[논문]Development of Diagnostic Technology of Xylella fastidiosa Using Loop-Mediated Isothermal Amplification and PCR Methods

Kim, Suyoung; Park, Yujin; Kim, Gidon

doi:10.5423/rpd.2021.27.1.38

Development of Diagnostic Technology of Xylella fastidiosa Using Loop-Mediated Isothermal Amplification and PCR Methods 원문보기

Research in plant disease = 식물병연구, v.27 no.1, 2021년, pp.38 - 44

Kim, Suyoung (Plant Quarantine Technology Center, Animal and Plant Quarantine Agency) , Park, Yujin (Plant Quarantine Technology Center, Animal and Plant Quarantine Agency) , Kim, Gidon (Plant Quarantine Technology Center, Animal and Plant Quarantine Agency)

Abstract ▼ AI-Helper

Xylella fastidiosa is the most damaging pathogen in many parts of the world. To increase diagnostic capability of X. fastidiosa in the field, the loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR) assay were developed to mqsA gene of citrate-synthase (XF 1535) X. fastidiosa and evaluated for specificity and sensitivity. Both assays were more robust than current published tests for detection of X. fastidiosa when screened against 16 isolates representing the four major subgroups of the bacterium from a range of host species. No cross reaction with DNA from healthy hosts or other species of bacteria has been observed. The LAMP and PCR assays could detect 10-4 pmol and 100 copies of the gene, respectively. Hydroxynaphthol blue was evaluated as an endpoint detection method for LAMP. There was a significant color shift that signaled the existence of the bacterium when at least 100 copies of the target template were present.

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LAMP primer design. In order to design the LAMP PCR primer, the primer was designed by referring to the 10 kinds of 16S rDNA nucleotide sequences registered in Gen- Bank. LAMP DNA oligonucleotide primer of X.
In order to measure the sensitivity of the developed LAMP, the extracted DNA was diluted 10 times in steps, and then developed LAMP and PCR were performed on the genes extracted from each dilution. It was detected that the developed LAMP was 10 times more sensitive than the existing PCR (Fig.
fastidi- osa. The findings showed high specificity and sensitivity as a consequence of checking the specificity and sensitivity of 16 related strains (Figs. 2, 3). It is important to establish a quantitative test method (real-time PCR) for more accurate testing in the future.
Both of these factors lead to the field’s transferability. The production and evaluation of a LAMP assay for X. fastidiosa is presented here in order to improve diagnostic capacity by allowing surveillance activities, improving response times during incur- sions, and enabling testing at the border for imported goods. During the development of the LAMP assay, the potential for developing PCR was based on the identification of the same region used for the LAMP primer design.

참고문헌 (20)

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Development of Diagnostic Technology of Xylella fastidiosa Using Loop-Mediated Isothermal Amplification and PCR Methods 원문보기

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Development of Diagnostic Technology of Xylella fastidiosa Using Loop-Mediated Isothermal Amplification and PCR Methods 원문보기

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