Comparison of three different PCR protocols for the detection of ferlaviruses

Abstract

Background Ferlaviruses are important pathogens in snakes often associated with respiratory and neurological disease. The detection of ferlaviral RNA by PCR is considered to be the most reliable method for the diagnosis of infection. The PCRs that have been used most commonly for this purpose have not been properly assessed to determine their sensitivity, specificity and ability to detect the known genetic diversity of this group of viruses. The aim of this study was to compare three published PCR protocols so that a single method could be recommended to laboratories that perform this testing. Results Comparisons were carried out using cell culture isolates and tissues from snakes infected with specific virus genotypes. A single round PCR targeting a short segment of the viral polymerase (L) gene provided the highest sensitivity and specificity, and detected isolated ferlaviruses from all four described genogroups, as well as from tissues of infected snakes. Conclusion A broadly-reactive PCR for the detection of all known ferlaviruses was found to provide a good combination of detection limit, specificity and speed. Based on these criteria, this method is recommended for the diagnosis of ferlavirus infections. Electronic supplementary material The online version of this article (10.1186/s12917-019-2028-0) contains supplementary material, which is available to authorized users.

Detection and molecular epidemiology of ferlaviruses in farmed snakes with respiratory disease in Guangxi Province, China

“…7,19 In order to ensure true positives, samples were further screened for ferlaviruses by another conventional PCR method. 7,11 PCR was performed in a final reaction volume of 25 μL, comprised of 12.5 µL of 2× DNA polymerase master mix (CW Bio), 0.4 µM (1 µL) of each primer, 2 µL of DNA or cDNA template, and 8.5 µL of RNase-free water (CW Bio). The PCR procedure was as follows: initialization at 94°C for 10 min; denaturation at 94°C for 30 s; annealing for 30 s at 45°C, 45°C, 47°C, and 46°C for ferlavirus, sunshine virus, reovirus, and adenovirus, respectively; extension at 72°C for 60 s; 35 cycles of denaturation, annealing, and extension steps; and final elongation at 72°C for 5 min.…”

“…However, 70 of 104 snakes were found to be ferlavirus positive after a conventional PCR (PCR II), suggesting that PCR II is more sensitive for ferlavirus detection than PCR I, which is consistent with the findings of a previous report. 11 The 70 ferlavirus-positive snakes included 49 oriental rat snakes, 19 cobras, and 2 king rat snakes, which were obtained from 34 of the 52 snake farms in Guangxi Province (Table 2). The positive detection rate of snake samples and farms was generally ≥ 50%.…”

“…7,19 In order to ensure true positives, samples were further screened for ferlaviruses by another conventional PCR method. 7,11…”

“…2,16 The amplicon acquired using PCR II was149 bp, which is relatively short and not suitable for phylogenetic studies. 11 Therefore, we used 566 bp of L gene sequences obtained by PCR I for sequence analysis and describing the phylogenetic tree. Sequence analysis of 58 PCR I products revealed that ferlaviruses collected from different snakes from the same farm were identical.…”

The Role of Host Species in Experimental Ferlavirus Infection: Comparison of a Single Strain in Ball Pythons (Python regius) and Corn Snakes (Pantherophis guttatus)

“…Briefly, the PCR targeted a 566 bp portion of the L-gene using primers F5-outer and R6-outer followed by F7-inner and R8-inner in a nested format as described by Ahne et al [14] using the specific methods described by Kolesnik et al [15]. This PCR has been shown to be highly specific and able to detect between 5 × 10 −1 and 5 × 10 −3 ng/µL viral RNA [15]. RNA prepared from a ferlavirus cell culture isolate was used as a positive control and HPLC water was used as a negative control.…”