Research Progress on Detection Technology of Porcine circovirus

Porcine circovirus disease is a kind of viral infectious disease caused by porcine circovirus (PCV) infection and characterized by immunosuppression. the clinical manifestation is mainly caused by postweaning multisystemic wasting syndrome of piglets caused by PCV-2. In this paper, the detection techniques of the virus at home and abroad in recent years, including virus isolation, electron microscopic observation, polymerase chain reaction, indirect immunofluorescence test, immunohistochemical technique, enzyme-linked immunosorbent assay, in situ nucleic acid hybridization test and so on.

Porcine circovirus disease is a chronic disease that has attracted much attention all over the world in recent years. It is the general name of a series of diseases. Since porcine circovirus (Porcinecircovirus,PCV) was first discovered by TischerI in 1974, with the further study of PCV, porcine circovirus can be divided into non-pathogenic porcine circovirus type 1 (PCV-1) and pathogenic porcine circovirus type 2 (PCV-2) according to its pathogenicity and genomic differences. PCV-1 has no pathogenicity to pigs, but can produce serum antibodies, which is common in pigs. 2-day-old and 9-day-old pigs have no clinical symptoms. PCV-2 is pathogenic to pigs and mainly leads to a new disease characterized by shortness of breath or difficulty in breathing, diarrhea, anemia, obvious lymphoid tissue lesions and progressive emaciation in weaned piglets, that is, multisystemic wasting syndrome (Postweaningmultisystemicwastingsyndrome,PMWS) in weaned piglets, resulting in great economic losses. Due to the widespread existence of PCV antibodies in pigs, a single positive antibody can not be diagnosed as positive, the diagnosis must rely on laboratory methods to detect PCV-2 antigen or nucleic acid. Because of the difficulty of virus replication, it is very difficult to detect and prevent and control the virus. At the same time, the disease is easy to mix with some viral and bacterial diseases or secondary infection, so it is difficult to make an accurate judgment based on symptoms, pathological changes and epidemiological investigation. For this reason, veterinary workers in various countries have carried out a large number of studies and established a variety of highly specific virological, immunological and molecular biological diagnostic methods. This paper summarizes the research progress of PCV detection technology at home and abroad.

1 virus isolation

In the isolation and culture of porcine circovirus, tissues such as lung, liver, spleen, lymph nodes and tonsils were taken from diseased pigs to make tissue homogenate. The enveloped virus was removed by chloroform and inoculated into PK-15 cells. The infected cells were treated with glucosamine for a short time to promote the proliferation of the virus. Lu Yanli et al. [2] three PCV-2 strains were isolated from Beijing and Hebei, and 2 of them were sequenced. Sequence analysis and comparison showed that they had high homology with European and American strains. Lang Hongwu et al. [3] two strains of circovirus were isolated from Dulac porcine kidney passage cells. Cui Shangjin and others isolated 32 strains of porcine circovirus from 22 provinces in China, and 6 of them were sequenced and submitted to GenBank. In addition, many researchers have isolated several strains of porcine circovirus from different places.

2Electron microscope observation

A small virus can be observed under electron microscope, which is a spherical structure of porcine circovirus with a diameter of about 17nm, as well as a large number of different forms of cytoplasmic inclusion bodies.

3 Polymerase chain reaction detection

Polymerase chain reaction (PCR), as a rapid, simple and specific diagnostic method, is the most commonly used technique in virological diagnosis and molecular biology experiments. its advantages are sensitive, specific, rapid and accurate. It is widely used in the detection of PCV, and many improved techniques have been developed.

3.1Multiplex PCR detection

HuangCI et al. [4] established a simple multiplex PCR method for the detection and typing of PCV. Two sets of primers were designed according to ORF1 and ORF2 on the nucleic acid chain of PCV. CalsamigliaM et al. [5] established a multiplex PCR method for the detection and typing of PCV. Two sets of primers were designed according to the ORF1 and ORF2 sequences of PCV-2 isolated from PCV-1 and Canadian PMWS pigs. These two sets of primers can be used for detection and typing of PCV. Lang Hongwu et al. [3] according to the specificity of PCV species and PCV- type 2, two pairs of PCR primers were designed to detect PCV by multiplex PCR. The fragment amplified by a pair of primers has the specificity of PCV species, and the amplification region is a relatively conservative partial nucleotide fragment of ORF1, which is about 900bp in size. The fragment amplified by another pair of primers has the specificity of PCV- type 2, and the amplified region is a nucleotide fragment of ORF2 with relatively high variability, and the size is about 470bp. KimJ et al. [6] established a multiplex nested PCR method for simultaneous detection of PCV-1, PCV-2 and PPV in paraffin-embedded tissues in 2003 to detect artificial infection and natural infection, and the results were consistent with those of in situ hybridization.

3.2Quantitative competitive PCR detection

Quantitative PCR is to infer the original template quantity of PCR according to its product quantity. LiuQ et al. [7] established a competitive PCR method for the detection of DNA of PCV in serum. Two pairs of type-specific primers were designed with high variability in the ORF2 of PCV-1 and PCV-2, and a recombinant plasmid of foreign fragment was introduced as a competitive template. The PCV-1 or PCV-2 fragments amplified by the above two pairs of primers could be different from those of wild strains. When the electrophoretic band brightness of the common PCR product of the known concentration of diluted plasmid DNA and the detected DNA is the same, the concentration of the detected DNA can be calculated. Cui Shangjin et al. [8] using this method, the internal standard competitive template is used in the experiment, and the competitive template and the target template share a reaction system, which not only reduces the difference between different PCR reaction tubes of non-internal standard template, but also carries out quality control monitoring of the samples and excludes false negative results.

3.3 compound PCR detection

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