pH 7.2. The lower limits of HAV detection were at initial seeding levels of 14 PFU/g of tomato sauce and 33 PFU/g of blended strawberries. Unlike TaqMan RT-PCR, nested RT-PCR was not inhibited by undiluted final RNA extracts of tomato sauce or blended strawberries. The successful adaptation of the AEC method for enteric and other virus recovery, quantitation and detection in processed foods demonstrates its potential for use in the investigation of foodborne outbreaks of viral etiology and for validation of virus disinfection and sanitary processing procedures used by the food industry.
Enhancement of the Molecular and Serological Assessment of Hepatitis E Virus in Milk Samples
Microorganisms, 2020
Hepatitis E virus (HEV) infection is endemic in developing and developed countries. HEV was reported to be excreted in the milk of ruminants, raising the possibility of transmission of HEV infection through the ingestion of contaminated milk. Therefore, the detection of HEV markers in milk samples becomes pivotal. However, milk includes inhibitory components that affect HEV detection assays. Previously it was reported that dilution of milk matrix improves the performance of HEV molecular assay, however, the dilution of milk samples is not the best strategy especially when the contaminated milk sample has a low HEV load. Therefore, the objective of this study is to compare the effect of extraction procedures on the efficiency of HEV RNA detection in undiluted milk samples. In addition, we assessed the effect of the removal of milk components such as fats and casein on the performance of the molecular and serological assays of HEV. Phosphate buffered saline (PBS) and different milk ma...
Evidence of Hepatitis E Virus in Goat and Sheep Milk
Viruses, 2020
Hepatitis E virus (HEV) is the etiological agent behind hepatitis E infection. Domestic pigs and wild boars are the main animal reservoirs of HEV. Very few papers describe HEV infection in goats and sheep. As the data pertaining to the presence of HEV virus in the milk of small ruminants in Europe are lacking, the aim of this paper was to examine a representative number of milk samples from these animals. The detection of HEV genome (HEV RNA) was performed using reverse transcriptase real-time polymerase chain reaction (RT-qPCR). HEV RNA was found in 2.8% of the examined samples. Positivity ranged from 101 to 103 genome equivalents/mL (GE/mL) with a median of 9.99 × 102 GE/mL. On the basis of these results, the milk of small ruminants could represent a source of HEV infection to consumers.
Food Control, 2008
The objective of this study was to detect HAV antigen in raw cow's milk supplied in Mashhad, Iran using ELISA technique. To ascertain the best recovery method for detecting HAV antigen in raw milk, different dilutions of HAV antigen (0.00, 1.0, 10 À3 , 10 À6 and 10 À9 ml/l) were added to UHT milk. Results obtained from the recovery of HAV antigen from treated milk showed that milk with acidic coagulation followed by filtration with paper filters and membrane filters, had the most optical density and considered to be the best recovery method. Then, the detection of HAV in raw milk in two seasons spring and summer was investigated. HAV antigen was detected in 13.3% and 34.48% of milk samples of spring and summer, respectively.
Monitoring of Foodborne Hepatitis A virus Outbreaks in the Fresh Foods
Egyptian Journal of Botany, 2017
H EPATITIS A virus (HAV) is one of the most widespread foodborne pathogens and the cause of viral hepatitis. Fresh foods can be considered as a vector of transmission for HAV when contaminated by spoiled irrigation water or when prepared by infected food handlers. To improve microbiological detection and to increase insights into the contribution of fruit and vegetables to foodborne viral transmission, sensitive and standardized methods are needed. Two outlet drainage water samples from El-Mariotia and El-Gable Elasfar canals (Nile River) were collected in each month starting from March 2015 over a period of 12 months. As well as vegetables and fruit samples (lettuce, green onion and strawberry) were irrigated and washed with drainage water were also collected. Samples were extracted and concentrated for viral analysis. Strawberry and lettuce foods collected from El-Mariotia and lettuce from El-Gable Elasfar gave positive serological results by enzyme-linked immunosorbent assay (ELISA) test. Reverse transcription polymerase chain reaction (RT-PCR) was successfully used to detect the virus in strawberry and lettuce samples using HAV-specific primers, designed to amplify a 500 bp fragment covering VP1/2A gene in HAV. The VP1/2A gene was sequenced and the nucleotide sequence similarity was in the range of 95-99.1% between HAV-Eg isolate and 30 HAV sequences retrieved from GenBank. Phylogenetic analysis revealed that HAV-Eg isolate was grouped into a clade comprising Egyptian HAV isolates sub-genotype IB. Phylogenetic tree of nucleotides sequence showed that HAV sub-genotype IB is the circulating strain.
Elsevier eBooks, 1990
Sensitive and specific methods are needed to detect hepatitis A virus (HAV) and other human enteroviruses in environmental samples such as drinking water and fobds. Clones of cDNA encoding the S-most 1 kb of the HAV and coxsackievirus B3 (CB3) genomes were subcloned into T7/SP6 RNA transcription vectors. In vitro transcribed RNA from the T7 promoter detected their respective HAV or CB3 genomic RNA. Conversely, SP6 transcripts detected viral negative-stranded RNA but not the genome. When both ssRNA probes were tested at high temperature (65"(Z), they did not hybridize with intracellular RNAs from 6 primate cell cultures used for isolation of HAV and other enteroviruses. The HAV probe did not hybridize with 13 different enteroviruses but detected as little as 500-1000 infectious units of the 7 strains >of HAV tested. Conversely, t&e CB3 probe showed strong homology with all 13 enteroviruses tested but not HAV. The probes were used to detect HAV and other enteroviruses in water samples after virus amplification in cell culture. HAV was detected in water samples obtained during a waterborne hepatitis outbreak using the ssRNA probe. These samples were negative for HAV by direct solid phase radioimmunoassay and were not positive by immunoassays of inoculated cell cultures until several weeks of propagation. The CB3 ssRNA probe detected
Tarım Bilimleri Araştırma Dergisi, 2016
Bovine enteroviruses are thought to be mild pathogenic or non pathogenic viral gastroenteretis agents. However there are some cases where they are identified from cattle having different symptoms. They can also be used as markers of environmental contamination. Enterovirus family has fast replication capacity and can be used as potential vectors. For this purpose isolation and molecular studies concerning Enteroviruses are currently increasing. In this study 10 cattle feces samples were used for evaluation of 4 different extraction methods for investigation of bovine enteroviruses. These methods comprise of TRIzol LS (Life Technologies), QIAamp Viral RNA mini kit (Qiagen), NucleoSpin RNA (Macherey-Nagel) and phenol-chloroform extraction. All procedures performed in BSL-2 cabinet and RNA yield of extracts were analysed in Bio-spec (Shimadzu) spectrophotometer. This RNA templates were then tested by Verso 1 step RT PCR (Thermo Scientific) kit with spesific for Enterovirus 5'UTR primers. And observed for band formation. While QIAmp Viral RNA kit had the shortest protocol, it did not have as much sensitivity as TRIzol LS and TRIzol was determined as the most sensitive method to make extraction of bovine enteroviruses from feces.
Preventive Veterinary Medicine, 2008
Bovine viral diarrhoea virus (BVDV) is an important pathogen of dairy cattle. In this study, bulk milk samples representing a total of 4105 milking cows, from 18 dairy cattle herds in the suburb of Mashhad-Iran, were tested for presence of BVDV by the use of a nested reverse transcription polymerase chain reaction (Nested RT-PCR) assay. Non of the cows in the herds had been vaccinated against BVDV. RNA was extracted from somatic cell pellets of bulk milk tank samples. Oligonucleotide primers were selected based on the 5ú ntranslated region of the BVD virus genome. BVD virus was detected in 2 (11.1%) out of 18 samples, representing 742 lactating cows. These results indicate that nested RT-PCR analysis of bulk milk samples may provide a rapid and sensitive screening method for the detection of BVDV infections in non-vaccinated dairy cattle herds.
Iranian Journal of Biotechnology, 2007
Bovine viral diarrhoea virus (BVDV) is an important pathogen of dairy cattle. In this study, bulk milk samples representing a total of 4105 milking cows, from 18 dairy cattle herds in the suburb of Mashhad- Iran, were tested for presence of BVDV by the use of a nested reverse transcription polymerase chain reaction (Nested RT- PCR) assay. Non of the cows in the herds had been vaccinated against BVDV. RNA was extracted from somatic cell pellets of bulk milk tank samples. Oligonucleotide primers were selected based on the 5´ untranslated region of the BVD virus genome. BVD virus was detected in 2 (11.1%) out of 18 samples, representing 742 lactating cows. These results indicate that nested RT-PCR analysis of bulk milk samples may provide a rapid and sensitive screening method for the detection of BVDV infections in non-vaccinated dairy
Applied and environmental microbiology, 1995
In this study we developed a concentration and purification procedure to facilitate reverse transcription (RT)-PCR detection of enteric viruses in water sample concentrates obtained by conventional filter adsorption-elution methods. One liter of beef extract-glycine eluate with or without humic acid and seeded with poliovirus type 1, hepatitis A virus, and Norwalk virus was used as a model system, and the eluent was further processed for RT-PCR compatibility. The sample concentration and purification procedures which we used included polyethylene glycol precipitation, Pro-Cipitate precipitation, a second polyethylene glycol precipitation, spin column chromatography, and ultrafiltration. The sample volumes were reduced from 1 liter to 20 to 50 microliters, and the samples were purified enough so that viruses could be detected by the RT-PCR. The ability to detect low levels of enteric viruses by molecular techniques was compared directly with the ability to detect enteric viruses by c...