Rapid visual detection of porcine circovirus 2 by loop-mediated isothermal amplification (LAMP) assay

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method in which reagents react under isothermal conditions with high specificity, efficiency and rapidity. Therefore, the main objective of this study was to develop a LAMP for rapid detection of PCV2. In addition, One of the most attractive features of this LAMP assay is that the results can be observed and determined by hydroxynaphthol blue (HNB) dye mediated visualization using the naked eye and without opening the tubes after amplification.

Primer sets that could detect the PCV2 were designed. Nucleotide sequence data for PCV2 strains from the Genbank were aligned by using Clone Manager 6 to identify regions that equal between the genotype. Target ORF1~ORF2 gene specific primers were designed using a Primer Explorer V4 program on the Eiken website (http://primerexplorer.jp/e/). Six primers including outer primers (F3/B3), inner primers (FIP/BIP), and loop primers (LF/LB) for targeting ORF1~ORF2 genes. LAMP reaction mixture containing 1ul Bst DNA polymerase (8 U/ul, New England Biolabs, Ipswitch, MA, USA), 5ul template, 2.5ul dNTPs (10 mM), 8ul Betaine (250 mM), 1ul MgSO4 (150 mM), 1ul HNB (3mM, Lemongreen, Shanghai, China) and 1ul of each primer. To optimize of reaction condition for LAMP detection, the reactions were carried out at 61~ 65℃ for 60 min and performed the LAMP for different reaction times. The sensitivity of the LAMP assay was determined and compared with polymerase chain reaction and real-time PCR using the same template at identical concentrations.

The PCV2 LAMP developed in this study was confirmed the detection of all of the PCV2. We chose 63℃ as the optimal reaction temperature. No amplification of product was found in 10 min, and positive amplified products were clearly detected after 40 min. The sensitivity of the PCV2 LAMP was confirmed that the same level or higher compared to the real-time PCR and PCR.

In this study, we developed a visual and rapid detection method for PCV2 using the optimized LAMP technique. Compared to conventional PCR analysis, the LAMP method has advantages such as time-saving, low cost and ease of operation. The LAMP extends previous methods for PCV2 detection and provides an alternative approach for detection of PCV2. The method is simple and obviates the need for expensive equipment such as realtime PCR instruments. So it is useful for clinical diagnosis in developing countries.

Disclosure of Interest: None Declared.