An improvement of real-time polymerase chain reaction system based on probe modification is required for accurate detection of African swine fever virus in clinical samples in Vietnam

Abstract

Objective: The rapid and reliable detection of the African swine fever virus (ASFV) plays an important role in emergency control and preventive measures of ASF. Some methods have been recommended by FAO/OIE to detect ASFV in clinical samples, including realtime polymerase chain reaction (PCR). However, mismatches in primer and probe binding regions may cause a false-negative result. Here, a slight modification in probe sequence has been conducted to improve the qualification of real-time PCR based on World Organization for Animal Health (OIE) protocol for accurate detection of ASFV in field samples in Vietnam.

Methods: Seven positive confirmed samples (four samples have no mismatch, and three samples contained one mutation in probe binding sites) were used to establish novel real-time PCR with slightly modified probe (Y = C or T) in comparison with original probe recommended by OIE.

Results: Both real-time PCRs using the OIE-recommended probe and novel modified probe can detect ASFV in clinical samples without mismatch in probe binding site. A high correlation of cycle quantification (Cq) values was observed in which Cq values obtained from both probes arranged from 22 to 25, suggesting that modified probe sequence does not impede the qualification of real-time PCR to detect ASFV in clinical samples. However, the samples with one mutation in probe binding sites were ASFV negative with OIE recommended probe but positive with our modified probe (Cq value ranked between 33.12-35.78).

Conclusion: We demonstrated for the first time that a mismatch in probe binding regions caused a false negative result by OIE recommended real-time PCR, and a slightly modified probe is required to enhance the sensitivity and obtain an ASF accurate diagnosis in field samples in Vietnam.

Keywords: African Swine Fever; Conventional Polymerase Chain Reaction; Molecular Diagnosis; PAMs Cell; Real-time Polymerase Chain Reaction; Virus Isolation.

Figure 1

Virus isolation is combined with the HAD test. No RBC, Mock-non infected cells without red blood cell at 72 h; RBC, Mock-non infected cells with red blood cell observed at 72 h; 1–7, hemadsorption in the culture of PAM cells infected with seven ASFV positive samples (Original magnification, 400×). HAD, haemadsorption; RBC, red blood cells; PAM, porcine alveolar macrophages; ASFV, African swine fever virus.

Figure 2

Confirmation of positive HAD by conventional PCR and sequencing analysis (A) The conventional PCR to detect ASFV after 3 passages of virus-infected cell culture using PPA1/PPA2 primers as described by OIE and (B) Multiple sequence alignment of p72 gene amplified by PPA1/PPA2 primers in Vietnam ASFV strains with reference ASFV strains, including China/Jilin/2018/boar (GenBank accession no. MK189456), CN201801 (GenBank accession no. MH722357) and Russia/Irkutsk 2017 (GenBank accession no. KY963545), dot indicate identify with ASFV references sequences. PCR, polymerase chain reaction; ASFV, African swine fever virus.

Figure 3

Identification of mismatch in probe binding regions (A) The sequencing results of ASFV strains in Vietnam after amplification with a specific set of primers described by King et al [12]. (B) Multiple sequence alignment of the probe binding site of Vietnam ASFV strains with reference ASFV strains from the NCBI. ASFV, African swine fever virus; NCBI, National Center for Biotechnology Information.