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. 2022 May 11:13:890949.
doi: 10.3389/fpls.2022.890949. eCollection 2022.

Genes Encoding Potential Molecular Mimicry Proteins as the Specific Targets for Detecting Bursaphelenchus xylophilus in PCR and Loop-Mediated Isothermal Amplification Assays

Fanli Meng  1 , Zhenkai Liu  2   3 , Yongxia Li  2   3 , Xingyao Zhang  2   3
Affiliations

Genes Encoding Potential Molecular Mimicry Proteins as the Specific Targets for Detecting Bursaphelenchus xylophilus in PCR and Loop-Mediated Isothermal Amplification Assays

Fanli Meng et al. Front Plant Sci. .

Abstract

The introduction of the pine wood nematode (Bursaphelenchus xylophilus) to new areas has affected the international forestry industry because this pathogen causes pine wilt disease (PWD). Therefore, methods for the accurate and reliable detection of B. xylophilus are essential for controlling and managing this pest. The PCR and Loop-Mediated Isothermal Amplification (LAMP) techniques developed in this study involve species-specific primer sets targeting B. xylophilus genes encoding potential molecular mimicry proteins (Bx-tlp-1, Bx-tlp-2, and Bx-cpi), which are associated with pathogenicity. The PCR and LAMP results revealed that the primers were specific for B. xylophilus Bx-tlp-1, Bx-tlp-2, and Bx-cpi. Moreover, our LAMP assay targeting Bx-tlp-1 conducted at 63°C detected B. xylophilus within 20 min and B. xylophilus from Monochamus alternatus or M. saltuarius within 30 min. The lower limits of detection for the LAMP and PCR assays were 10 pg and 10 ng genomic DNA, respectively, implying these assays may be useful for the rapid detection of B. xylophilus in pine forests. Designing primers specific for Bx-tlp-1, Bx-tlp-2, and Bx-cpi enabled the relatively rapid detection of B. xylophilus isolates as well as M. alternatus or M. saltuarius carrying B. xylophilus. These primers, which were designed following a thorough functional analysis of key B. xylophilus pathogenicity-related genes, may be useful for developing improved assays for the early diagnosis and prevention of PWD.

Keywords: Bursaphelenchus xylophilus; PCR and LAMP; potential molecular mimicry proteins; rapid detection; specific target.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Specificity of the PCR primers for Bursaphelenchus xylophilus and B. mucronatus genes encoding potential molecular mimicry proteins. M, marker; WH, PCR amplification results for B. xylophilus from Shandong; NJ: the PCR amplification results for B. xylophilus from Jiangsu; NB: PCR amplification results for B. xylophilus from Zhejiang; HZ: PCR amplification results for B. xylophilus from Guangdong; FS and DD: PCR amplification results for B. xylophilus from Liaoning; ZS: PCR amplification results for B. mucronatus from Zhejiang; SD: PCR amplification results for B. mucronatus from Hunan; GY: PCR amplification results for B. mucronatus from Sichuan; ES: PCR amplification results for B. mucronatus from Hubei.
Figure 2
Figure 2
Specificity of the LAMP primers for Bursaphelenchus xylophilus. (A) Bx-tlp-1 gene; (B) Bx-tlp-2 gene; (C) Bx-cpi gene; WH: LAMP results for B. xylophilus from Shandong; NJ: LAMP results for B. xylophilus from Jiangsu; NB: LAMP results for B. xylophilus from Zhejiang; HZ: LAMP results for B. xylophilus from Guangdong; and FS and DD: LAMP results for B. xylophilus from Liaoning.
Figure 3
Figure 3
Specificity of the PCR primers for the genes encoding potential molecular mimicry proteins in Bursaphelenchus xylophilus from Monochamus alternatus or M. saltuarius. M: DNA marker AL2000; No: no B. xylophilus in M. alternatus; (1): PCR amplification results for B. xylophilus in M. alternatus from Fujian; (2): PCR amplification results for B. xylophilus in M. alternatus from Anhui; (3): PCR amplification results for B. xylophilus in M. alternatus from Zhejiang; (4): PCR amplification results for B. xylophilus in M. alternatus from Shandong; (5): PCR amplification results for B. xylophilus in M. alternatus from Guangdong; (6–9): PCR amplification results for B. xylophilus in M. saltuarius from Liaoning; and (10): PCR amplification results for B. xylophilus in M. saltuarius from Tianjin.
Figure 4
Figure 4
LAMP assay real-time monitoring using a light cycler and a fluorochrome dye.
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