# Real-Time PCR Confirms Infection with _Lagovirus europaeus_
## 2 Real-Time PCR in _Lagovirus europaeus_ GI.1 and GI.2 Studies  
Real-time PCR is a routine diagnostic tool in _L. europaeus_ GI.1 and GI.2 infection [4]. [PERSON] et al. [13] were one of the first research teams to use real-time PCR in the diagnosis of _L. europaeus_ GI.1. The analysis of the obtained results revealed 100% sensitivity of the method, and the possibility of detecting 10 copies of the virus per well and linearity in the range of \(10^{1}\) to \(10^{10}\) copies [13]. The usefulness of real-time PCR was noticed by [PERSON] et al. [14], who used this method to analyze the differentiation of HBGA (histo-blood group antigen binding capacity) among strains showing different pathogenicity to confirm the presence of viral RNA in the liver of experimentally infected rabbits. The potential of real-time PCR has also been verified by [PERSON] et al. [4], who in their studies determined the specificity of virus detection in the livers of rabbits, experimentally infected with _L. europaeus_ GI.1 17 strains, showing different biological characteristics such as antigenicity, hemagglutination capacity, and induced mortality. It was reported that real-time PCR was also used by [PERSON] et al. [15], who analyzed the distribution of the virus (strain XA/2010 _L. europaeus_ GI.1) at various sites in infected rabbits. In this study, the liver, heart, trachea, spleen, kidneys, lungs, small intestine, and body fluids (urine, saliva, and intestinal contents) were subjected to the presence of viral RNA. Analysis of the results revealed that the most copies of the virus were in the spleen, while the smallest amounts were found in the small intestine. So far, it has been found that four different lagoviruses circulate in Australia, of which three are in the GI.1 group. Thus, there was a need to develop a test to detect all of these factors, which is why [PERSON] et al. [16] described a highly specific real-time PCR. The method validation revealed that the developed tests allow for wide and effective detection of all lagoviruses circulating in Australia, and for determination of the number of viral copies in the gastrointestinal tract with high accuracy, thus confirming the high utility of real-time PCR in virological research [16].  
The appearance of _L. europaeus_ GI.2 virus in 2010 was associated with the need to develop a rapid and effective diagnostic method. Identification of this factor has proved to be a key tool in limiting the occurrence of epizootics. Thus, [PERSON] et al. [17] proposed using real-time PCR for this purpose, which allows detection of the presence of _L. europaeus_ GI.2 strains in less than three hours. Analysis of the obtained results showed that only nine viral RNA molecules were detected, and the average viral load was \(1.5\times 10^{7}\) virus copies in \(1\) mg of tissue [17]. The obtained results were also used for the phylogenetic analysis of \(L\). _europaeus_ GI.2. It was found that despite the detection of the virus in 2016, it had already circulated in the environment in 2013; however, its identification had not been possible without the use of molecular methods [18]. RHD epidemiological studies are an important point in monitoring the current situation. [PERSON] et al. [19] conducted a study using real-time PCR, which showed the presence of the virus in the found corpse of a Mediterranean vole (_[PERSON] duodecimcostatus_) and two shrews (_Croclatura russula_). Therefore, it has been shown that within \(L\). _europaeus_ GI.2 there may be passive transmission of the virus to non-Lagomorpha species [19].  
In this work, we use the real-time PCR method in the qualitative diagnosis of _Lagovirus europaeus_ GI.1 strain 237/04, _Lagovirus europeaus_ GI.1a strain 72V/2003, and recombinant strain Hartmannsdorf. Reassessing the effectiveness of this method seems to be necessary in view of the increasing spectrum of RHD-inducing factors. In our research, we have confirmed that real-time PCR is a quick and effective diagnostic method for pathogens causing RHD.