Highly sensitive PCR assay for routine diagnosis of African swine fever virus in clinical samples

Abstract

This work provides a novel, highly sensitive, hot start PCR method for rapid and specific detection of African swine fever virus (ASFV) that can be used as a routine diagnostic test for ASFV in surveillance, control, and eradication programs. A confirmatory test of the specificity of this method based on restriction endonuclease analysis was also developed.

FIG. 1.

Sensitivity of the ASFV PCR assay using the OIE primer pair or novel PPA-1/2 primer set. DNAs extracted from serial dilutions, in serum, of a suspension of ASFV strain Spain 70 (Spa70) with a titer of 1.6 × 10⁶ UHAD₅₀/ml were employed as templates in the PCR assays under reaction conditions described in the text using OIE primers (left) or novel PPA-1/2 primers (right). M, molecular weight marker VI (Roche Molecular Biochemicals).

FIG. 2.

BsmAI restriction endonuclease analysis of amplification products of different ASFV strains. Lanes 1 and 1′, Brazil 78; 2 and 2′, Haiti 78; 3 and 3′, Spain 70; 4 and 4′, Lisbon 60; 5 and 5′, Cape Verde Islands 97; 6 and 6′, Ivory Coast 99; 7 and 7′, Nigeria 2001. Lanes 1, 2, 3, 4, 5, 6, and 7 are amplification products. Lanes 1′, 2′, 3′, 4′, 5′, 6′, and 7′ are amplification products after digestion with BsmA. Reaction conditions are described in the text. M, molecular weight marker V (Roche Molecular Biochemicals).

FIG. 3.

ASFV detection by PCR in blood (B) or serum (S) samples from experimentally infected animals using OIE (a) or PPA-1/2 (b) primer pairs. Samples of pigs were obtained at different dpi, as described in the text. M, molecular weight marker VI (Roche Molecular Biochemicals).

FIG. 4.

ASFV detection by PCR in blood and pig tissue samples. Samples of blood (treated with EDTA), kidney, liver, lung, lymph nodes, spleen, and tonsils were obtained from a pig experimentally inoculated with Lisbon 60 at 5 dpi and analyzed by PCR using PPA-1/2 primers. M, molecular weight marker VI (Roche Molecular Biochemicals).

FIG. 5.

PCR detection of ASFV on badly preserved samples. Pieces of kidney from a pig infected with ASFV isolate Lisbon 60 were homogenized and analyzed by PCR using either the PPA-1/2 or OIE primer set after 0, 14, or 28 days of storage at room temperature. M, molecular weight marker VI (Roche Molecular Biochemicals).