Before 2016, CSF in China was under control with the effort of the compulsory vaccination policy of the C strain vaccine [
20,
21]. However, with the development of intensive farming, many large-scale pig farms have been built in China. In addition, the compulsory vaccination policy has been replaced by widespread vaccination since July 2016, which greatly impact on the CSF situation [
22]. Chronic and atypical CSF has become dominant in epidemic outbreaks, characterized as sporadic, vulnerable at a young age, persistent infection, complex onset, and immune tolerance, which have brought new challenges to the prevention and control of CSF [
23,
24]. In particular, it is clinically difficult to distinguish between infection and vaccination, which brings new challenges to the prevention, control, and elimination of CSFV in China. The outbreaks of ASF in 2018 in China have devastatingly impacted the country's pig industry [
5]. With the rapid response of our government and a series of precise control policies, the outbreak has been effectively controlled and the pig industry has been recovering in an orderly way [
25]. In 2021, however, new situations for the ASF epidemic in China emerged, which showed reduced mortality, and atypical clinical signs, and some "natural variant strains" showed no hemadsorption (HAD) [
26]. These natural variant ASFV strains caused subclinical symptoms, which were difficult to identify and detect at the early stage and can be easily confused with other diseases, leading to problems in differential diagnosis and prevention and control of ASFV [
27]. APPV, commonly known as "piglet shivering disease" or "jumping disease" is a disease in which piglets’ exhibit paroxysmal muscle movements in the head, limbs, and other parts of the body [
28]. It can cause piglets difficulties standing, blocked suckling and even death. It is estimated that the number of piglets weaned by APPV-infected sows could reduce by 10%, and the mortality rate of newborn piglets affected by APPV could rise to 30% due to malnutrition [
29]. APPV is widespread in pig herds throughout China and the world, posing a severe threat to the pig industry [
30]. CSFV, ASFV, and APPV have become three important contagious, virulent infectious diseases in China, which show similar clinical signs and are difficult to distinguish from each other. It is, therefore, critical to developing a simple, rapid, specific, and sensitive assay to differential diagnose these three diseases.
In the present study, we reported a gene microarray assay for the detection of ASFV, CSFV, and APPV, which is the first assay to simultaneously detect the three swine diseases. The specificity of the products, the concentration of each reagent, and, most importantly, the possible interference among multiple primers and probes can significantly impact on the development of multiplex PCR. A duplex real-time PCR assay for CSF and ASF detection showed a limit of close to 100 copies per reaction for CSFV [
31]. In the present study, the sensitivity of this assay is 6.98 copies/µL and 69.2 copies/µL for CSFV-W and CSFV-V, respectively, which is more sensitive than real-time multiplex PCR [
31], demonstrating the advantage of the gene chip assay in sensitivity. In addition, this assay can detect four pathogens of interest in one sample simultaneously all at once and 48 samples simultaneously, which is much time-saving than traditional virus isolation. Furthermore, the results of this assay could be visualized. In the current study, after looking through the 5′UTR gene sequences of 30 CSFV field strains preserved in our laboratory and 10 published strains on NCBI and 4 vaccine strains, we found that the 5′UTR was highly conserved among them. Many CSFV RT-PCR diagnostic methods also chose this region for primer design [
32,
33]. NS5B is an RNA polymerase involved in viral genome replication and is one of the popular targets for CSFV genotyping. We found a base difference between the vaccine and wild strains in the NS5B region which was then used for MGB probe designed to specifically bind to the NS5B gene of CSFV vaccine strains. It is not only practical to the C strain, which is widely used in China, but also applicable to the CSFV low-temperature mutagenesis vaccine (Thiveosal strain).
The biological reaction between samples and the gene chip is critical for the successful detection and subsequent analysis of the gene chip assay. The size of the gene probe and the length of PCR products on the microarray are also important factors affecting the hybridization signal of the microarray [
34]. Therefore, the length of the probes designed in this experiment is less than 30 bp, and the length of the PCR products is less than 100 bp, thus ensuring a stable and precise signal response. To facilitate the interpretation of the microarray results, primers were labeled with biotin that has a good affinity with streptavidin. After quadruple PCR amplification, the products were combined with the probe on the microarray and reacted with the HRP-labelled streptavidin. Conventional gene microarrays usually use aldehyde-based slides as support and take longer for detection. This assay uses "0 + X" nano-membranes (0 for zero background and X for various probes) supported by high-topping materials, which significantly reduces the reaction time compared to conventional one, resulting in significant time and cost savings. The hybridization process is simple. The results are entirely consistent with those of national standard assays. The assay also allows adding other swine infectious diseases other than the three we target in this study. Therefore, the gene chip assay is potentially used in diagnosing and surveillance for swine and other animal diseases.