A multi-channel CRISPR-based method for rapid, sensitive detection of four diseases of Brassica rapa in the field

Abstract

Pathogens significantly restrict the production of Brassica rapa (B. rapa L. ssp. Pekinensis), with climate change and evolving planting patterns exacerbating disease prevalence. Multichannel rapid diagnostic methods in the field can facilitate the early detection and control of diseases in B. rapa. Here, we established a multichannel lateral flow biosensor (LFB) combined with a CRISPR/Cas12a cleavage assay for the simultaneous detection of four B. rapa diseases. Key innovations of this study include: (1) High specificity and sensitivity, down to pathogen concentrations of 1.5 pg/μl—due to the optimization of crRNA secondary structure: the more stable the crRNA, the higher its detection sensitivity. (2) Optimized visual detection parameters. We identified ideal concentration ratios for the visual fluorescence detection system: 50 nM Cas12a, 50 nM crRNA, and 500 nM ssDNA fluorescent probe. Furthermore, the optimal concentrations of components on the LFB detection system were 3 μl SA-GNPs, 500 nM ssDNA test strip probe, 0.5 mg/ml biotin-BSA as the test line, and 1 mg/ml anti-FITC as the control line. (3) Field-Ready Cas-AIRPA Platform. We developed the on-site Cas-AIRPA platform for the simultaneous detection of B. rapa pathogens by combining rapid nucleic acid extraction and a four-channel lateral flow biosensor (4-LFB), which quickly provides disease-related information through a specific 2D barcode. Analysis of B. rapa samples in the field confirmed the suitability of the Cas-AIRPA platform for rapid (~25 min) and simultaneous on-site detection of four diseases of B. rapa. This platform can also be adapted to detect other plant diseases in the field.

Acknowledgements

This work was supported by grants from the National Key Research and Development Program of China (2023YFD1201504), the Innovation and Capacity-Building Project of BAAFS (KJCX20230221), the National Natural Science Foundation of China (32302550, 32072567, 32102400), the Beijing Nova Program (20220484052), China Agriculture Research System of MOF and MARA (CARS-A03), the Foundation for Reform and Development of BVRC (KYCX202302), and the Innovation Team Project of Modern Agricultural Technology System in Hebei Province (HBCT2021200207).

Author contributions

S.Y., F.Z., and M.W. conceptualized the study; X.L. and S.Y. performed experiments and wrote the manuscript; T.S., X.X., P.L., W.W., C.S., X.Z., D.Z., N.L., and J.W. contributed to data collection and data analysis.

Data availability

Data and Materials Availability. All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. All materials generated in this study are available from the corresponding author S.C.Y.

Conflict of interest statement

The authors declare no conflicts of interest.

Supplementary data

Supplementary data is available at Horticulture Research online.

References

[1] Dong H, Xiong R, Liang Y. et al. Development of glycine-copper(II) hydroxide nanoparticles with improved biosafety for sustain-able plant disease management. RSC Adv. 2020;10: 21222-7

[2] Su T, Wang W, Wang Z. et al. BrMYB108 confers resistance to verticillium wilt by activating ROS generation in Brassica rapa. Cell Rep. 2023;42:112938

[3] Sundelin T, Jensen DF, Lübeck M. Identification of expressed genes during infection of Chinese cabbage (Brassica rapa subsp. pekinensis) by Plasmodiophora brassicae. J Eukaryot Microbiol. 2011;58: 310-4

[4] Zhang B, Su T, Xin X. et al. Wall-associated kinase BrWAK1 con-fers resistance to downy mildew in Brassica rapa. Plant Biotechnol J. 2023;21: 2125-39

[5] Zhang B, Li P, Su T. et al. BrRLP48, encoding a receptor-like protein, involved in downy mildew resistance in Brassica rapa. Front Plant Sci. 2018;9:1708

[6] Singh D, Dhar S, Yadava DK. Genetic and pathogenic variability of Indian strains of Xanthomonas campestris pv. campestris causing black rot disease in crucifers. Curr Microbiol. 2011;63: 551-60

[7] Ahmed A, Munir S, He P. et al. Biocontrol arsenals of bacte-rial endophyte: an imminent triumph against clubroot disease. Microbiol Res. 2020;241:126565

[8] Khater M, de la Escosura-Muñiz A, Merkoçi A. Biosensors for plant pathogen detection. Biosens Bioelectron. 2017;93: 72-86

[9] Querci M, Van den Bulcke M, ˇZel J. et al. New approaches in GMO detection. Anal Bioanal Chem. 2010;396: 1991-2002

[10] Datukishvili N, Kutateladze T, Gabriadze I. et al. Newmultiplex PCR methods for rapid screening of genetically modified organ-isms in foods. Front Microbiol. 2015;6:757

[11] Patwardhan S, Dasari S, Bhagavatula K. et al. Simultaneous detection of genetically modified organisms in a mixture by multiplex PCR-Chip capillary electrophoresis. JAOACInt. 2015;98: 1366-74

[12] Dobnik D, Spilsberg B, Bogoˇzalec Koˇsir A. et al. Multiplex quan-tification of 12 European Union authorized genetically modified maize lines with droplet digital polymerase chain reaction. Anal Chem. 2015;87: 8218-26

[13] Berg T, Tesoriero L, Hailstones DL. A multiplex real-time PCR assay for detection of Xanthomonas campestris from brassicas. Lett Appl Microbiol. 2006;42: 624-30

[14] Treml D, Venturelli GL, Brod FCA. et al. Development of an event-specific hydrolysis probe quantitative real-time polymerase chain reaction assay for Embrapa 5.1 genetically modified com-mon bean (Phaseolus vulgaris). J Agric Food Chem. 2014;62: 11994-2000

[15] Venturelli GL, Brod FCA, Rossi GB. et al. A specific endogenous reference for genetically modified common bean (Phaseolus vulgaris L.) DNA quantification by real-time PCR targeting lectin gene. Mol Biotechnol. 2014;56: 1060-8

[16] Chen X, Wang X, Jin N. et al. Endpoint visual detection of three genetically modified rice events by loop-mediated isothermal amplification. Int J Mol Sci. 2012;13: 14421-33

[17] Huang X, Zhai C, You Q. et al. Potential of cross-priming amplification and DNA-based lateral-flow strip biosensor for rapid on-site GMO screening. Anal Bioanal Chem. 2014;406: 4243-9

[18] Wu H, Zhang X, Wu B. et al. Rapid on-site detection of genetically modified soybean products by realtime loop-mediated isother-mal amplification coupled with a designed portable amplifier. Food Chem. 2020;323:126819

[19] Zhu Z, Li R, Zhang H. et al. PAM-free loop-mediated isother-mal amplification coupled with CRISPR/Cas12a cleavage (Cas-PfLAMP) for rapid detection of rice pathogens. Biosens Bioelectron. 2022;204:114076

[20] Liu H, Wang J, Zeng H. et al. RPA-Cas12a-FS: a frontline nucleic acid rapid detection system for food safety based on CRISPR-Cas12a combined with recombinase polymerase amplification. Food Chem. 2021;334:127608

[21] Zeng H, Wang J, Jia J. et al. Development of a lateral flow test strip for simultaneous detection of BTCry1Ab, BT-Cry1Ac and CP4 EPSPS proteins in genetically modified crops. Food Chem. 2021;335:127627

[22] Son C-Y, Haines BB, Luch A. et al. Identification of the transgenic integration site in 2C T cell receptor transgenic mice. Transgenic Res. 2018;27: 441-50

[23] Tengs T, Kristoffersen AB, Berdal KG. et al. Microarray-based method for detection of unknown genetic modifications. BMC Biotechnol. 2007;7:91

[24] Evangelista VO, Pelegrini PB, Mulinari F. et al. Anovel immunocromatographic strip test for rapid detection of Cry1Ac and Cry8Ka5 proteins in genetically modified crops. Anal Meth-ods. 2015;7: 9331-9

[25] Li Y, Li S, Wang J. et al. CRISPR/Cas systems towards next-generation biosensing. Trends Biotechnol. 2019;37: 730-43

[26] Sashital DG. Pathogen detection in the CRISPR-Cas era. Genome Med. 2018;10:32

[27] Fonfara I, Richter H, BratoviˇcM. et al. The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA. Nature. 2016;532: 517-21

[28] He Q, Yu D, Bao M. et al. High-throughput and all-solution phase African swine fever virus (ASFV) detection using CRISPR-Cas12a and fluorescence based point-of-care system. Biosens Bioelectron. 2020;154:112068

[29] Huifeng X, Rui P, Weihua H. et al. Label-free dual-mode sens-ing platform based on target-regulated CRISPR-Cas12a activity for ochratoxin A in Morinda officinalis. Anal Methods. 2023;15: 4518-23

[30] Gootenberg JS, Abudayyeh OO, Lee JW. et al. Nucleic acid detec-tion with CRISPR-Cas13a/C2c2. Science. 2017;356: 438-42

[31] Shen J, Zhou X, Shan Y. et al. Sensitive detection of a bac-terial pathogen using allosteric probe-initiated catalysis and CRISPRCas13a amplification reaction. Nat Commun. 2020;11:267

[32] Smargon AA, Cox DBT, Pyzocha NK. et al. Cas13b is a type VI-B CRISPR-associated RNA-guided RNAse differentially regu-lated by accessory proteins Csx27 and Csx28. Mol Cell. 2017;65: 618-630.e7

[33] Li S-Y, Cheng Q-X, Wang J-M. et al. CRISPR-Cas12a-assisted nucleic acid detection. Cell Discov. 2018;4:20

[34] Chen JS, Ma E, Harrington LB. et al. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Sci-ence. 2018;360: 436-9

[35] Wang M, Liu X, Yang J. et al. CRISPR/Cas12a-based biosensing platform for the on-site detection of single-base mutants in gene-edited rice. Front Plant Sci. 2022;13:944295

[36] Zetsche B, Gootenberg JS, Abudayyeh OO. et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPRCas system. Cell. 2015;163: 759-71