The developed RT-qPCR was able to detect the viral RNA in all bovine pestiviral strains tested and showed high specificity. A major advantage, is the ability to detect Pestivirus H strains, which can be useful in areas where Pestivirus H are co-circulating with Pestivirus A and B. Further, complete agreement was observed between RT-qPCR and viral isolation results of all clinical samples tested. In pooled sera, the test was able to detect a positive animal among 30 sera, which means a significant advantage when carrying out BVD control in low-prevalence herds since it allows reducing the number of assays. However, if pool size is too large, there is an increased chance that any single pool will test positive, requiring additional testing to identify the viremic individuals (Muñoz-Zanzi et al. 2000; Smith et al. 2008). Thus, prevalence estimation should be considered when laboratories use the pooling-strategy, especially in regions with high BVD prevalence (Emmanuel et al. 1988). This research has provided an in-house validated RT-qPCR that can replace commercially available kits, which represent a great cost in trading for some countries, and can be useful for veterinary diagnostic laboratories equipped with the infrastructure to perform qPCR assays.
Because of the SYBR Green-based detection system, the developed RT-qPCR presents some advantages: it is sensitive, easy to use and inexpensive. However, it is important to recognize the presence of any double-stranded DNA (e.g., primer-dimers) that can cause false positive results. This inconvenient can be sorted including an analysis after the amplification run, using the melting temperature of the expected amplicon, which allows the discrimination of the target amplicon to undesired products that can interfere with the results (Ririe et al. 1997). Also, using One-Step for retro-transcription and PCR reaction simultaneously reduces the costs, time-consumed and possibilities of contamination of the assay.
Diagnostic laboratories routinely use blood or serum for BVD analyses. In terms of practicability in the use of filter papers at the point of care, whole blood is more suitable than serum since it avoids the centrifugation step. Although semen is not used as a sample of choice to diagnose bovine pestiviruses, it is useful for the control of BVD in breeding herds (Saliki and Dubovi 2004), however, our group tested the same methodology using semen straw samples spotted in filter papers without achieving satisfactory results (Online Resource 2).
To the best of our knowledge, just two studies were published about preserving bovine pestiviral RNA by using solid carriers. The first article used several filter papers to preserve RNA up to six months in blood and sera samples from PI animals at RT, 4°C and -18°C (Vilcek et al. 2001). In this study, the authors evaluated four different types of papers: classical filter papers, Whatman paper No. 1, nitrocellulose membrane and HYBONDTM-M nylon membrane. Results were similar for all papers tested except for classical filter papers, which yielded lower PCR products. However, the authors used an end-point RT-PCR assay and RNA isolation was carried out directly from filter papers, avoiding the elution step. Unfortunately, that strategy did not yield good results in our experiments (data not shown). Another study was reported in FTA cards for the detection of several viral agents involved in the bovine respiratory complex, including bovine pestivirus, where viral RNA from respiratory tract swabs was stored 14 days at temperatures between -27 and 46°C. The article compared the RNA recovery from specimens in viral transport medium and FTA fixed samples, which proved 100% agreement (Liang et al. 2014). Unlike the former, in this paper authors used a qPCR assay and carried out an elution protocol before RNA extraction.
Our study suggests that chromatographic papers are a useful alternative for collecting and shipping blood samples for BDV diagnostic purposes, as a cost-effective method. However, for longer storage time (6 and 12 months), the recovery percentages decreased, although RNA was still detected, meaning that the biobanking purposes are not suitable for more than 12 months storage at any temperature with the protocols evaluated in this experiment. Nevertheless, the fact that pestiviral RNA was amplified in PI animals’ blood stored up to 12 months in chromatographic paper, suggests that, although the recovery percentage was lower as storage time progressed, it was still possible to recover RNA in blood samples from PI animals kept at refrigeration temperature.
FTA cards contain chelating agents and a free-radical trap designed to deal with atmospheric pollutants, thus protecting the entrapped nucleic acids for at least six years at room temperature (Ahmed et al. 2011). However, in our work, chromatographic paper -which lacks these components-, yielded similar results. Moreover, for 12 months storage, drops spotted in chromatographic paper showed slightly better results than in FTA cards, considering the number of positive repetitions.
When collecting samples, we recommend drying the drop spotted in filter papers at room temperature for at least four hours (depending on ambient humidity), and sending to the laboratory in individual zipped bags with a desiccant, like silica gel. For shipping, refrigeration is not necessary.
The possibility to store viral nucleic acids in filter papers means a breakthrough in veterinary practice. The chance to collect samples directly in filter papers from herds where the disease is suspected could be very helpful, since in this kind of matrix, samples do not require immediately shipping or refrigeration and more important, they do not need to be processed instantly (Choi et al. 2014). This methodology is mostly advantageous when performing molecular studies, since it guarantees nucleic acid stability at a wide range of temperatures and filter papers can be used with a variety of samples. Comparing with the traditional method for collecting, shipping and storing samples, filter papers methodology represents a major improvement specially in large countries with poor infrastructure for shipping samples and also for those which lack laboratories who run this type of assays in every state or province.
Our study demonstrated that an economical filter paper, like chromatographic paper, is an appropriate alternative for collecting, shipping and storing blood samples for BVD diagnosis, giving results as good as using FTA specialized cards. The aforementioned is valid, as long as it is accompanied by a highly sensitive technique as the RT-qPCR reported in this study.
Further studies using this methodology applied to other viruses are granted, especially for diseases relevant to bovine health, which require mandatory control and are economically important for local production in order to improve sampling, shipping and laboratory processing conditions.