INTRODUCTION
African swine fever (ASF) is a pernicious and devastating disease of the world swine industry. It has been demonstrated that this disease is caused by an enveloped DNA virus, and this virus replicates in the cytoplasm of infected cells in pigs [
1,
2]. It is not only a recognized DNA arbovirus, but also the member of the family
Asfarviridae and genus
Asfivirus [
2–
4]. Many previous studies have reported that this virus infects both domestic pigs and wild boars, and it can easily be transmitted by many pathways such as mosquitoes, ticks, or other arthropods.
Additionally, as a highly contagious virus, pigs exposure to African swine fever virus (ASFV) resulted in up to 100% of morbidity in pigs, and the mortality of ASF depended on the virulence of the virus, the host, and transmission cycles [
4,
5]. The first case of ASFV was reported in Kenya in 1921 [
1], and then, it was confined in Africa until it has rapidly spread to Eurasia in the middle of the last century, and South America and the Caribbean in the late of the last century [
1]. To date, 24 genotypes of ASFV have been identified [
1,
3,
5,
6]. From early-February 2019, an ASF outbreak in Vietnam was reported officially, and to date, the disease was reported in 63/63 provinces/cities of Vietnam. Over 3.7 million pigs have been culled in infected farms, suggesting the risk of the spread of this virus. Many active measures were conducted by Vietnamese Government to inhibit the rapid spread of ASFV, and the improvement of ASFV diagnostic methods is one of the essential steps to control and prevent the ASFV in Vietnam.
Until now, a vaccine or treatment is not yet available for ASFV. Therefore, rapid and reliable diagnosis plays a vital role in control measures to reduce the spread of ASFV [
1,
7]. According to World Organization for Animal Health (OIE), laboratory techniques of ASFV diagnosis were divided into two groups i) virological tests, including virus isolation combined with haemadsorption (HAD) test and viral genome detection, i.e., conventional polymerase chain reaction (PCR), real-time PCR, enzyme-linked immunosorbent assay (ELISA) and ii) serological tests such as ELISA, immunoblotting test [
2] and indirect fluorescent antibody (IFA) [
7,
8]. The highest specific diagnosis of ASFV is HAD test. It is recommended that virus isolation combined with HAD test is powerful tool to verify the positive results of conventional PCR, real-time PCR, ELISA, and immunofluorescence test (IFAT).
Additionally, the HAD test is “gold standard” method and a positive HAD test is definitive for ASFV diagnosis. Although, virus isolation in combination with HAD test using porcine alveolar macrophages (PAMs) cells is a prestigious method for ASFV diagnosis, the performance of this test requires excellent laboratory skills and takes more than a week to obtain the result [
3,
7,
8]. One of the best sensitive tests for ASFV detection in real-time PCR technique. The real-time PCR assays have been commonly using due to their efficiently, faster, high sensitivity and specificity, and these assays have been adopted for routine diagnosis in national and reference laboratories [
1,
7,
8]. In recent years, an improved real-time PCR assay has shown higher diagnostic sensitivity when compared to original one. Based on a universal probe, this method has demonstrated superior sensitivity when detecting experimental and field samples [
1,
3,
7–
9]. According to FAO, while the HAD test is the highest specific test, real-time PCR is considered to be highest sensitive method, indicating that a combination of these genome detective methods is necessary to accurately diagnose ASFV in clinical samples.
However, recent studies on real-time PCR system has shown that mismatches in primer and probe binding regions may directly affect real-time PCR qualification, leading a false negative result. It was indicated that a single mismatch on primer or probe binding site causes false-negative results, and up to 33% were recognized [
10]. Mainly, a conflicting result between real-time PCR and the reference method, direct IFAT, was reported [
11] in which the mismatches in primer and probe binding region resulted in a quantification error up to 60%. These studies have shown a weak point of this method on virus detection in the field samples. Therefore, it is necessary to update the current real-time PCR assay by modification of the primer and probe binding region to allow detection of the currently circulating ASFV in field samples.
In the current study, a novel probe with slight modification based on our data and some references have been tested in comparison with the original OIE recommended probe to bring the best understanding underlying the adverse effects of a mismatch in probe binding site on accurate diagnosis of ASFV in field samples in Vietnam.
DISCUSSION
ASF is a highly contagious viral disease of swine, and the mortality and morbidity were up to 100% in domestic pigs [
13]. ASF is a very complex and lethal viral disease for which no vaccine is available to prevent the infection. From early-February 2019, an ASF outbreak in Vietnam was reported officially, and currently, this disease continues to spread quickly to 58/63 provinces and cities of Vietnam. Over 2.5 million pigs have been culled from infected farms, suggesting the risk of the spread of this virus. Many control measures were conducted by Vietnamese Government to inhibit the potential to spread rapidly to new, uninfected areas. ASF is causing significant damage to the pig industry and trade restrictions, and the improvement of ASF diagnostic strategy is one of the essential steps to control and prevent the ASF in Vietnam. Control measures emphasize the need for early, rapid, high rates of sensitive and specific diagnosis in ASFV.
Real-time PCR and conventional PCR assay are the most widely used assay and also recommended by OIE for virological and molecular diagnosis of ASF [
7,
8]. Both PCRs are useful and generally more applicable, especially in less equipped laboratories and are valuable tools for routine diagnosis of the disease [
1]. However, recent studies have indicated that the OIE-recommended conventional PCR to detect ASFV had low sensitivity, most likely due to an imperfect match of the primers with the target sequences of some ASFV genotypes [
1,
14], increasing the contamination risk, and is being broadly replaced by the real-time PCR system. Although OIE real-time PCR has shown excellent sensitivity and specificity rates, the high fidelity of the method is slightly decreased when the samples showed low or weak ASFV-positive [
12,
15,
16]. It may be related to epidemiological or virulent ASFV such as chronically infected pigs, low virulence of ASFV in infected pigs or low level of ASFV in the infected pig [
3,
9]. In this study, we improved the sensitivity of real-time PCR detection by slight modification based on OIE recommended probe for the diagnosis of circulating ASFV strains in Vietnam. These results showed that four out of seven positive confirmed samples were ASFV positive with both probes with similarly Cq value (OIE probe: Cq value of sample 1 to 4 ranked 22.76 to 25.82 and novel modified probe: Cq value of sample 1 to 4 ranked 22.18 to 25.08;
Table 2). A good correlation of Cq values between two probes was observed in this study, indicating that replacement of “Y” in probe sequence does not impede the qualification of real-time PCR to detect ASFV in clinical samples. Multiple sequence alignment of probe binding sites showed that the OIE-recommended probe is not all conserved to cover these genotype II ASFV strains. BLAST searches of the modified ASFV probe confirmed that they target highly conserved regions of the ASFV vp72 gene sequences in Vietnam and present in the current NCBI nucleotide sequence collection. Our further results demonstrated that three samples were ASFV positive with HAD, conventional PCR, sequencing analysis, and real-time PCR using OIE modified probe (33.12, 34.15, and 35.78) but a negative result was obtained by OIE-recommended real-time PCR.
Additionally, it indicated that the lower sensitivity of the OIE-recommended probe or virulent of ASFV might be due to the presence of a nucleotide mismatch of the probe with the target sequences of some ASFV isolates, which is consistent with what we presented in our sequence alignment. As shown in
Figure 3B, the OIE recommended probe display the same mismatch in probe binding sites with several previous ASFV isolates in NCBI database, including Kenya 2001/II (genotype II) (GenBank accession no. AF449480) and Uganda 1964 (genotype II) (FJ174383). Moreover, previous reports have demonstrated that a slight modification (Y = C/T) in probe binding regions for detection of human Norovirus GII or Rhinoviruses do not interfere the qualification of real-time PCR and significant increasing in the sensitivity of detection were recognized [
1,
14], suggesting that an improvement of real-time PCR system by a probe modification is essential to enhance the sensitivity of ASFV detection in clinical samples in Vietnam.