# Real-Time PCR Confirms Infection with _Lagovirus europaeus_
## 5 Discussion  
The real-time PCR method is a technique that successfully replaces classic PCR in virological research. Due to its high efficiency and the possibility of quantifying a small number of viral particles, it is widely used in research on _L. europaeus_, including its diagnostics [25]. The list of primers used in published analyses using the real-time PCR method is presented in Table 2.  
Figure 4: Percentage distribution of Ct values. Figure 3: Exemplary analysis of melting curves and temperature for _L. europaeus_ GL1 on the example of strain 72V/2003 (red curve: sample 1, blue curve: sample 2, green curve: sample 3). \begin{table}
\begin{tabular}{c c c} \hline \hline
**Primer** & **Genogroup** & **Reference** \\ \hline vp60-7\_for—ACYTGACTGAACTYATTGACG & & [13] \\ vp60-8\_rev—TCAGACATAAGAAAAGCCATT GG & & [13] \\ \hline RHDV-P33(F)—CCACCCCAACACTTCAGGT & & [26] \\ RHDV-P34(R)—CAGGTTGAACACAGGTGTGC & & [27] \\ \hline RHDV-RT2-fw—ACCCAGATACGGCACGRGCCTCCAACC & & [27] \\ RHDV-RT2-rv—CATATCCCATCAAACCAGATGCAAAGG & & [14] \\ \hline forward primer—TCTGTGTCGTCAGCCCCCC & & [4] \\ reverse primer—GACGATGATGATGATG & & [4] \\ \hline forward primer—ACYTGACTGAACTYATTGACG & & \\ reverse primer—TCAGACATAAGAACGCATTGG & & \\ \hline forward primer—ACCCAGATACGGCACGRGCCTCCAACC & & \\ reverse primer—CTATCTCCATGAAACCAGATGCAAAGG & & \\ \hline forward primer—GTTGTATTCGGCCAACCGGAAG & & [28] \\ reverse primer—CTATGGAACACAAGCAAACATCAC & & \\ \hline forward primer—GGTGTTTATGGTGCCTGTAA & & \\ reverse primer—GCACAATTACCTTGTCCCAAAGTTT & & \\ \hline forward primer—ACCCAGATACGGCACGRGCCTCCAACCAC & & [29] \\ reverse primer—CTATCCCATCGAAACCAGATGCAAAGG & & [16] \\ \hline VP60-D-Is—GTTCCCACACCTGTCCTTAG & & [15] \\ VP60-D-Ia—GTAGAGACTGGGGTCGAG & & [16] \\ \hline GI\_qRTPCR_Fw—TTGACTACCCCTGGGACC & & \\ GI\_qRTPCR_Rw—TCAGACATAAGAAGCCATTTGYTG & & \\ \hline GI1-Aus\_fwd—GCGTGCGCATTGTGCCAGCATCG1.Ia-Aus\_rev— & & \\ TGTTGGTAAAGCCATAATACGACG & & [16] \\ \hline GI1c\_fwd—AGCAAGACCTGGTACACATTCG & & [17] \\ \hline GI1c\_rev—AGCCCTGCCACACGTCAGT & & \\ \hline GI1a-K5\_fwd—TTTTATAGATGTATCCCGCTCAAC & & \\ GI1a-K5\_rev—CCGTTCGAGTTCCTTCCGAG & & \\ \hline RHDVXa2010-F1—GCACCCGGCAGTATTCTC & & [30] \\ RHDVXa2010-R1—CCACAGCCCAGCGTCACATCTG & & [30] \\ \hline RHDV2-F—TGGAAACTTGCTTGAGGTGTTG & & [16] \\ RHDV2-R—ACAAGCCGTGTGTGTGGACGG & & \\ \hline RHDV2.F1—GTGTCTCGGTAGTACCTG & & \\ RHDV2.R1—GTGTCCGTCGGTGTGTAC & & \\ \hline RHDV2.F2—ACAATTCCCCTCATGTTGTCATTG & & \\ RHDV2.R2—GGGTGTCAGCTGCAGACC & & [18] \\ \hline RHDV2.3F—GCCGTTTTCCCCCCATTGC & & \\ RHDV2.3R—GTTAGACCTGAACCGGCCTCAG & & GL2 \\ \hline RHDV2.4F—CAGCCAAACAGAGATTGTCAATG & & \\ RHDV2.4R—CCTCGAAGTCCAATTCCAACC & & \\ \hline
27F—CCATGCCAGACATTGGCGTCCC & & \\
986R—AACCATCTGGGACAATTGGG & & \\ \hline
717F—CCCAGATCTCCTCACAACCC & & [31] \\ RC10R—GCCCTGCAAGTCCAATCC & & \\ \hline RHDVDbf–GACCAGACAGAGGTAAACG & & \\ RHDVDbr—TGTCAGGTCGACCCG & & [32] \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Characterization of primers used in real-time PCR in \(L\). _europaeus_ GI.1 and GI.2 studies. One of the first research teams who used real-time PCR in the diagnosis of _L. europaeus_ GL1 virus was [PERSON] et al. [13], who developed a highly specific real-time PCR method. These studies were then based on the analysis of 13 strains responsible for the RHD outbreaks in Germany in 1989-2004. The analysis of the obtained results revealed 100% sensitivity of the method and the ability to detect as many as 10 copies of the virus per well, as well as linearity in the range of \(10^{1}\) to \(10^{10}\) copies [13]. In routine diagnostics, real-time PCR was used by [PERSON] et al. [36] to diagnose the presence of the virus in 19 liver samples collected from rabbits from three Egyptian provinces (Giza, Kalubia, and Kafr-El-Sheikh). The analysis used specific primers developed in 1995 by [PERSON] et al. [26], based on the gene encoding the VP60 protein. The analysis of the results showed that real-time PCR is a highly efficient and effective method for diagnosing _L. europaeus_ GL1 infections [36]. [PERSON] et al.