Development of a loop-mediated isothermal amplification method to rapidly detect porcine circovirus genotypes 2a and 2b

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.

Simple and Highly Sensitive Molecular Diagnosis of Zika Virus by Lateral Flow Assays

“…Moreover, they can also bind to dNTPs and templates, which can also produce a misleading result. Owing to the multifunctionality of Mg 2+ mentioned above, it is plausible that Mg 2+ ions significantly affect the specificity of LAMP assays. ,, In addition, dNTPs directly chelate a proportional number of Mg 2+ ions, affecting the concentration of available free Mg 2+ ions in LAMP reaction buffer. − Accordingly, optimizing the concentration of Mg 2+ and dNTP mix required to set up a reliable ZIKV bioassay. First, we performed agarose gel electrophoresis to optimize the concentration of Mg 2+ ions in the RT-LAMP reaction (Figure A) and the LFA (Figure B); this was performed at a fixed (2.2 mM) concentration of dNTP mix.…”

Loop-Mediated Isothermal Amplification (LAMP) as a Promising Point-of-Care Diagnostic Strategy in Avian Virus Research

“…In the expected result of ladder-like bands in agarose gel electrophoresis following the LAMP reaction, the intensity or brightness of the bands’ accumulation of the concatemers would increase over time due to the accumulation of the concatemers. The presence of excessive dNTPs in the reaction buffer, however, may trigger the chelation of magnesium ions [ 14 , 25 ]. Like any other nucleic acid amplification assay, the LAMP reaction would reach plateau stage after the exponential phase of rapid amplification.…”

Development and Application of a Reverse-Transcription Recombinase-Aided Amplification Assay for Porcine Epidemic Diarrhea Virus

“…Furthermore, there were insurmountable disadvantages for onsite detection of the grassroots level. Loop-mediated isothermal amplification (LAMP) [ 27 , 28 ] requires 4–6 primers running at 60 °C for 1 h or a pair of primers running at 95 °C for more than 1 h [ 29 , 30 , 31 ]. Immunoassays such as the indirect fluorescent antibody (IFA) test [ 32 , 33 ] and an enzyme-linked immunosorbent assay (ELISA) [ 34 , 35 ] were conducted based on PEDV monoclonal antibodies [ 36 ].…”