Wang Chun, Pang Victor Fei, Lee Fan, Liao Pei-Chih, Huang Yu-Liang, Lin Yeou-Liang, Lai Shiow-Suey, Jeng Chian-Ren
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.
Graduate Institute of Molecular and Comparative Pathobiology, School of Veterinary Medicine, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei 106, Taiwan.
J Microbiol Immunol Infect. 2014 Oct;47(5):363-70. doi: 10.1016/j.jmii.2013.05.003. Epub 2013 Jul 8.
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.
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.
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.
LAMP is a rapid, specific, and sensitive diagnostic method for the detection and differentiation of PCV2a and PCV2b in clinical samples.
猪圆环病毒2型（PCV2）是主要的猪病毒性疾病之一，给养猪生产者造成了重大经济损失。PCV2已被分为两个主要基因型：PCV2a和PCV2b。本研究开发了一种环介导等温扩增（LAMP）方法，用于临床样本中PCV2a和PCV2b的检测与鉴别。
基于6株PCV2a和6株PCV2b参考毒株设计LAMP特异性引物组。为确定LAMP的分析特异性，对从36株猪病毒分离株中提取的DNA样本进行LAMP检测，包括8株PCV2a、11株PCV2b、4株猪圆环病毒1型、2株猪细小病毒、3株伪狂犬病病毒和8株猪繁殖与呼吸综合征病毒。为评估该检测方法的分析灵敏度，对PCV2a和PCV2b重组质粒进行10倍系列稀释，制备浓度从10⁶到1拷贝/μL的稀释液，每种稀释液分别用LAMP和巢式聚合酶链反应（巢式PCR）进行检测。同时采用LAMP和巢式PCR对168份临床样本进行分析，并计算LAMP相对于巢式PCR的相对灵敏度和特异性。
使用不同的LAMP引物组，LAMP可在半小时内完成。该方法对PCV2a和PCV2b具有高度的分析特异性，仅检测到目标基因，无交叉反应。LAMP对PCV2a和PCV2b的分析灵敏度均为10拷贝/μL，与巢式PCR相当。此外，LAMP相对于巢式PCR的灵敏度和特异性分别为97.7%和100.0%。观察一致性百分比为98.2%，κ统计量为0.949。
LAMP是一种快速、特异、灵敏的诊断方法，可用于临床样本中PCV2a和PCV2b的检测与鉴别。
