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Detection of amplicons 510 bp by RT-PCR method using undiluted and diluted RNA samples extracted from the liver of experimentally infected rabbit (strain KGM RHDV). From left to right: DNA molecular weight marker Promega) – 1,000, 750, 500, 300, 150, and 50 bp, RNA dilutions: 10 -1 ,
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A real-time RT–PCR method for the rapid detection of the rabbit haemorrhagic disease virus (RHDV) in the liver and serum samples of rabbits was described. A primer set that targets 3’ part of VP60 gene and TaqMan probe specific for the conserved region in RHDV genome was used in the method. The assay was able to detect genetic material in rabbits i...
Context in source publication
Context 1
... conventional RT-PCR and the set of specific primers flanking 5’ portion of RHDV capsid structural protein, the viral RNA was detected in 26 liver homogenates of rabbits infected with the analysed RHDV strains. The 510 bp fragments of KGM isolate -1 -5 were detected at RNA dilution from 10 to 10 ( Fig. 2). The agarose gel analysis of PCR products demonstrated the presence of 510 bp fragments in two liver homogenates (L3/RI/2008 and L5/IV/2008) of healthy rabbits with corresponding mean C T values of 32 and 40, respectively. No 510 bp fragment was detected in S1 serum sample taken 10 d post challenge (data not shown) from control rabbits, which survived the experimental infection The aim of the presented study was to assess the potential use of the single one-step TaqMan rRT-PCR method for virological diagnosis of RHD. Although international diagnostic methods have not been validated and there is no uniform pattern of RHD virus, many serological and virological tests are currently used in routine diagnosis. For virological purpose, different ELISAs with specific monoclonal or polyclonal antibodies, haemagglutination assay (HA), direct immunoflurescence, and immune electron microscopy are used (1). In addition, several RT-PCR assays were developed and evaluated for the detection of RHDV based on nucleotide sequences of different genome fragments (3, 7, 11, 13, 14). In recent years, various real-time PCR methods have been implemented and applied to the diagnosis of human and animal diseases (15). Real-time RT-PCR (rRT-PCR) offers certain advantages over conventional RT-PCR. It avoids the use of agarose gel electrophoresis, therefore decreasing the risk of contamination, and is suitable for large scale testing and ...
Citations
... A comparison between real-time RT-PCR and conventional RT-PCR highlights some of its advantages, namely the reduced risk of cross-contamination. To this greatly contributes the use of a one-tube protocol that avoids post-PCR handling, which in turn significantly shortens the reaction and the diagnostic times [7,156,158,159]. RNA virus degradation is less challenging as the target amplicons are usually smaller, thus also increasing sen-sitivity [7,158], and intra and inter-assay variability is low [154,156]. ...
... To this greatly contributes the use of a one-tube protocol that avoids post-PCR handling, which in turn significantly shortens the reaction and the diagnostic times [7,156,158,159]. RNA virus degradation is less challenging as the target amplicons are usually smaller, thus also increasing sen-sitivity [7,158], and intra and inter-assay variability is low [154,156]. Detection limits of RHDV real-time RT-PCRs range from 9-100 copies [7,27,156,160] and have been shown to be similar to those of nested PCR [155]. ...
Since the early 1980s, the European rabbit (Oryctolagus cuniculus) has been threatened by the rabbit hemorrhagic disease (RHD). The disease is caused by a lagovirus of the family Caliciviridae, the rabbit hemorrhagic disease virus (RHDV). The need for detection, identification and further characterization of RHDV led to the development of several diagnostic tests. Owing to the lack of an appropriate cell culture system for in vitro propagation of the virus, much of the methods involved in these tests contributed to our current knowledge on RHD and RHDV and to the development of vaccines to contain the disease. Here, we provide a comprehensive review of the RHDV diagnostic tests used since the first RHD outbreak and that include molecular, histological and serological techniques, ranging from simpler tests initially used, such as the hemagglutination test, to the more recent and sophisticated high-throughput sequencing, along with an overview of their potential and their limitations.
... The analysis of the results showed that real-time PCR is a highly efficient and effective method for diagnosing L. europaeus GI.1 infections [36]. Fitzner et al. [37] decided to perform a comparative analysis of the results obtained by conventional PCR and real-time PCR in the diagnosis of infection in the livers of rabbits experimentally infected with 26 strains of L. europaeus GI.1. Analysis of the obtained results revealed linearity ranging from 10 1 to 10 10 copies, which was consistent with the expected values. ...
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Our experience with real-time PCR, together with several different studies in this field show that this is a very useful method for rapid, specific and effective virus detection.
Abstract
Lagovirus europaeus GI.1/GI.2 is an etiological agent causing the highly dangerous rabbit hemorrhagic disease (RHD). Molecular research is the basic tool today that can help solve epidemic problems related to the expansion of pathogens in the world. By using the real-time polymerase chain reaction technique (PCR), we detected three different strains of Lagovirus europaeus/GI.1, which is an RNA virus infecting mainly rabbits. The results showed that the method used was fast, very specific, and effective.
... This technique is known to be more efficient than conventional PCR in the detection and quantification of small numbers of viral particles. 59,111 It has been employed successfully for detection of RHDV strains circulating in rabbit populations in various European countries 1,16,22,30,35,38,86,87 and Australia 77 or for studying virus distribution in tissues of experimentally infected rabbits. 71 Broadly reactive primers within the conserved 3'-region of the VP60 RHDV gene were designed and used in the Taq-Man 35 and SYBR Green 71,86 RT-rtPCR methods. ...
... 71 Broadly reactive primers within the conserved 3'-region of the VP60 RHDV gene were designed and used in the Taq-Man 35 and SYBR Green 71,86 RT-rtPCR methods. When compared with conventional RT-PCR, RT-rtPCR was 100 times more sensitive, 35 with its detection level established at 6.09 genome copies per reaction. 71 The emergence of new RHDV-2 strains in rabbits initiated research toward the development of strain-specific TaqMan RT-rtPCR methods. ...
Various PCR-based assays for rabbit viruses have gradually replaced traditional virologic assays, such as virus isolation, because they offer high-throughput analysis, better test sensitivity and specificity, and allow vaccine and wild-type virus strains to be fully typed and differentiated. In addition, PCR is irreplaceable in the detection of uncultivable or fastidious rabbit pathogens or those occurring in low quantity in a tested sample. We provide herein an overview of the current state of the art in the molecular detection of lagomorph viral pathogens along with details of their targeted gene or nucleic acid sequence and recommendations for their application. Apart from the nucleic acids–based methods used for identification and comprehensive typing of rabbit viruses, novel methods such as microarray, next-generation sequencing, and mass spectrometry (MALDI-TOF MS) could also be employed given that they offer greater throughput in sample screening for viral pathogens. Molecular methods should be provided with an appropriate set of controls, including an internal amplification control, to confirm the validity of the results obtained.
... Some inconvenience of this system is the fact that this fluorochrome non-specifically binds to double-stranded DNA, yet in order to exclude the possibility of wrong interpretation, the analysis of product melting curves must be performed (Kubista et al. 2006, Watzinger et al. 2006, Lipiński et al. 2008). Real-time PCR method, as an element of viral diagnostics, was applied to assessment of viruses of DNA from the Parvoviridae (Dzieciątkowski et al. 2007), Herpesviridae (Grabarczyk et al. 2003, Radkowski et al. 2005, Dzieciątkowski et al. 2008, Poxviridae (Bacławski (Bae et al. 2003, Pejsak et al. 2006, Gurukumar et al. 2009), Retroviridae (Kuźmiak et al. 2006) and Caliciviridae (Nowaczyk 2007, Fitzner et al. 2011, Teixeira et al. 2011 families. Within the last family, it was used for assessment of the volume of viral copies in the peripheral blood of rabbits infected with EBHS (European Brown Hare Syndrome), which is a pathogenic factor for hares (Nowaczyk 2007). ...
... Research performed by Teixeira et al. (2011), referred to the use of a new method for isolation of the RHD virus from liver tissue, namely centrifugation at high rpm in iodixanol gradient, which can be very useful for further "processing", e.g. using real-time PCR. Also Fitzner et al. (2011) applied the real-time PCR method for assess-ment of 26 strains of the RHD virus (KGM 1988, SGM 1988, PD 1989, BLA 1994, MAL 1994, PRB 1995, BDG 1/1996, BDG 2/1997, BDG 3/1997, BDG 4/1998, GSK 1998, PIA 1999, POZ 199, ZD0 2000, SIZ 202, ZDU 2003, OPO 2004, GRZ 2004, ROK 2004, CB 2005, KRY 2005, ZKA 2005, DCE 2006, in which not only the presence of RHD alone was assessed in particular liver samples. It was also shown that the limit of detection of viral RNA extracted from livers is at the 10 -7 dilution. ...
... Thus the optimalisation and validation of the method was appropriate as it confirms the clinical observations (mortality of infected animals). Firstly, the conservative region primers, which were also applied in the study by Fitzner et al. (2011), permitted detection of all analysed strains of the RHD virus, and the result was not affected by differences recorded among the seventeen analysed strains of the RHD virus as regards their biological properties, such as haemagglutination capacity or formation of antigen variants, and time and place of isolation of such strains. Furthermore, various pathogenicity, measured using the mortality percentage of rabbits infected with the analysed strains, also did not affect the positive result of the real-time PCR reaction, which points to the fact that the method is unusually sensitive, and is not dependent on the volume of material to be detected, in this case the number of viral particles in the liver. ...
The paper concerns the use of a novel, very effective diagnostic method, a real-time PCR for diagnosis of a viral agent causing viral haemorrhagic disease in rabbits - RHDV. Until now, the method was widely used for detecting many different viruses, both DNA, and RNA, but as far as RHDV is concerned, there are not many records of such use. This study aimed at the detection of 17 different strains from different European regions, differing in biological features and mortality. The study confirmed that real-time PCR is an applicable and effective method for diagnosis of RHDV, irrespective of the stains' features.
