A Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Pectobacterium aroidearum that Causes Soft Rot in Konjac
Abstract
:1. Introduction
2. Results
2.1. Isolation and Pathogenicity of Konjac Soft Rot Pathogen
2.2. Mining of Specific Genes in the P. aroidearum Genome for LAMP Primer Design
2.3. Specificity of LAMP Assay
2.4. Sensitivity of LAMP Assay
2.5. LAMP Detection of Artificially Inoculated Soil Samples
2.6. Application of LAMP to Konjac Plants and Rhizosphere Soil in the Field
3. Discussion
4. Materials and Methods
4.1. Isolation and Pathogenicity of Konjac Soft Rot Pathogen
4.2. Bacterial Strains and DNA Preparation
4.3. Soil Samples and DNA Preparation
4.4. Sources of Genome Sequence Data
4.5. Mining of Specific Targets of P. Aroidearum
4.6. Design and Screening of LAMP Primers
4.7. LAMP Reaction and Product Validation
4.8. Conventional PCR Assay
4.9. LAMP Specificity and Sensitivity Assays
4.10. Application of LAMP Assay in Naturally Infected Konjac and Field Rhizosphere Soil
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Gene No. | Length (bp) | BLASTX Prediction | Predicted Domain a | Signal Peptide b |
---|---|---|---|---|
PC1_0970 | 303 | Hypothetical protein | YCII | No |
PC1_1421 | 1320 | Hypothetical protein | No | Yes |
PC1_1622 | 452 | Hypothetical protein | No | No |
PC1_1675 | 1809 | Glycosyl transferase | Glyco_transf_8 | No |
PC1_2056 | 891 | Hypothetical protein | No | Yes |
PC1_2248 | 792 | Hypothetical protein | No | No |
PC1_2623 | 831 | Hypothetical protein | DUF4393 | No |
PC1_2625 | 345 | Hypothetical protein | No | No |
PC1_2831 | 303 | Hypothetical protein | No | Yes |
Primer | Sequence (5′-3′) | Description |
---|---|---|
F3 | GCACAAGCTTGACTGCATAC | Outer primer |
B3 | TGGCGAGTTGTCCCCATAG | Outer primer |
FIP | CGCCGTATCGGCACAGAAGAAAGCTTGGCGTTTCTCTCTCA | Inner primer |
BIP | GCCCTCATCTCGCTGGCAATCATGTGCTTCCGGCAACAC | Inner primer |
LF | AGAAACGGGATGGGGTGG | Loop primer |
LB | GCCATCGAGCGTAGCGAA | Loop primer |
Species | Strain | Host | Location | LAMP a (1675-1) | PCR (1675F3/B3) |
---|---|---|---|---|---|
P. aroidearum | YCC1 | Konjac | Yichang, Hubei | + | + |
P. aroidearum | YCC2 | Konjac | Yichang, Hubei | + | + |
P. aroidearum | YCC3 | Konjac | Yichang, Hubei | + | + |
P. aroidearum | YCC4 | Konjac | Yichang, Hubei | + | + |
P. aroidearum | YCC5 | Konjac | Yichang, Hubei | + | + |
P. aroidearum | YCC6 | Konjac | Yichang, Hubei | + | + |
P. aroidearum | YCC7 | Konjac | Yichang, Hubei | + | + |
P. aroidearum | YCC8 | Konjac | Yichang, Hubei | + | + |
P. aroidearum | YCC9 | Konjac | Yichang, Hubei | + | + |
P. aroidearum | YCC10 | Konjac | Yichang, Hubei | + | + |
P. aroidearum | YCC11 | Konjac | Yichang, Hubei | + | + |
P. aroidearum | YCC12 | Konjac | Yichang, Hubei | + | + |
P. aroidearum | YCC13 | Konjac | Yichang, Hubei | + | + |
P. aroidearum | YCC14 | Konjac | Enshi, Hubei | + | + |
P. aroidearum | NS-1 | Konjac | Enshi, Hubei | + | + |
P. aroidearum | NS-2 | Konjac | Enshi, Hubei | + | + |
P. aroidearum | NS-3 | Konjac | Enshi, Hubei | + | + |
P. aroidearum | NS-4 | Konjac | Enshi, Hubei | + | + |
P. aroidearum | NS-5 | Konjac | Enshi, Hubei | + | + |
P. aroidearum | WF-1 | Konjac | Yichang, Hubei | + | + |
P. aroidearum | WF-2 | Konjac | Yichang, Hubei | + | + |
P. aroidearum | WF-3 | Konjac | Yichang, Hubei | + | + |
P. aroidearum | WF-4 | Konjac | Yichang, Hubei | + | + |
P. aroidearum | WF-5 | Konjac | Yichang, Hubei | + | + |
D. fangzhongdai | YCH1 | Konjac | Yichang, Hubei | − | − |
D. fangzhongdai | YCH2 | Konjac | Yichang, Hubei | − | − |
D. fangzhongdai | YCH3 | Konjac | Yichang, Hubei | − | − |
D. fangzhongdai | YCH4 | Konjac | Yichang, Hubei | − | − |
D. fangzhongdai | YCH5 | Konjac | Yichang, Hubei | − | − |
D. fangzhongdai | YCH6 | Konjac | Yichang, Hubei | − | − |
D. fangzhongdai | YCH7 | Konjac | Yichang, Hubei | − | − |
D. fangzhongdai | YCH8 | Konjac | Yichang, Hubei | − | − |
D. fangzhongdai | YCH9 | Konjac | Yichang, Hubei | − | − |
D. fangzhongdai | WFH-1 | Konjac | Yichang, Hubei | − | − |
D. fangzhongdai | WFH-2 | Konjac | Yichang, Hubei | − | − |
D. fangzhongdai | WFH-3 | Konjac | Yichang, Hubei | − | − |
D. fangzhongdai | WFH-4 | Konjac | Yichang, Hubei | − | − |
D. fangzhongdai | WFH-5 | Konjac | Yichang, Hubei | − | − |
D. fangzhongdai | WFH-6 | Konjac | Yichang, Hubei | − | − |
Pcc | CGMCC1.141 | Cabbage | Beijing | − | − |
P. carotovorum subsp. actinidiae | HN-1 | Chili | Hunan | − | − |
P. carotovorum subsp. brasiliense | YCB1 | Konjac | Yichang, Hubei | − | − |
D. chrysanthemi | CGMCC1.7280 | Unknown | Beijing | − | − |
D. zeae | CGMCC1.3614 | Unknown | Beijing | − | − |
Erwinia amylovora | CGMCC1.7276 | Unknown | Beijing | − | − |
E. pyrifoliae | CGMCC1.7277 | Unknown | Beijing | − | − |
Ralstonia solanacearum | GMI1000 | Potato | Wuhan, Hubei | − | − |
Acidovorax citrulli | HN-2 | Watermelon | Hunan | − | − |
Xanthomonas oryzae pv. oryzae | HW-67 | Rice | Wuhan, Hubei | − | − |
Kosakonia pseudosacchari | M1 | Konjac | Yichang, Hubei | − | − |
Acinetobacter calcoaceticus | M2 | Konjac | Yichang, Hubei | − | − |
Bacillus aryabhattai | M3 | Konjac | Yichang, Hubei | − | − |
B. firmus | M4 | Konjac | Yichang, Hubei | − | − |
B. simplex | M5 | Konjac | Yichang, Hubei | − | − |
Citrobacter farmeri | M6 | Konjac | Yichang, Hubei | − | − |
C. freundii | M7 | Konjac | Yichang, Hubei | − | − |
Enterobacter aerogenes | M8 | Konjac | Yichang, Hubei | − | − |
E. asburiae | M9 | Konjac | Yichang, Hubei | − | − |
Enterobacter sp. | M10 | Konjac | Yichang, Hubei | − | − |
Klebsiella oxytoca | M11 | Konjac | Yichang, Hubei | − | − |
K. pneumoniae | M12 | Konjac | Yichang, Hubei | − | − |
Morganella morganii | M13 | Konjac | Yichang, Hubei | − | − |
Paenibacillus polymyxa | M14 | Konjac | Yichang, Hubei | − | − |
Pantoea agglomerans | M15 | Konjac | Yichang, Hubei | − | − |
Providencia rettgeri | M16 | Konjac | Yichang, Hubei | − | − |
Pseudomonas plecoglossicida | M17 | Konjac | Yichang, Hubei | − | − |
P. putida | M18 | Konjac | Yichang, Hubei | − | − |
Raoultella ornithinolytica | M19 | Konjac | Yichang, Hubei | − | − |
R. planticola | M20 | Konjac | Yichang, Hubei | − | − |
R. terrigena | M21 | Konjac | Yichang, Hubei | − | − |
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Sun, M.; Liu, H.; Huang, J.; Peng, J.; Fei, F.; Zhang, Y.; Hsiang, T.; Zheng, L. A Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Pectobacterium aroidearum that Causes Soft Rot in Konjac. Int. J. Mol. Sci. 2019, 20, 1937. https://doi.org/10.3390/ijms20081937
Sun M, Liu H, Huang J, Peng J, Fei F, Zhang Y, Hsiang T, Zheng L. A Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Pectobacterium aroidearum that Causes Soft Rot in Konjac. International Journal of Molecular Sciences. 2019; 20(8):1937. https://doi.org/10.3390/ijms20081937
Chicago/Turabian StyleSun, Miaomiao, Hao Liu, Junbin Huang, Jinbo Peng, Fuhua Fei, Ya Zhang, Tom Hsiang, and Lu Zheng. 2019. "A Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Pectobacterium aroidearum that Causes Soft Rot in Konjac" International Journal of Molecular Sciences 20, no. 8: 1937. https://doi.org/10.3390/ijms20081937