RESUMO
A rapid, simple, and sensitive diagnostic technique for the detection of African swine fever virus (ASFV) nucleic acid was developed for testing clinical samples in the field or resource-constrained settings. In the current study, the saltatory rolling-circle amplification (SRCA) technique was used for the first time to detect ASFV. The technique was developed using World Organization for Animal Health (WOAH)-approved primers targeting the p72 gene of the ASFV genome. The assay can be performed within 90 minutes at an isothermal temperature of 58°C without a requirement for sophisticated instrumentation. The results can be interpreted by examination with the naked eye with the aid of SYBR Green dye. This assay exhibited 100% specificity, producing amplicons only from ASFV-positive samples, and there was no cross-reactivity with other pathogenic viruses and bacteria of pigs that were tested. The lower limits of detection of SRCA, endpoint PCR, and real-time PCR assays were 48.4 copies/µL, 4.84 × 103 copies/µL, and 4.84 × 103 copies/µL, respectively. Thus, the newly developed SRCA assay was found to be 100 times more sensitive than endpoint and real-time PCR assays. Clinical tissue samples obtained from ASFV-infected domestic pigs and other clinical samples collected during 2020-22 from animals with suspected ASFV infection were tested using the SRCA assay, and a 100% accuracy rate, negative predictive value, and positive predictive value were demonstrated. The results indicate that the SRCA assay is a simple yet sensitive method for the detection of ASFV that may improve the diagnostic capacity of field laboratories, especially during outbreaks. This novel diagnostic technique is completely compliant with the World Health Organization's "ASSURED" criteria advocated for disease diagnosis, as it is affordable, specific, sensitive, user-friendly, rapid and robust, equipment-free, and deliverable. Therefore, this SRCA assay may be preferable to other complex molecular techniques for diagnosing African swine fever.
Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Suínos , Animais , Vírus da Febre Suína Africana/genética , Febre Suína Africana/diagnóstico , DNA Viral/genética , Sensibilidade e Especificidade , Sus scrofa , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Reação em Cadeia da Polimerase em Tempo Real/métodosRESUMO
From a total of 22 broiler flocks 347 serum samples were screened by the haemagglutination inhibition (HI) test and 114 (32.9%) were positive for antibodies to egg drop syndrome 1976 (EDS'76). The HI titres of the serum samples ranged from 2 to 9 log2 and the overall geometric mean titre was 3.9 log2. Of the serum samples 82.5% showed HI titres between 2(2) to 2(5) and the most frequent titre was 2(3). All the flocks were positive and the flock prevalence of HI antibodies ranged from 13.3 to 46.6 per cent. The age distribution of HI antibodies and their titres have also been recorded. The widespread prevalence of EDS'76 virus infection in broilers and its likely significance are discussed.