In the present study, a new one-step real-time reverse transcription-polymerase chain reaction (RT-PCR) strategy with minor-groove-binder (MGB) technology for the detection of EAV from 40 semen ...samples of Slovenian carrier stallions was tested. A novel MGB probe (EAVMGBpr) and a reverse primer (EAV-R) based on the multiple sequence alignment of 49 different EAV strain sequences of the highly conserved ORF7 (nucleocapsid gene) were designed. The performance of the assay was compared with different molecular detection methods. Three different primer pairs targeting the ORF1b and ORF7 were used, respectively. The real-time RT-PCR assay was at least 2 log
10 more sensitive than the classical RT-PCR and at least 1 log
10 more sensitive than the primer set used in the semi-nested PCR. The specificities of the amplification reactions were confirmed with biotinylated probes in the PCR-enzyme-linked immunosorbent assay (PCR-ELISA). Under the conditions described in our study, the sensitivity of the real-time RT-PCR was found to be superior to the PCR-ELISA assay. Thus, while the PCR-ELISA method was found to be both relatively demanding and time consuming, better sensitivity coupled with high specificity and speed of the assay makes the real-time RT-PCR a valuable tool for diagnosis of EAV infection.
Abstract Rotaviral RNA was detected in the stool sample of an asymptomatic fattening pig at a Slovenian pig farm. To characterize the rotavirus, RT-PCR was used, employing primers specific for the ...VP7, VP4 and NSP4 genes. Specific products were purified and the sequencing reaction was performed for the molecular analysis of amplified genes. Nucleotide and amino acid sequences of the VP7 gene were found highly identical (85.3–88.1% and 90.7–91.6%) to G1 genotype strains. Phylogenetic and molecular analyses of the VP7 antigen regions revealed the sample to be from a new lineage of G1 genotype. In the molecular analysis of the VP4 gene, only 70.9% nucleotide (76.2% amino acid) identity was found with the most related rotavirus VP4 gene from GenBank. Following this, the NSP4 gene was also analyzed. After the phylogenetic analysis, it clustered with the NSP4 B genotype, but also seemed to represent a new lineage of this genotype. This new rotavirus strain, named P21-5, differed greatly from all rotaviruses characterized so far in all three genes analyzed. The virulence of this strain is not clear yet and has to be investigated.
Infectious hematopoietic necrosis virus (IHNV) is the causative agent of infectious hematopoietic necrosis (IHN) a lethal disease of salmonid fish. Mortality in juvenile fish populations can reach up ...to 100% and in adult fish between 30-70% mortality has been reported. IHN is an economically serious disease of salmonid aquaculture. In Slovenia IHN is widespread and was first isolated in our laboratory in 1996. Following isolation by cell culture, a one-step RT-PCR was performed to confirm the presence of the virus. In this research study a comprehensive genetic characterization of 17 Slovene IHNV isolates obtained from 1997 to 2006 was performed using a 303bp nucleotide fragment of the glycoprotein (G) gene. Probably due to an intensive trade of infected eggs and fish, IHNV was introduced into Europe from USA. In 1987, IHN was detected in France and Italy for the first time. Consequently, IHNV was then distributed to other European countries, however the route of IHNV introduction to Slovenia is unknown. European IHN viruses within M genogroup could be distinguished in 2 subgroups: M-Eur1 compises isolates that originally stem from France, whereas M-Eur2 includes more isolates of Italian origin. The phylogenetic analysis revealed that 13 of 17 IHNV Slovene isolates cluster together with other European IHNV isolates in two subgroups (M-Eur1 and M-Eur2) with genogroup M and that all the Slovene isolates analysed expressed a low genetic diversity. It is important to note that 4 Slovenian isolates within genetic subgroup M-Eur2 were clearly separated from other European IHNV isolates inside M-Eur2.
Rotavirus G9 genotype was thought to be the fifth most common genotype circulating amongst the population. In previous studies in Slovenia, only G1, G3 and G4 genotypes were detected.
To determine G ...and P genotypes of rotaviruses causing dehydrating gastroenteritis in children hospitalised at the University Medical Centre Ljubljana during the winter season 2001–2002. Some data obtained in previous years are included, too.
For the G and P genotypes determination, we selected 99 of the total of 565 rotavirus positive samples. RT-PCR was carried out for G gene or partial P gene amplification. The RT-PCR product was used as a template for multiplex nested PCR using genotype-specific primers. In untypable samples, a sequence analysis of a short segment of G or P gene was performed. From the period before July 2001, 183 stool samples were examined using the same methods.
Genotype G1 was determined in 37, G4 in 6, and G9 in 28 samples out of 99. Only one sample showed a mixed infection with G1G4 genotype specifics. Following the sequence analysis of the short segment of G gene in 11 G9 genotypes, 2 different clusters of G9 genotype were determined. All samples had the same P genotype—P8. G9 genotype had not been detected prior to July 2001.
Rotavirus G9 genotype emerged in Slovenia in the year 2001. Two different clusters were determined which have to be further characterised in detail.
Infectious haematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) are widely distributed fish pathogens in Europe. A reverse transcriptase–polymerase chain reaction ...(RT–PCR) assay was developed for the detection of both viruses as an alternative method to virus assay in cell culture. Oligonucleotide primers corresponding to highly conserved regions of glycoprotein G‐gene sequences were used for IHNV. For the detection of IPNV the VP2‐coding region was selected for RT–PCR amplification. Products of the expected size were amplified from total ribonucleic acid (RNA) extracts of infected cells. The optimized RT–PCR methods successfully detected viral RNA from ovarian and seminal fluids and other organs. To enhance the sensitivity and specificity of RT–PCR, a semi‐nested PCR assay was tested using additional specific inner primers for reamplification of products obtained by RT–PCR. Because of the possibility of template carry‐over contamination, a closed one step RT–PCR method was tested. This technically simplified approach was then combined with the PCR–enzyme linked immunosorbent assay (ELISA) method for the detection of amplification products and verification using specific biotinylated probes. The test provides an additional tool for the detection of IHNV and IPNV which is rapidly and easily performed and is highly sensitive, especially for the detection of IHNV in fish samples coinfected with IPNV. The PCR–ELISA method for the detection of RT–PCR products enables the screening of large numbers of samples and offers the possibility for automatisation of diagnostic work.
Because secondary transmission masks the connection between sources and outbreaks, estimating the proportion of foodborne norovirus infections is difficult. We studied whether norovirus genotype ...frequency distributions (genotype profiles) can enhance detection of the sources of foodborne outbreaks. Control measures differ substantially; therefore, differentiating this transmission mode from person-borne or food handler-borne outbreaks is of public health interest. Comparison of bivalve mollusks collected during monitoring (n = 295) and outbreak surveillance strains (n = 2,858) showed 2 distinguishable genotype profiles in 1) human feces and 2) source-contaminated food and bivalve mollusks; genotypes I.2 and I.4 were more frequently detected in foodborne outbreaks. Overall, approximately 21% of all outbreaks were foodborne; further analysis showed that 25% of the outbreaks reported as food handler-associated were probably caused by source contamination of the food.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, ODKLJ, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
During a period of 5 years (1997–2001) several outbreaks of classical swine fever (CSF) were recorded in Croatia. For genetic typing, fragments of 150 nucleotides within the 5′-non-translated region ...(5′-NTR) and 190 nucleotides within the E2 glycoprotein coding gene of nine field isolates that were derived from domestic pigs and wild boars were used. For better epizootiological understanding, isolates from other European countries were included in the study. The results show that the isolates belong to subgroups 2.1 and 2.3 of CSF virus. Isolates from subgroup 2.1 were collected from domestic pigs during sporadic outbreaks in June 1997 and are genetically closely related. A genomic similarity between these isolates and CSF virus isolates from pigs in other European countries from the same year could also be confirmed. In contrast, the isolate from October 1997 was found to be a member of subgroup 2.3, and is closely related to European CSF virus isolates from outbreaks in the last decade in Western and Central European countries. These results show that two different sources of CSF virus caused outbreaks in Croatia during the same year. Furthermore, a close relationship was found between an isolate from a domestic pig in 1999 and isolates of subgroup 2.3 that originated from Croatian wild boars.
A selection of 43 bovine viral diarrhoea viruses isolated from mainly persistently infected cattle on 23 Slovenian farms between 1997 and 2001 were characterised genetically. Viral RNA was extracted ...from infected cell cultures, reverse transcribed and amplified by PCR with primers targeting the 5′-UTR and the N
pro gene, followed by direct sequencing of purified PCR products obtained for both genomic regions. The N
pro sequences provided the best genetic resolution, and gave also higher statistical support for phylogenetic classification of the viruses. Thirty-eight of the Slovenian isolates were of genetic subtypes 1d and 1f, four were 1b, and one subtype 1g. No BVDV type 2 viruses were found. This genetic prevalence matched those previously reported for neighbouring countries, as opposed to findings reported for more distant European countries, e.g. France, Spain and the UK. From eight cattle herds several virus isolates were analysed; with one exception all isolates from each herd were of the same genetic group. Extended sequencing of the N
pro and part of the C gene of virus isolates with identical 5′-UTR sequences allowed differentiation between isolates obtained at different times from one herd.
Real-time PCR is an accurate, rapid and reliable method that can be used for the detection and also for the quantitation of specific DNA molecules. The basic principle is the recurring measurement of ...a fluorescent signal, which is proportional to the amount of amplification product. In our trial two detection systems were tested for classical swine fever virus (CSFV) detection and for its discrimination from other pestiviruses; non-specific dsDNA-binding dye SYBR Green and specific fluorogenic TaqMan MGB probes. Real-time RT-PCR assays were evaluated for diagnostic sensitivity and specificity by different pestiviral reference and field strains. With both approaches, SYBR Green and TaqMan probes, respectively, all of the CSFV strains isolated on cell culture were detected and also clearly distinguished from other pestiviruses. However, the established one-step real-time TaqMan RT-PCR assay was shown to be more appropriate for pestivirus quantitation, it reduces the risk of contamination and is less time consuming.
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