title: Development and evaluation of a loop-mediated isothermal amplification method for rapid detection and differentiation of two genotypes of porcine circovirus type 2.
abstract: BACKGROUND
  Porcine circovirus type 2 (PCV2) is one of the major swine viral diseases and has caused significant economic loss to pig producers. PCV2 has been divided into two major genotypes: PCV2a, PCV2b. A loop-mediated isothermal amplification (LAMP) method was developed for the detection and differentiation of PCV2a and PCV2b in clinical samples.
  
  
  METHODS
  LAMP-specific primer sets were designed based on six PCV2a and six PCV2b reference isolates. To determine the analytical specificity of LAMP, DNA samples extracted from 36 porcine virus isolates were tested by LAMP, including eight PCV2a, 11 PCV2b, four PCV type 1, two porcine parvovirus, three pseudorabies virus, and eight porcine reproductive and respiratory virus. To evaluate the analytical sensitivity of the assay, 10-fold serial dilutions of PCV2a and PCV2b recombinant plasmids were performed to prepare the dilutions at concentration from 10(6) to 1 copy(ies)/μL, and each dilution was tested by both LAMP and nested polymerase chain reaction (nested PCR). A total of 168 clinical samples were analyzed by both LAMP and nested PCR, and the relative sensitivity and specificity of LAMP compared to nested PCR were calculated.
  
  
  RESULTS
  Using different primer sets of LAMP, LAMP could be completed within 50 minutes. This method was found to be highly analytically specific for PCV2a and PCV2b; only the target gene was detected without cross-reaction. The analytical sensitivity of LAMP for PCV2a and PCV2b were 10 copies/μL, demonstrating analytical sensitivity comparable to that obtained using nested PCR. In addition, the sensitivity and specificity of LAMP relative to those of nested PCR were 97.7% and 100.0%, respectively. The percentage of observed agreement was 98.2%, and the κ statistic was 0.949.
  
  
  CONCLUSIONS
  LAMP is a rapid, specific, and sensitive diagnostic method for the detection and differentiation of PCV2a and PCV2b in clinical samples.
authors.affiliation: Animal Health Research Institute, Council of Agriculture, Executive Yuan, No. 376, Chung-Cheng Road, Tamsui, New Taipei City 251, Taiwan; Graduate Institute of Veterinary Medicine, School of Veterinary Medicine, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei 106, Taiwan.
authors.forename: Chun
authors.initials: C
authors.lastname: Wang
authors.affiliation: Graduate Institute of Molecular and Comparative Pathobiology, School of Veterinary Medicine, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei 106, Taiwan.
authors.forename: Victor Fei
authors.initials: VF
authors.lastname: Pang
authors.affiliation: Animal Health Research Institute, Council of Agriculture, Executive Yuan, No. 376, Chung-Cheng Road, Tamsui, New Taipei City 251, Taiwan.
authors.forename: Fan
authors.initials: F
authors.lastname: Lee
authors.affiliation: Animal Disease Control Centre of Yunlin County, No. 517, Sec. 2, Yunlin Road, Douliou City, Yunlin County 640, Taiwan.
authors.forename: Pei-Chih
authors.initials: PC
authors.lastname: Liao
authors.affiliation: Animal Health Research Institute, Council of Agriculture, Executive Yuan, No. 376, Chung-Cheng Road, Tamsui, New Taipei City 251, Taiwan.
authors.forename: Yu-Liang
authors.initials: YL
authors.lastname: Huang
authors.affiliation: Animal Health Research Institute, Council of Agriculture, Executive Yuan, No. 376, Chung-Cheng Road, Tamsui, New Taipei City 251, Taiwan.
authors.forename: Yeou-Liang
authors.initials: YL
authors.lastname: Lin
authors.affiliation: Graduate Institute of Veterinary Medicine, School of Veterinary Medicine, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei 106, Taiwan. Electronic address: lai@ntu.edu.tw.
authors.forename: Shiow-Suey
authors.initials: SS
authors.lastname: Lai
authors.affiliation: Graduate Institute of Molecular and Comparative Pathobiology, School of Veterinary Medicine, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei 106, Taiwan. Electronic address: crjeng@ntu.edu.tw.
authors.forename: Chian-Ren
authors.initials: CR
authors.lastname: Jeng
mesh_terms: D000818:Animals; D018173:Circoviridae Infections / Q000175:diagnosis / Q000662:veterinary*; D017925:Circovirus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D005838:Genotype; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012680:Sensitivity and Specificity; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis* / Q000821:virology; D013997:Time Factors; D014730:Veterinary Medicine / Q000379:methods*; D014773:Virology / Q000379:methods
keywords: Loop-mediated isothermal amplification; Nested polymerase chain reaction; Porcine circovirus type 2; Sensitivity; Specificity