Rapid and Visual Detection of Heterodera schachtii Using Recombinase Polymerase Amplification Combined with Cas12a-Mediated Technology

Heterodera schachtii is a well-known cyst nematode that causes serious economic losses in sugar beet production every year. Rapid and visual detection of H. schachtii is essential for more effective prevention and control. In this study, a species-specific recom...

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Autores principales:	Ke Yao, Deliang Peng, Chen Jiang, Wei Zhao, Guangkuo Li, Wenkun Huang, Lingan Kong, Haifeng Gao, Jingwu Zheng, Huan Peng
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	recombinase polymerase amplification (RPA) sugar beet cyst nematode <i>Heterodera schachtii</i> lateral flow dipstick (LFD) Cas12a Biology (General) QH301-705.5 Chemistry QD1-999
Acceso en línea:	https://doaj.org/article/71ee63c96c634dd1980b2031931c65eb
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spelling	oai:doaj.org-article:71ee63c96c634dd1980b2031931c65eb2021-11-25T17:58:01ZRapid and Visual Detection of <i>Heterodera schachtii</i> Using Recombinase Polymerase Amplification Combined with Cas12a-Mediated Technology10.3390/ijms2222125771422-00671661-6596https://doaj.org/article/71ee63c96c634dd1980b2031931c65eb2021-11-01T00:00:00Zhttps://www.mdpi.com/1422-0067/22/22/12577https://doaj.org/toc/1661-6596https://doaj.org/toc/1422-0067<i>Heterodera schachtii</i> is a well-known cyst nematode that causes serious economic losses in sugar beet production every year. Rapid and visual detection of <i>H. schachtii</i> is essential for more effective prevention and control. In this study, a species-specific recombinase polymerase amplification (RPA) primer was designed from a specific <i>H. schachtii</i> sequence-characterized amplified region (SCAR) marker. A band was obtained in reactions with DNA from <i>H. schachtii</i>, but absent from nontarget cyst nematodes. The RPA results could be observed by the naked eye, using a lateral flow dipstick (LFD). Moreover, we combined CRISPR technology with RPA to identify positive samples by fluorescence detection. Sensitivity analysis indicated that 10<sup>−4</sup> single cysts and single females, 4<sup>−3</sup> single second-stage juveniles, and a 0.001 ng genomic DNA template could be detected. The sensitivity of the RPA method for <i>H. schachtii</i> detection is not only higher than that of PCR and qPCR, but can also provide results in <1 h. Consequently, the RPA assay is a practical and useful diagnostic tool for early diagnosis of plant tissues infested by <i>H. schachtii</i>. Sugar beet nematodes were successfully detected in seven of 15 field sugar beet root samples using the RPA assay. These results were consistent with those achieved by conventional PCR, indicating 100% accuracy of the RPA assay in field samples. The RPA assay developed in the present study has the potential for use in the direct detection of <i>H. schachtii</i> infestation in the field.Ke YaoDeliang PengChen JiangWei ZhaoGuangkuo LiWenkun HuangLingan KongHaifeng GaoJingwu ZhengHuan PengMDPI AGarticlerecombinase polymerase amplification (RPA)sugar beet cyst nematode<i>Heterodera schachtii</i>lateral flow dipstick (LFD)Cas12aBiology (General)QH301-705.5ChemistryQD1-999ENInternational Journal of Molecular Sciences, Vol 22, Iss 12577, p 12577 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	recombinase polymerase amplification (RPA) sugar beet cyst nematode <i>Heterodera schachtii</i> lateral flow dipstick (LFD) Cas12a Biology (General) QH301-705.5 Chemistry QD1-999
spellingShingle	recombinase polymerase amplification (RPA) sugar beet cyst nematode <i>Heterodera schachtii</i> lateral flow dipstick (LFD) Cas12a Biology (General) QH301-705.5 Chemistry QD1-999 Ke Yao Deliang Peng Chen Jiang Wei Zhao Guangkuo Li Wenkun Huang Lingan Kong Haifeng Gao Jingwu Zheng Huan Peng Rapid and Visual Detection of <i>Heterodera schachtii</i> Using Recombinase Polymerase Amplification Combined with Cas12a-Mediated Technology
description	<i>Heterodera schachtii</i> is a well-known cyst nematode that causes serious economic losses in sugar beet production every year. Rapid and visual detection of <i>H. schachtii</i> is essential for more effective prevention and control. In this study, a species-specific recombinase polymerase amplification (RPA) primer was designed from a specific <i>H. schachtii</i> sequence-characterized amplified region (SCAR) marker. A band was obtained in reactions with DNA from <i>H. schachtii</i>, but absent from nontarget cyst nematodes. The RPA results could be observed by the naked eye, using a lateral flow dipstick (LFD). Moreover, we combined CRISPR technology with RPA to identify positive samples by fluorescence detection. Sensitivity analysis indicated that 10<sup>−4</sup> single cysts and single females, 4<sup>−3</sup> single second-stage juveniles, and a 0.001 ng genomic DNA template could be detected. The sensitivity of the RPA method for <i>H. schachtii</i> detection is not only higher than that of PCR and qPCR, but can also provide results in <1 h. Consequently, the RPA assay is a practical and useful diagnostic tool for early diagnosis of plant tissues infested by <i>H. schachtii</i>. Sugar beet nematodes were successfully detected in seven of 15 field sugar beet root samples using the RPA assay. These results were consistent with those achieved by conventional PCR, indicating 100% accuracy of the RPA assay in field samples. The RPA assay developed in the present study has the potential for use in the direct detection of <i>H. schachtii</i> infestation in the field.
format	article
author	Ke Yao Deliang Peng Chen Jiang Wei Zhao Guangkuo Li Wenkun Huang Lingan Kong Haifeng Gao Jingwu Zheng Huan Peng
author_facet	Ke Yao Deliang Peng Chen Jiang Wei Zhao Guangkuo Li Wenkun Huang Lingan Kong Haifeng Gao Jingwu Zheng Huan Peng
author_sort	Ke Yao
title	Rapid and Visual Detection of <i>Heterodera schachtii</i> Using Recombinase Polymerase Amplification Combined with Cas12a-Mediated Technology
title_short	Rapid and Visual Detection of <i>Heterodera schachtii</i> Using Recombinase Polymerase Amplification Combined with Cas12a-Mediated Technology
title_full	Rapid and Visual Detection of <i>Heterodera schachtii</i> Using Recombinase Polymerase Amplification Combined with Cas12a-Mediated Technology
title_fullStr	Rapid and Visual Detection of <i>Heterodera schachtii</i> Using Recombinase Polymerase Amplification Combined with Cas12a-Mediated Technology
title_full_unstemmed	Rapid and Visual Detection of <i>Heterodera schachtii</i> Using Recombinase Polymerase Amplification Combined with Cas12a-Mediated Technology
title_sort	rapid and visual detection of <i>heterodera schachtii</i> using recombinase polymerase amplification combined with cas12a-mediated technology
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/71ee63c96c634dd1980b2031931c65eb
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Rapid and Visual Detection of <i>Heterodera schachtii</i> Using Recombinase Polymerase Amplification Combined with Cas12a-Mediated Technology

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