| African swine fever (ASF) is an acute, highly contagious disease in pigs caused by African swine fever virus (ASFV). The disease was originally discovered in Kenya in 1921 and became prevalent in Africa, Europe and Southern America in 1950s and 1960s. Recently, the disease has spread to our neighbor countries inclusing Georgia, Armenia, Azerbaijan and Russia, which presents a great risk for pig industry in China. Presently, there is no effective vaccine against the disease due to poor understanding of its pothagenic mechanisms. Therefore, quick and accurate diagnosis is vital for control and elimination of the disease. In this study three techniques, including ASFV nucleic acid gold nanoparticle amplification, recombinant antigen-based indirect ELISA and colloidal gold indirect immunochromatography, were established for laboratory diagnostic of ASFV.1. ASFV nucleic acid gold nanoparticle amplificationAccording to the sequence alignment of p72 genes of 22 ASFV strains, a conserved sequence was selected to design universal PCR primers and nucleic acid hybridization probes. The capture probes were labelied with 5’-biotin and the detection probes were modified with 3’-alkylthiol, which were then labeled with gold nanoparticles. The highly conserved 651-bp p72 sequence was amplified by PCR and hybridized with the probes. The hybridized products were captured in streptavidin-coated ELISA plates and the signal was amplified using siliver staining, which was easily observable by naked eyes. The result showed that the sensitivity for detection of target sequence was up to 10 fM. To further improve the specificity and sensitivity of the diagnostic method, the highly conserved sequence among the p72 sequences of 100 ASFV strains were selected to design two additional sets of PCR primes and hybridization probes. By systemic optimization of the conditions for PCR amplification and nucleic acid hybridization, the detection sensitivity up to 1 fM was acieved. To verify the specifity of the detection method,11 different ASFV strains and 5 porcine viruses (PRV, PPV, PCV-2, CSFV and PRRSV) were submitted to PCR amplification and gold particle amplification, confirming the high sensitivity and specificity of the detection method.To futher verify the usability of the diagnostic method for detection of the viral genome in the clinical samples, a recombinant pseudorabies virus (PRV) expressing ASFV p72 gene was constructed for infection of mice. The tissues including heart, liver, spleen, lung, kidney, blood, brain, intestine and muscle were collocted 24 h post-infection and the DNAs were extracted for p72 sequence gold nanoparticle amplification. The result showed high consisant with PCR detection.2. Recombinant antigen-based ELISA for ASFV antibody detectionThe recombinant plasmid pET30a-A104R, pET30a-B602L, pET30a-p54 and pET30a-K205R were constructed and transformed into BL21 (DE3) E.coli. After IPTG induction, the recombinant proteins pA104R, pB602R, p54 and pK205R of expected molecular weights of 19KDa,75 KDa,21 KDa and 25KDa were detected in the lysate of the transformants. The recombinant antigens were purified using affinity chromatography and were recognized by anti-ASFV sera in Western-blotting assay. Using the recombiant proteins to detecting the sera from pigs collected at different time post infected with ASFV, the data indicated that the proteins pB602R, p54 and pK205R were good antigens,for indirect ELISA. Then a multi-antigen enzyme-linked immunosorbent assay (MA-ELISA) was developed by using the three proteins as a mixed antigen. For testing the sera from experimentally ASFV infected pigs (N=214), the MA-ELSA had a specificity of 92.1%(95% CI,85.0% to 96.5%) and a sensitivity of 98.2% (95% CI,93.8% to 99.8%). The performance of the MA-ELISA was compared with that of Office International des Epizooties (OIE)-approved ELISA (OIE-ELISA) and two commercial ELISA kits (hereafter ID Vet-ELISA and Ingenasa-ELISA). For testing the experimental sera (n=120), the MA-ELISA had an almost perfect agreement (93.3% agreement, κ=0.86) with OIE-approved Western blotting assay, compared to substantial agreements for OIE-ELISA (88.3%, κ=0.74) and Ingenasa-ELISA (84.2%, κ=0.64), and a slight agreement for ID Vet-ELISA (66.7%, κ=0.18). The main reason for the higher sensitivity of the MA-ELISA was due to its capability to detect ASFV antibodies in the weakly positive sera. For testing the field positive sera from Congo (n=48), the MA-ELISA was significantly consistent with Western blotting recommended by OIE. The results above indicate that the MA-ELISA developed here may become an alternative method for routine serodiagnosis of ASF, in ASF-free countries in particular.3. Colloidal gold indirect immunochromatography for ASFV antibody detectionTwo healthy pigs were immunized with the recombinant protein p54 with adjuvant for 3 times and the sera were collected on day 7 after final boosting with ELISA titer 105-6. Colloidal gold particles of 20-30nm in diameter was labeled with protein P54 and was adsorbed onto fiberglass. As the testing and quality control lines, respectively, SPA and p54 antibody were spraied on nitrocellulose membranes at the concerntrations of 0.2mg/ml and 1mg/ml for colloidal gold strip assembly. In clinical detection, the data indicated that the strips were specific for ASFV positive serum, and no cross reactions for positive sera to CSFV, PRV, PRRSV, PPV and PCV-2. In detection of 141 porcine sera, the colloidal gold strip was compared with OIE recommended indirect ELISA and Western blotting, and the results showed that the strip was better than OIE ELISA in early antibody detection, and there was a higher positive coincidence rate to Western blotting in detecting sera from epidemic area by the strip. Which indicated that the colloidal gold strip was highly specific and sensitive, and had a very good prospect of application in ASF detection. |
