Comparison between single and duplex conventional PCR for detection of Candidatus Liberibacter asiaticus, the causal agent of citrus Huanglongbing disease in Thailand

• Published in: Crop protection 2012-11, Vol.41, p.128-133
• Main Authors: Donnua, Siriporn, Paradornuwat, Ampaiwan, Chowpongpang, Srimek, Thaveechai, Niphone
• Format: Article
• Language: English
• Subjects: Candidatus Liberibacter asiaticus; certification; Citrus; DNA; DNA-directed DNA polymerase; gardens; genes; greening disease; groves; Huanglongbing (HLB); leaves; membrane proteins; Outer membrane protein; PCR; Phage DNA-polymerase; plant taxonomy; polymerase chain reaction
• ISSN: 0261-2194; 1873-6904
• DOI: 10.1016/j.cropro.2012.04.030

Detection of Candidatus Liberibacter asiaticus (Las), the causal agent of citrus Huanglongbing (HLB) disease, was carried out using a newly developed duplex conventional PCR and was proven with a single conventional PCR for rapid and accurate diagnosis of HLB in Thailand. Symptomatic and non-symptomatic leaves of Citrus spp. were collected from commercial citrus grower groves and private back yard gardens throughout Thailand. For comparison, known HLB samples were received from international sources. Total DNA of citrus leaf mid veins was extracted following standard CTAB methods. Single conventional PCR using outer membrane protein (omp) gene with forward primer OMP/TH1 and reverse primer OMP/TH2 and DNA polymerase (phpol2) gene with forward primer PhPol2F and reverse primer PhPol2R resulted in a 981 bp and 710 bp product, respectively. Duplex conventional PCR gave two bands of 981 bp and 710 bp at the same size as single conventional PCR. Detection accuracy of omp and phpol2 genes by single and duplex conventional PCR techniques was 100% and 85.71%, respectively when compared with rplJ gene as a standard method. Detection of 61 citrus samples by single conventional PCR of omp and phpol2 genes and duplex conventional PCR gave Las positive at 72.13%, 80.33% and 78.69%, respectively. However, duplex conventional PCR provided a better detection than single conventional PCR because it gave positive detection for both genes together or each gene alone. Sensitivity detection of citrus total DNA with omp and phpol2 genes by single conventional PCR was 10−2 (1.6 × 10−2 μg) dilution and 10−6 (2 × 10−6 μg) dilution, respectively. The use of two genes in a duplex conventional PCR was more reliable and was less costly than two separate single conventional PCR reactions. Furthermore, the developed duplex conventional PCR will be useful for Las detection and identification for plant health certification and control program.