# Real-Time PCR Confirms Infection with _Lagovirus europaeus_
## 5 Discussion
The identification of this factor has proved to be a key tool to limit the emerging epizootics, due to the ineffectiveness of the commonly used vaccines against the GI.1 group. Thus, [PERSON] et al. [17] proposed the use of real-time PCR, which enables the detection of _L. europaeus_ GI.2 strains in less than three hours. The analysis showed that the applied real-time PCR is a highly specific and sensitive diagnostic method, as only nine viral RNA particles were detected, and the average viral load was \(1.5\times 10^{7}\) virus copies in 1 mg of tissue [17]. The application of this method allowed for the diagnosis of _L. europaeus_ GI.2 infection among dead rabbits found on the islands of Garciosa, Flores, Sao Jorge, and Terceir. The conducted tests of samples taken from the livers and lungs showed an increased viral load, which amounted to \(1.5\times 10^{8}\) in 1 mg of tissue, but this was explained by an error resulting from the dehydration of the corpses or the age of the examined individuals [39]. Real-time PCR was used by [PERSON] et al. [18], thanks to which the presence of _L. europaeus_ GI.2 virus was confirmed in Sweden. The obtained results were also used for the phylogenetic analysis of _L. europaeus_ GI.2, and it was possible to state that despite the presence of the virus in 2016, it had already been circulating in the environment in 2013, but its identification had not been possible without the use of molecular methods [18]. Epidemiological research on RHD is an important point in monitoring the current situation. [PERSON] et al. [19] carried out studies using real-time PCR, which showed the presence of the virus in the found carcasses of Mediterranean voles (_Microtus duodenecimcostatus_) and in two specimens of shrews (_Crocidura russula_). Thus, it has been shown that within _L. europaeus_ GI.2, there can be passive transmission of the virus to species beyond the Lagomorph order [19]. The appearance of the _L. europaeus_ GI.2 virus caused a great deal of confusion in many countries, making it necessary to monitor the evolution and spread of this pathogen in wild populations. For this reason, [PERSON] et al. [34] conducted an analysis to assess the picture of the RHD epidemic caused by GI.2 strains in southern Spain in 2013-2017. The analysis of the obtained results showed that the GL.2 strains predominate in the study area, which in turn confirms the usefulness and efficiency of the real-time PCR method in epidemiological analyses [34]. The wide application possibilities of real-time PCR in multidirectional research on _L. europaeus_ GI.1 and GI.2 certainly confirm the advantages of this method. Nevertheless, it has serious limitations. A definite disadvantage of this method is the use of expensive specialized equipment, without which it is impossible to carry out the reaction. The preparation of the sample itself can be tedious as well. Moreover, in real-time PCR, PCR product increases exponentially and variation increases with cycle number [40]. In some cases, overlapping of the emission spectra can be observed, causing an increased risk of false negative results [40]. Currently, more methods are available that allow for the rapid diagnosis of pathogen infection, such as reverse transcription loop-mediated isothermal amplification (RT-LAMP) and clustered regularly-interspaced short palindromic repeats (CRISPR) [41], so you should consider limiting the use of real-time PCR for other analyses not necessarily aimed at diagnostics. Real-time PCR is a very sensitive diagnostic method and therefore false-positive results may be obtained when testing vaccinated rabbits. [PERSON] et al. [31] conducted an experiment in which they detected RNA from vaccines using real-time PCR. The obtained results revealed that RNA vaccine becomes undetectable 15 days after inoculation, which is a very important aspect when interpreting the results of diagnostic analyses. Simultaneous occurrence of various _L. europaeus_ viruses, including group GL2, as well as their recombinants, is observed in many places.