Molecular Diagnosis of African Swine Fever by a New Real-Time PCR Using Universal Probe Library

Abstract

A highly sensitive and specific real-time PCR method was developed for the reliable and rapid detection of African swine fever virus (ASFV). The method uses a commercial Universal Probe Library (UPL) probe combined with a specifically designed primer set to amplify an ASFV DNA fragment within the VP72 coding genome region. The detection range of the optimized UPL PCR technique was confirmed by analysis of a large panel (n = 46) of ASFV isolates, belonging to 19 of the 22 viral p72 genotypes described. No amplification signal was observed when closely clinically related viruses, such as classical swine fever, or other porcine pathogens were tested by this assay. The detection limit of the UPL PCR method was established below 18 DNA copies. Validation experiments using an extensive collection of field porcine and tick samples (n = 260), coming from Eastern and Western African regions affected by ASF, demonstrated that the UPL PCR technique was able to detect over 10% more positive samples than the real-time TaqMan PCR test recommended in the OIE manual, confirming its superior diagnostic sensitivity. Clinical material collected during experimental infections with different ASFV p72 genotypes was useful for assuring both the capacity of the UPL PCR for an early viral DNA detection and the competence of the technique to be applied in any ASF diagnostic target sample. The reliability and robustness of the UPL PCR was finally verified with a panel of ASFV-infected clinical samples which was repeatedly tested at different times. Additionally, an internal control PCR assay was also developed and standardized using UPL probes within the endogenous β-actin gene. Finally, the complete study offers a new validated real-time PCR technique, by means of a standardized commercial probe, providing a simple, rapid and affordable test, which is ready for application in the routine diagnosis of ASF.

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“…Although no virus was recovered from these samples, the presence of antibodies indicates ASFV exposure in these wild boars and confirms the specificity of the results obtained. Overall, the comparative PCR results presented in this study agree with previous work in which the UPL-PCR was able to detect more positive samples than the OIE-PCR tests, thereby confirming its superior diagnostic sensitivity in the detection of survivors and its ability to rapidly detect the disease even when the typical clinical signs are as yet not evident (38).…”

“…In the data obtained, 3.3% more samples were shown to be ASFV positive with the UPL-PCR method (38) than with the OIE conventional PCR (33,36). The DNA was easily detected by both PCRs when high levels of virus were present in blood and tissues in the clinical phase of infection.…”

“…Three PCR assays for the detection of the ASFV genome (33,(36)(37)(38) were tested in parallel using 785 field and experimental samples obtained from genotype II-infected pigs. In the data obtained, 3.3% more samples were shown to be ASFV positive with the UPL-PCR method (38) than with the OIE conventional PCR (33,36).…”

“…The field and experimental samples were tested to detect the ASFV genome using the OIE conventional PCR (33,36), the OIE real-time PCR (33,37), and the Universal ProbeLibrary (UPL) real-time PCR (38). Briefly, 10% (wt/vol) clarified homogenized tissue suspensions were prepared in phosphate-buffered saline using field and experimental tissue samples.…”

“…These PCRs are the most widely used at the national reference laboratory (NRL) level within the EU. New real-time PCRs developed in recent years have been shown to provide greater sensitivity for detecting animals that have survived infection (38,39). Other assays, such as antigen detection enzymelinked immunosorbent assay (ELISA), which allows for largescale testing of samples, are also available at the NRL level but have been reported as having lower analytical sensitivity than that of PCR tests (34).…”