# Real-Time PCR Confirms Infection with _Lagovirus europaeus_
## 5 Discussion
[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_ GL1. Analysis of the obtained results revealed linearity ranging from \(10^{1}\) to \(10^{10}\) copies, which was consistent with the expected values. The high efficiency of the technique was confirmed, as well as a high sensitivity, because it was possible to obtain a positive result using a fivefold dilution of the tested material. The potential of real-time PCR was also verified by [PERSON] et al. [4], who in their research determined the specificity of the virus detection in the livers of rabbits experimentally infected with 17 strains of _L. europaeus_ GL1, showing different biological characteristics such as antigenicity, hemagglutination capacity of the strain, and induced mortality level. In the tests, the result indicating the presence of the virus in the sample was evidenced by the increase in fluorescence generated by the use of SYBRGreen dye, while the specificity of the reaction was confirmed by the analysis of the melting point of the products, which ranged from 87.23 \({}^{\circ}\)C to 87.65 \({}^{\circ}\)C. Thus, it was confirmed that real-time PCR is a highly effective method for the assessment of infection caused by _L. europaeus_ GL1 with different biological properties [4]. Interesting research using real-time PCR was conducted by [PERSON] et al. [28], who compared the replication of the virus in young rabbits experimentally infected with two strains (Czech and Korean) which have been recognized as having potential utility in reducing the abundance of _Oryctolagus_ (_O_). _cuniculus_ in the environment. In the study, real-time PCR analysis was performed using the fluorescent SYBRGreen dye and primers specific for both strains. Thanks to the conducted study, it was possible to determine that the material derived from rabbits infected with the _L. europaeus_ GL1a strain contained twelve times more virus than in the CAPM V-351 strain [28]. Real-time PCR was also used by [PERSON] et al. [29] in studies on the change in the level of virulence (mortality rate, disease duration, number of progeny virions propagated in the host) of four _L. europaeus_ GL1 isolates circulating in the natural environment in Australia since 2006. In the studies, it was shown that the analyzed \begin{table}
\begin{tabular}{c c c} \hline \hline
**Primer** & **Genogroup** & **Reference** \\ \hline RHDV2.F1—GTCGGTCTCGGTAGACTG & & \\ RHDV2.R1—GTGTGTGTCCGGTGTGC & & \\ \hline RHDV2.F2—ACAATTCCCTCATTGTCATTG & & \\ RHDV2.R2—GGGTGTCAGCTCGAGACC & & [33] \\ \hline RHDV2.3F—GCGGTTTTCCGCCGACTTGAC & & \\ RHDV2.3R—GTAGCTGAAACCGGCCTCAG & & \\ \hline RHDV2.4F—CAGCCAAACAGGATTGCAAATG & & \\ RHDV2.4R—CCTGCAAGTCCAATCCAAC & & \\ \hline forward primer—TCCAGATGTTYCCTGACATG & & [34] \\ reverse primer—GCGGTAGGGAGGGTGTGTG & & \\ \hline GI\_qRTPCR\_Fw—TTGACRTACGCCCTGTGGGACC & GL1 and GL2 & [35] \\ GI\_qRTPCR\_Rv—TCAGACATAAGAACGCCATTRGYTG & & \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Cont. samples contained at least \(2.5\times 10^{8}\) copies of the virus, which confirms the high sensitivity of the method used [29]. [PERSON] et al. [38] optimized the real-time PCR method in 2015 with the aim of introducing it as a routine diagnostic measure in the Azores. Thus, its repeatability, reproducibility, specificity, and limit of detection were determined. The performed analysis showed that the reaction efficiency was 94.5% [38]. Real-time PCR was also used by [PERSON] et al. [15], who analyzed the distribution of viral particles of the XA/2010 _L. europaeus_ GI.1 strain in various parts of the organism in infected rabbits. The analysis revealed that the spleen contained the largest amounts of the virus, while the smallest amounts were found in the contents of the small intestine [15]. The appearance of _L. europaeus_ GI.2 in 2010 was associated with the need to develop a quick and effective diagnostic method.