Rapid and Visual Detection of Heterodera schachtii Using Recombinase Polymerase Amplification Combined with Cas12a-Mediated Technology
Abstract
:1. Introduction
2. Results
2.1. Primer Design
2.2. Detection and Confirmation of RPA Products
2.3. Specificity Test
2.4. Sensitivity Test
2.5. Direct Detection of Heterodera schachtii in Artificially Inoculated Roots
2.6. Detection of Heterodera schachtii in Natural Field Samples
3. Discussion
4. Materials and Methods
4.1. Nematode Populations
4.2. DNA Extraction
4.3. RPA Primer and Probe Design
4.4. RPA Reaction
4.5. Lateral Flow Dipstick (LFD) Assay
4.6. RPA/Cas12a Detection
4.7. RPA Assay Specificity Test
4.8. RPA Assay Sensitivity Test
4.9. Direct Detection of Heterodera schachtii in Inoculated Plant Roots and Soil
4.10. Detection of Heterodera schachtii in Natural Field Roots
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Primers | Sequences | Purpose | References |
---|---|---|---|
HsRPA-F1 | 5′-GAGAATTGAATTTATCTCCTTTTCACCTCAGCTC-3′ | RPA amplification | This study |
HsRPA-R1 | 5′-CACAGAGACAACACGAAGGAGCGAGCTCAATG-3′ | ||
HsRPA-F2 | 5′-TATCATCCAAATTTTGAGCGGTTCAAAGCG-3′ | ||
HsRPA-R2 | 5′-GGCCTTTTGCGTACACAGCGAAACTTGCGAATC-3′ | ||
HsRPA-F3 | 5′-CAACAACTACCACCACAATTACCGAACCACCG-3′ | ||
HsRPA-R3 | 5′-CAACTCCTTCAATGATTTGTCTAAGAATTCTC-3′ | ||
ssDNA | 5′-FAMTTTTT-3′BHQ1 | Cas12a degradation | |
Hs-probe | 5′-FAMCACCCGAGAATTGAATTTATCTCCTTTTCAC(THF)TCAGCTCGGCTATGTGTTG-3′ | ||
Hs-gRNA | 5′-AAUUUCUACUGUUGUAGAUGAAUUUAUCUCCUUUUCACCUCA-3′ | ||
SHF6 | 5′-GTTCTTACGTTACTTCCA-3′ | PCR amplification | [8] |
rDNA2 | 5′-TTTCACTCGCCGTTACTAAGG-3′ |
Species Code | Species | Population Origin | Host | Sampling Date | References |
---|---|---|---|---|---|
Hs1 | H. schachtii | Germany | Sugar beet | - a | [30] |
Hs2 | H. schachtii | Belgium | Sugar beet | - a | [9] |
Hs3 | H. schachtii | Bozova, anliurfa Province, Turkey | Sugar beet | - | [31] |
Hs4 | H. schachtii | Karakprü anliurfa Province,Turkey | Sugar beet | - | [31] |
Hs5 | H. schachtii | Siverek, anliurfa Province, Turkey | Sugar beet | - | [31] |
Hs6 | H. schachtii | Xinyuan county Xinjiang, China | Sugar beet | Sept. 2019 | This study |
Hs7 | H. schachtii | Xinyuan county Xinjiang, China | Sugar beet | Sept. 2019 | This study |
Hs8 | H. schachtii | Xinyuan county Xinjiang, China | Sugar beet | Sept. 2019 | This study |
Hs9 | H. schachtii | Emin county, Xinjiang, China | Sugar beet | Sept. 2019 | This study |
Hg1 | H. glycines | Langfang, Hebei, China | Soybean | Oct. 2019 | [32] |
Hg2 | H. glycines | Heilongjiang, China | Soybean | Oct. 2019 | [32] |
Hg3 | H. glycines | Japan | - | - b | This study |
Hso | H. sojae | Jiangxi, China | Soybean | Sept. 2017 | [33] |
Hc | H. cruciferae | Hubei, China | Oilseed rape | Sept. 2017 | This study |
Ha | H. avenae | Norway | Wheat | Sept. 2016 | [34] |
Hf | H. filipjevi | Tangyin. Henan, China | Wheat | July 2019 | [35] |
Hl | H. latinpones | Belgium | Wheat | - a | [36] |
He | H. elachista | Hunan, China | Rice | Dec. 2016 | [37] |
Hh | H. humuli | Guizhou, China | Weeds | July 2018 | This study |
Hr | H. ripae | Guizhou, China | Weeds | July 2018 | This study |
Hz | H. zeae | Laibin, Guangxi, China | Maize | Mar. 2018 | [38] |
Gr | Globodera rostochiensis | Belgium | Potato | - a | [39] |
Cc | Cactoderacacti | Liaoning, China | Weeds | July 2018 | [40] |
Samples | Host | Location | Cyst Nematode Density a | Detection Results | ||
---|---|---|---|---|---|---|
RPA/LFD (Detections/Trials) | RPA/CRISPR (Detections/Trial) | PCR | ||||
1 | Sugar beet | Huocheng County, Xinjiang, China | 0 | 0/3 | 0/3 | − |
2 | Zhaosu County, Xinjiang, China | 0 | 0/3 | 0/3 | − | |
3 | Xinyuan county-1, Xinjiang, China | 22 ± 7 | 3/3 | 3/3 | + | |
4 | Xinyuan county-2, Xinjiang, China | 46 ± 4 | 3/3 | 3/3 | + | |
5 | Xinyuan county-3, Xinjiang, China | 38 ± 5 | 3/3 | 3/3 | + | |
6 | Xinyuan county-4, Xinjiang, China | 20 ± 5 | 3/3 | 3/3 | + | |
7 | Gongliu county, Xinjiang, China | 0 | 0/3 | 0/3 | − | |
8 | Gongliu county, Xinjiang, China | 0 | 0/3 | 0/3 | − | |
9 | Emin county, Xinjiang, China | 11 ± 4 | 3/3 | 3/3 | + | |
10 | Emin county, Xinjiang, China | 19 ± 4 | 3/3 | 3/3 | + | |
11 | Keyouqian Banner, Inner Mongolia, China | 0 | 0/3 | 0/3 | − | |
12 | Urad Front Banner, Inner Mongolia, China | 0 | 0/3 | 0/3 | − | |
13 | Linxi county, Inner Mongolia, China | 0 | 0/3 | 0/3 | − | |
14 | Zhangbei county, Hebei province, China | 5 ± 2 | 3/3 | 3/3 | + | |
15 | Kangbao county, Hebei province, China | 0 | 0/3 | 0/3 | − |
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Yao, K.; Peng, D.; Jiang, C.; Zhao, W.; Li, G.; Huang, W.; Kong, L.; Gao, H.; Zheng, J.; Peng, H. Rapid and Visual Detection of Heterodera schachtii Using Recombinase Polymerase Amplification Combined with Cas12a-Mediated Technology. Int. J. Mol. Sci. 2021, 22, 12577. https://doi.org/10.3390/ijms222212577
Yao K, Peng D, Jiang C, Zhao W, Li G, Huang W, Kong L, Gao H, Zheng J, Peng H. Rapid and Visual Detection of Heterodera schachtii Using Recombinase Polymerase Amplification Combined with Cas12a-Mediated Technology. International Journal of Molecular Sciences. 2021; 22(22):12577. https://doi.org/10.3390/ijms222212577
Chicago/Turabian StyleYao, Ke, Deliang Peng, Chen Jiang, Wei Zhao, Guangkuo Li, Wenkun Huang, Lingan Kong, Haifeng Gao, Jingwu Zheng, and Huan Peng. 2021. "Rapid and Visual Detection of Heterodera schachtii Using Recombinase Polymerase Amplification Combined with Cas12a-Mediated Technology" International Journal of Molecular Sciences 22, no. 22: 12577. https://doi.org/10.3390/ijms222212577