Rapid Detection of Phytophthora cambivora Using Recombinase Polymerase Amplification Combined with CRISPR/Cas12a
Abstract
:1. Introduction
2. Materials and Methods
2.1. Isolate Preservation and DNA Extraction
2.2. Screening Phytophthora cambivora Target Genes
2.3. Designing Primers, crRNA, and ssDNA Reporter
2.4. RPA-CRISPR/Cas12a Assay
2.5. Specificity and Sensitivity of the RPA-CRISPR/Cas12a Assay
2.6. Detection of P. cambivora in Artificially Inoculated Apple Fruits Using the RPA-CRISPR/Cas12a Detection System
3. Results
3.1. Optimization of the RPA-CRISPR/Cas12a Assay for the Detection of Phytophthora cambivora
3.2. Verification of the Specificity of the RPA-CRISPR/Cas12a Assay
3.3. Sensitivity of the RPA-CRISPR/Cas12a Assay
3.4. Detection of P. cambivora in Artificially Inoculated Apples Using the RPA-CRISPR/Cas12a Detection System
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
References
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Number | Species | Isolate 1 | Origin | RPA-CRISPR/Cas12 2 | |
---|---|---|---|---|---|
Source | Host/Substrate | ||||
1 | Phytophthora cambivora | Pc1 | Shanghai, China | Malus domestica Borkh | + |
2 | Phytophthora cambivora | Pc2 | Shanghai, China | Malus domestica Borkh | + |
3 | Phytophthora cambivora | Pc3 | Shanghai, China | Malus domestica Borkh | + |
4 | Phytophthora cambivora | Pc4 | Shanghai, China | Malus domestica Borkh | + |
5 | Phytophthora cambivora | Pc5 | Shanghai, China | Malus domestica Borkh | + |
6 | Phytophthora cambivora | Pc6 | Shanghai, China | Malus domestica Borkh | + |
7 | Phytophthora cambivora | CBS 248.60 | Boston, MA, USA | Castanea sativa | + |
8 | Phytophthora boehmeriae | Pb3 | Nanjing, China | Boehmeria nivea | − |
9 | Phytophthora cactorum | C1 | Nanjing, China | Malus pumila | − |
10 | Phytophthora capsici | Pc1 | Nanjing, China | Capsicum annuum | − |
11 | Phytophthora castaneae | CBS587.85 | Taiwan | Soil | − |
12 | Phytophthora citrophthora | Pcit | Nanjing, China | Citrus reticulata Blanco | − |
13 | Phytophthora cinnamomi | Pci1 | Anqing, China | Pinus sp. | − |
14 | Phytophthora cryptogea | Pcr1 | Nanjing, China | Solanum lycopersicum | − |
15 | Phytophthora citricola | Pcit | Nanjing, China | Rhododendron pulchrum | − |
16 | Phytophthora drechsleri | CBS 292.35 T | CA, USA | Beta vulgaris var. altissima | − |
17 | Phytophthora fragariae | CBS 209.46 | England, UK | Fragaria × ananassa | − |
18 | Phytophthora hibernalis | CBS 270.31 | Richmond, VA, USA | Citrus sinensis | − |
19 | Phytophthora infestans | Pi1 | Fuzhou, China | Solanum tuberosum | − |
21 | Phytophthora lateralis | CBS 168.42 | Vancouver, CA, USA | Cedrus deodara | − |
22 | Phytophthora megasperma | CBS305.36 | CA, USA | Matthiola incana | − |
23 | Phytophthora melonis | PMNJHG1 | Nanjing, China | Cucumis sativus | − |
24 | Phytophthora mississippiae | 57J3 | Commonwealth of Virginia, USA | Irrigation water | − |
25 | Phytophthora nicotianae | Pn1 | Fuzhou, China | Nicotiana tabacum | − |
26 | Phytophthora palmivora | Pp1 | Lijiang, China | Iridaceae | − |
27 | Phytophthora parvispora | CBS132771 | Basilicata, Italy | Arbutus unedo | − |
28 | Phytophthora pini | Ppini1 | Nanjing, China | Rhododendron pulchrum | − |
29 | Phytophthora plurivora | Pplu1 | Haikou, China | Manihot esculenta | − |
30 | Phytophthora quercina | CBS 789.95 | Brisbane, Australia | Quercus petraea | − |
31 | Phytophthora sojae | Psy1 | Lijiang, China | Glycine max | − |
32 | Phytophthora syringae | 9099 | Shanghai, China | Malus domestica Borkh | − |
33 | Phytophthora ramorum | EU1 2275 | Exeter, UK | Quercus palustris | − |
34 | Phytophthora rubi | CBS 967.95 | Scotland, UK | Rubus idaeus | − |
35 | Phytophthora vignae | CPHSTBL 30 | M. D. Coffey | Vigna sp. | − |
36 | Phytopythium litorale | PC-dj1 | Nanjing, China | Rhododendron simsii | − |
37 | Phytopythium helicoides | PH-C | Nanjing, China | Rhododendron simsii | − |
38 | Phytopythium vexans | DT10 | Nanjing, China | Rhododendron simsii | − |
39 | Pythium ultimum | Pul1 | Nanjing, China | Citrus sinensis | − |
40 | Pythium spinosum | Psp1 | Nanjing, China | Oryza sativa L. | − |
41 | Pythium aphanidermatum | Pap1 | Nanjing, China | Nicotiana tabacum | − |
42 | Fusarium oxysporium | Fox1 | Nanjing, China | Gossypium sp. | − |
43 | Fusarium solani | Fso1 | Nanjing, China | Gossypium sp. | − |
44 | Fusarium circinatum | A045-1 | Shanghai, China | Pinus sp. | − |
45 | Fusarium fujikuroi | Ffu1 | Nanjing, China | Oryza sativa | − |
46 | Fusarium graminearum | Fgr1 | Nanjing, China | Triticum aestivum | − |
47 | Fusarium acuminatum | Fac1 | Chengdu, China | Rhizophora apiculata | − |
48 | Fusarium asiaticum | Fas1 | Nanjing, China | Triticum aestivum | − |
49 | Fusarium avenaceum | Fav1 | Nanjing, China | Glycine max | − |
50 | Fusarium culmorum | Fcu1 | Suining, China | Glycine max | − |
51 | Fusarium commune | Fco1 | Harbin, China | Soil | − |
52 | Fusarium equiseti | Feq1 | Nanjing, China | Glycine max | − |
53 | Fusarium lateritium | Flat1 | Nanjing, China | Soil | − |
54 | Fusarium moniforme | Fmo1 | Nanjing, China | Oryza sativa | − |
55 | Fusarium nivale | Fniv | Nanjing, China | Triticum aestivum | − |
56 | Fusarium proliferatum | Fpr1 | Nanjing, China | Pinus sp. | − |
57 | Fusarium incarnatum | IL3HQ | Nanjing, China | Medicago sativa | − |
58 | Colletotrichum truncatum | Ctr1 | Nanjing, China | Glycine max | − |
59 | Colletotrichum glycines | Cgl1 | Nanjing, China | Glycine max | − |
60 | Colletotrichum orbiculare | Cor1 | Nanjing, China | Citrullus lanatus | − |
61 | Neofusicoccum parvum | BJ3-1 | Nanjing, China | Fatsia japonica | − |
62 | Verticilium dahliae | Vda1 | Nanjing, China | Gossypium sp. | − |
63 | Rhizoctonia solani | Rso1 | Nanjing, China | Gossypium sp. | − |
64 | Magnaporthe grisea | Guy11 | Tsukuba, Japan | Oryza sativa | − |
65 | Endothia parasitica | Epa1 | Nanjing, China | Castanea mollissima | − |
66 | Bremia lactucae | Bla1 | Nanjing, China | Lactuca sativa | − |
67 | Aspergillus flavus | NJC03 | Nantong, China | Actinidia chinensis | − |
68 | Botrytis cinerea | Bci1 | Nanjing, China | Cucumis sativus | − |
69 | Alternaria alternata | Aal1 | Nanjing, China | Soil | − |
70 | Tilletia indica | Tin1 | Nanjing, China | Triticum aestivum | − |
71 | Diaporthe mahothocarpus | DT1 | Nanjing, China | Kerria japonica | − |
72 | Diaporthe sapindicola | WHZ3 | Nanjing, China | Sapindus mukorossi | − |
73 | Botryosphaeria dothidea | Bci1 | Nanjing, China | Koelreuteria paniculata | − |
74 | Bursaphelenchus xylophilus | Js-1 | Nanjing, China | Pinus thunbergii | − |
75 | Bursaphelenchus mucronatus | Bmucro | Nanjing, China | Pinus sp. | − |
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Zhou, J.; Dai, H.; Dai, T.; Liu, T. Rapid Detection of Phytophthora cambivora Using Recombinase Polymerase Amplification Combined with CRISPR/Cas12a. Forests 2023, 14, 2141. https://doi.org/10.3390/f14112141
Zhou J, Dai H, Dai T, Liu T. Rapid Detection of Phytophthora cambivora Using Recombinase Polymerase Amplification Combined with CRISPR/Cas12a. Forests. 2023; 14(11):2141. https://doi.org/10.3390/f14112141
Chicago/Turabian StyleZhou, Jing, Hanqian Dai, Tingting Dai, and Tingli Liu. 2023. "Rapid Detection of Phytophthora cambivora Using Recombinase Polymerase Amplification Combined with CRISPR/Cas12a" Forests 14, no. 11: 2141. https://doi.org/10.3390/f14112141
APA StyleZhou, J., Dai, H., Dai, T., & Liu, T. (2023). Rapid Detection of Phytophthora cambivora Using Recombinase Polymerase Amplification Combined with CRISPR/Cas12a. Forests, 14(11), 2141. https://doi.org/10.3390/f14112141