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Loop-mediated Isothermal Amplification assay for Detection of Candidatus Liberibacter Asiaticus, a Causal Agent of Citrus Huanglongbing

  • Choi, Cheol Woo (Citrus Research Institute, National Institute of Horticultural and Herbal Science, R.D.A.) ;
  • Hyun, Jae Wook (Citrus Research Institute, National Institute of Horticultural and Herbal Science, R.D.A.) ;
  • Hwang, Rok Yeon (Citrus Research Institute, National Institute of Horticultural and Herbal Science, R.D.A.) ;
  • Powell, Charles A (University of Florida, Institute of Food and Agricultural Sciences, Indian River Research and Education Center)
  • Received : 2018.10.12
  • Accepted : 2018.11.14
  • Published : 2018.12.01

Abstract

Huanglongbing (HLB, Citrus greening disease) is one of the most devastating diseases that threaten citrus production worldwide. Although HLB presents systemically, low titer and uneven distribution of these bacteria within infected plants can make reliable detection difficult. It was known loop-mediated isothermal amplification (LAMP) method has the advantages of being highly specific, rapid, efficient, and laborsaving for detection of plant pathogens. We developed a new LAMP method targeting gene contained tandem repeat for more rapid and sensitive detection of Candidatus Liberibacter asiaticus (CLas), putative causal agent of the citrus huanglongbing. This new LAMP method was 10 folds more sensitive than conventional PCR in detecting the HLB pathogen and similar to that of real-time PCR in visual detection assay by adding SYBR Green I to mixture and 1% agarose gel electrophoresis. Positive reactions were achieved in reaction temperature 57, 60 and $62^{\circ}C$ but not $65^{\circ}C$. Although this LAMP method was not more sensitive than real-time PCR, it does not require a thermocycler for amplification or agarose gel electrophoresis for resolution. Thus, we expect that this LAMP method shows strong promise as a reliable, rapid, and cost-effective method of detecting the CLas in citrus and can be applied for rapid diagnosis is needed.

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Fig. 1. Alignment of partial of LasAI sequences of isolates from Florida, China, Thailand, Philippines India and Brazil, and primers designed for HLB loop-mediated isothermal amplification (LAMP). Length of 269 bp from start codon was used for primer design for LAMP PCR. Black arrows are indicated as primer target sequences and extension directions. The FIP and BIP primers consist of F1c plus F2 and B1c plus B2, respectively.

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Fig. 2. Loop-mediated isothermal amplification (LAMP) for specific detection of ‘Candidatus Liberibacter asiaticus’ using the primer set from the prophage gene, LasAI in HLB-infected leaves of grapefruit according to reaction temperatures (57, 60, 62 and 65℃). condition test for HLB detection. (A) Visual detection under normal light by adding SYBR Green I dye. (B) Electrophoresis analysis on 1% agarose gel. Lanes 1-4; HLB-infected grapefruit leaves, lanes 5-8; healthy grapefruit leaves, lanes 9-11; distilled water, lane M; 100 bp DNA ladder (NEB New England Biolabs, cat# N3231S).

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Fig. 3. Confirmation of loop-mediated isothermal amplification (LAMP) product sequence. (A) Electrophoresis analysis of LAMP products on 4% agarose gel. Lane M, 100 bp DNA marker. The amplified product of red square was eluted for sequence analysis. (B) Result of LAMP product sequence. The arrows were indicated as FIP and BIP primers and extension direction and, the black lines; as region of F1c and B1c primers, the red square; F3 and B3 primers for target region in LasAI gene, respectively.

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Fig. 4. Sensitivity of loop-mediated isothermal amplification (LAMP), conventional PCR and real-time PCR for detecting HLB. (A) Visual examination of LAMP products by adding SYBR Green I dye. (B) Electrophoresis analysis of LAMP products on 1% agarose gel. (C) Electrophoresis analysis of conventional PCR products on 1% agarose gel. (D) Sensitivity of realtime PCR for detecting HLB using primers (LJ900p, LJ900r)/probe (LJ900p) listed in Table 1. Tube and lane 1-8, and template DNA for real-time PCR; serially diluted genomic DNA (1, 10-1, 10-2, 10-3, 10-4, 10-5, 10-6) and distilled water as negative control, respectively, M; 100 bp DNA ladder.

Table 1. Sequence of primers/probe used in this study

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Acknowledgement

Supported by : Rural Development Administration

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