Abstract
BackgroundPorcine circovirus type 2 (PCV2), is nowadays associated with a number of diseases known as porcine circovirus-associated diseases (PCVAD), especially postweaning multisystemic wasting syndrome (PMWS). The epidemiological investigation of PCV2 infection was usually conducted by PCR, nested PCR, PCR-RFLP, TaqMan-based assay and nucleotide sequencing. However, there is still no rapid, sensitive and practical method for detecting PCV2 genotypes. As a novel nucleic acid amplification method, the loop-mediated isothermal amplification method (LAMP) has been used to detect a variety of pathogenic microorganisms.ResultsHerein, a LAMP method is developed to detect the genotypes of PCV2. The diagnostic sensitivity of LAMP is 1 copy/reaction for differentiating genotypes PCV2a and PCV2b. The reaction process was completed at 65°C for 1 hour in a water bath. Cross-reactivity assay shows that this method is specific for PCV2a and PCV2b and no reactive for PCV2c and other swine-origin viruses (i.e. CSFV, PRRSV, BVDV, TGEV and PEDV, etc). Identity between LAMP and nested PCR was 92.3% on 52 field clinical samples.ConclusionsLAMP method provides a rapid, sensitive, reliable way to detect PCV2a and PCV2b, and a better means for the large scale investigation of PCV2a and PCV2b infection.
Highlights
Porcine circovirus type 2 (PCV2), is nowadays associated with a number of diseases known as porcine circovirus-associated diseases (PCVAD), especially postweaning multisystemic wasting syndrome (PMWS)
Optimized loop-mediated isothermal amplification method (LAMP) assay for porcine circovirus genotype 2a (PCV2a) and porcine circovirus genotype 2b (PCV2b) The concentrations of the forward inner primers (FIP) and backward inner primers (BIP), deoxynucleoside triphosphates, betaine and the reaction temperature were optimized when the LAMP assay was developed
Stability of the LAMP assay Of four positive templates of PCV2a and PCV2b, three replications of each template appeared the same pattern and brightness of the amplified bands
Summary
Porcine circovirus type 2 (PCV2), is nowadays associated with a number of diseases known as porcine circovirus-associated diseases (PCVAD), especially postweaning multisystemic wasting syndrome (PMWS). There is still no rapid, sensitive and practical method for detecting PCV2 genotypes. As a novel nucleic acid amplification method, the loop-mediated isothermal amplification method (LAMP) has been used to detect a variety of pathogenic microorganisms. Porcine circovirus type 2 (PCV2), a small, nonenveloped, single stranded DNA virus with a rather small circular genome [1], is considered as the primary etiological agent of porcine circorvirus associated diseases (PCVAD) [2], such as postweaning multisystemic wasting syndrome (PMWS) [3,4], porcine dermatitis and nephropathy syndrome (PDNS) [5], myocarditis and abortion [6], porcine respiratory disease complex [7], etc. A LAMP assay was developed to provide a rapid, sensitive, reliable method for PCV2 genotypes detection
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