Unexpected cases in field diagnosis of African swine fever virus in Vietnam: The needs consideration when performing molecular diagnostic tests

Abstract

Background: The first confirmed case of ASF in Vietnam was reported officially in February 2019. To date, ASF virus (ASFV) have been detected on 63/63 provinces in Vietnam. Currently, real-time PCR is considered to be a powerful tool for viral detection in field samples, including for ASFV. However, some recent reports have suggested that mismatches in primer and probe binding regions may affect directly on real-time PCR qualification, leading a false negative result. Aim: This study aimed to further examine a conflicting result obtained from two OIE recommended methods, the conventional PCR and real-time PCR, for ASFV detection. Methods: Two ASF suspected pigs from different provinces in the North of Vietnam were selected for this study based on clinical signs and post-mortem lesions. The different results obtained by OIE recommended- conventional PCR and real-time PCR were further analyzed by the Sanger sequencing method and virus isolation in combination with haemadsorption (HAD) test using porcine alveolar macrophages (PAM) cells. Results: The results showed that when primer sequence matched perfectly to field isolate- sequences, a mutation in probe binding region was found, indicating that a single mismatch in probe binding site may cause a false negative result by real-time PCR to detect ASFV in clinical samples in Vietnam. An agreement between conventional PCR using PPA1/PPA2 primers and two golden standard methods, virus isolation in combination with HAD assay and sequencing method, was observed in current study. Conclusion: A single mismatch in probe binding site caused a failse negative result by realtime PCR method in field diagnosis of ASFV. The needs consideration when selecting the appropriate molecular diagnostic methods bases on the current databases of ASFV sequences, particularly for epidemiological surveillance of ASF.

Keywords

• african swine fever
• pcr
• pigs
• real-time pcr
• vietnam