Name: Combined Recombinase Polymerase Amplification CRISPR/Cas12a Assay for Detecting Fusarium oxysporum f. sp. cubense Tropical Race 4 (Video) | JoVE Journal
Uploaded: 2025-11-14T13:35:00.000+00:00
Duration: 6 min 47 s
Description: Learn a rapid CRISPR/Cas12a detection assay for Fusarium oxysporum f. sp. cubense tropical race 4 using recombinase polymerase amplification. This method enables accurate, cost-effective pathogen detection in banana crops.

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5 minutes

We acknowledge funding from the National Research Foundation of South Africa, Grant Number: 138109, under the competitive support for unrated researchers&#39; program. We also acknowledge the European Union Horizon 2020 research and innovation program Marie Sklodowska-Curie Fellowship funding under the INDICANTS Project Grant Number: 890856.

1. Simplified DNA extraction from plant material
NOTE: See Figure 1 for an overview.
<ol>
	<li>Perform DNA extraction.
	<ol>
		<li>Transfer 250 mg of each piece of symptomatic pseudostem into a separate extraction bag (see Table of Materials).</li>
		<li>Add 2 mL of 0.5 M NaOH-PVP (Table of Materials) to bags with plant material. Grind infected vessels in an extraction bag using a manual grinder like a pestle. 
		NOTE: Add 1-2 mL supplementary 0.5 M NaOH-PVP if necessary to get enough liquid extract from the bag.</li>
		<li>Add 195 &#181;L of 100 mM Tris-HCL (pH 8.0) (Table of Materials) into 1.5 mL tubes.</li>
		<li>Pipette 5 &#181;L of extract from the bag into the 1.5 mL tube with 195 &#181;L of 100 mM Tris-HCL (pH 8.0), diluting the extract 40-fold.</li>
		<li>Vortex the tubes for 15-30 s and store at -20 &#176;C until use.</li>
	</ol>
	</li>
</ol>
2. crRNA synthesis and quality control
<ol fo:break-after="page">
	<li>Perform transcription.
	<ol>
		<li>Using the RNA transcription kit (Table of Materials), add the following to a 0.2 mL PCR tube: 1.5 &#181;L of 10x T7 Reaction buffer, 1.5 &#181;L of each dNTP, 1.5 &#181;L of T7 DNA polymerase mix, 1 &#181;L of nuclease-free water, and 10 &#181;L of 100 &#181;M T7 template DNA for a total volume of 20 &#181;L.</li>
		<li>Incubate the tube at 37 &#176;C in a water bath or PCR machine for at least 4 h.</li>
		<li>Use a spectrophotometer (Table of Materials) to determine the quantity of transcribed RNA. Dilute the RNA to 250 ng/&#181;L (10 &#181;g) in a volume of 40 &#181;L using nuclease-free water or TE buffer (Supplementary File 1). 
		NOTE: The transcribed RNA can be stored at -80 &#176;C until continuing with DNase treatments and cleaning steps.</li>
	</ol>
	</li>
	<li>Perform DNase treatment and crRNA cleanup. 
	NOTE: Perform all steps at room temperature. Keep RNA and DNase I enzyme on ice.
	<ol>
		<li>Treat the RNA sample with DNase I included in the RNA cleaning kit (Table of Materials) according to the manufacturer instructions22.</li>
		<li>Clean the RNA sample using the RNA cleaning kit according to manufacturer instructions, following Protocol II22.</li>
		<li>Nanodrop the cleaned crRNA to determine the quantity and quality present.&#160;Dilute the RNA to 100 ng/&#181;L using nuclease-free water and store at -80 &#176;C until use.</li>
		<li>Heat the crRNA with RNA loading dye (Table of Materials) to 70 &#176;C for 10 min in a water bath, and subject the crRNA to electrophoresis on a 1.5% agarose gel to visualize the crRNA synthesized (Figure 2).</li>
	</ol>
	</li>
</ol>
3. Recombinase polymerase amplification (RPA)-Cas12a one-tube reaction for in vitro Foc TR4 detection
NOTE: See Figure 3 for an overview.
<ol>
	<li>Prepare the RPA and Cas12a reaction mixes. 
	NOTE: Ensure that these steps are carried out in a DNA-free space on ice, with the reagents and materials pre-cooled. Wipe the working surface and pipettes with bleach and 70% ethanol before the experiment. See Table 1 for information on all oligonucleotides used in this study.
	<ol>
		<li>Prepare a 20 &#181;L Cas12a reaction mixture in a separate 0.5 mL tube (Table of Materials) with the following reagent volumes: 2 &#181;L of 10x NEBuffer r2.1 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 100 &#181;g/mL Recombinant Albumin, pH 7.9), 0.4 &#181;L of 10 &#181;M LbCas12a protein, 0.6 &#181;L of 100 ng/&#181;L crRNA, 0.4 &#181;L of 10 &#181;M reporter, and 16.6 &#181;L of deionized water. 
		NOTE: The final concentrations of LbCas12a protein, crRNA, and reporter are 200 nM, respectively.</li>
		<li>Prepare a 9.5 &#181;L RPA reaction mixture in a separate 0.5 mL tube with the following reagent volumes: 5 &#181;L of 2x Reaction Buffer (final concentration 1x according to manufacturer guidelines), 1.6 &#181;L of dNTP Mix (final concentration 1.6 mM), 1 &#181;L of 10x Basic E-Mix (final concentration 1x according to manufacturer guidelines), 0.5 &#181;L of each RPA primer at 10 &#181;M (final concentration 0.5 &#181;M each), 0.5 &#181;L of 20x Core Reaction Mix (final concentration 1x according to manufacturer guidelines), and 0.4 &#181;L of deionized water.</li>
		<li>Place an 8-tube strip into a tube rack (Table of Materials) on ice.</li>
		<li>Transfer the 20 &#181;L Cas12a reaction mix into the tube base.</li>
		<li>Transfer the 9.5 &#181;L RPA reaction mix into the tube strip lid. Add 0.5 &#181;L of 280 nM MgOAc (final concentration 14 nM) to the RPA reaction mix in the tube strip lid and mix by pipetting up and down.</li>
		<li>Add 0.5 &#181;L of deionized water to the non-template control (NTC) tube lid and close carefully to ensure that the RPA and Cas12a reaction mixtures are not combined. 
		NOTE: A non-template control (NTC) is included to ensure reliability of the results.</li>
	</ol>
	</li>
	<li>Add template DNA and start the reaction.
	<ol>
		<li>Add 0.5 &#181;L of template DNA to the 10 &#181;L RPA reaction mixture containing MgOAc in the tube strip lid, except for the NTC, with deionized water in place of DNA. Always add a known positive control Foc TR4 DNA in one well to ensure the assay worked as intended. Mix by pipetting up and down.</li>
		<li>Carefully close the tube strip lids to ensure the RPA and Cas12a reaction does not mix.</li>
		<li>Switch on the portable fluorometer (Table of Materials) and create a Run Profile for the RPA-Cas12a assay (Supplementary File 2). Open the saved Run Profile and place the tube strip into the portable fluorometer to incubate for 30 min at 37 &#176;C. 
		NOTE: Be careful not to touch the bottom of the tubes when handling the tube strip; instead hold the outer edges at the top left and right end.</li>
		<li>Remove the tube strip from the portable fluorometer after 30 min. Invert the tubes six times to mix the RPA and Cas12a reactions and spin down in a mini centrifuge (see Table of Materials) for 5 s.</li>
		<li>Place the tube strip back into the portable fluorometer and incubate for a further 30 min at 37 &#176;C using the saved Run Profile.</li>
	</ol>
	</li>
	<li>Visualize the results.
	<ol>
		<li>Interpret the average relative fluorescence units (RFUs) measured on the portable fluorometer (Supplementary File 2 and the user manual23).</li>
		<li>Visualize the tubes under LED blue light at 465 nm excitation wavelength using a blue light transilluminator (Supplementary File 3). Capture images with a phone camera.</li>
	</ol>
	</li>
</ol>
&#160; &#160;

<ol><li>Viljoen, A., Ma, L. J., Molina, A. B. Fusarium Wilt (Panama Disease) and Monoculture in Banana Production: Resurgence of a Century-Old Disease. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aViljoen%2C+A+%22Emerging+Plant+Diseases+and+Global+Food+Security%22">Emerging Plant Diseases and Global Food Security</a>. , The American Phytopathological Society. St. Paul, MN. (2020).</li>
<li>Viljoen, A., et al. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aViljoen%2C+A+%22Banana+Diseases+and+Pests%3A+Field+Guide+for+Diagnostics+and+Data+Collection%22">Banana Diseases and Pests: Field Guide for Diagnostics and Data Collection</a>. , IITA, Ibadan. (2017).</li>
<li>Dita, M. A., Waalwijk, C., Buddenhagen, I. W., Souza, M. T., Kema, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+molecular+diagnostic+for+tropical+race+4+of+the+banana%5BTitle%5D)+AND+%22Plant+Pathol%22%5BJournal%5D)">A molecular diagnostic for tropical race 4 of the banana.</a> Plant Pathol. 59 (2), 348-357 (2010).</li>
<li>Li, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+and+application+of+a+unique+genetic+locus+in+diagnosis+of+Fusarium+oxysporum+f.+sp.+cubense+tropical+race+4%5BTitle%5D)+AND+%22Can+J+Plant+Pathol%22%5BJournal%5D)">Identification and application of a unique genetic locus in diagnosis of Fusarium oxysporum f. sp. cubense tropical race 4.</a> Can J Plant Pathol. 35 (4), 482-493 (2013).</li>
<li>Aguayo, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+a+hydrolysis+probe-based+real-time+assay+for+the+detection+of+tropical+strains+of+Fusarium+oxysporum+f.+sp.+cubense+race+4%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Development of a hydrolysis probe-based real-time assay for the detection of tropical strains of Fusarium oxysporum f. sp. cubense race 4.</a> PLoS One. 12 (2), e0171767(2017).</li>
<li>Matthews, M. C., Mostert, D., Ndayihanzamaso, P., Rose, L. J., Viljoen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+detection+of+economically+important+Fusarium+oxysporum+f.+sp.+cubense+strains+in+Africa+in+plants%2C+soil+and+water%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Quantitative detection of economically important Fusarium oxysporum f. sp. cubense strains in Africa in plants, soil and water.</a> PLoS One. 15 (7), e0236110(2020).</li>
<li>Ordóñez, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+loop-mediated+isothermal+amplification+%28LAMP%29+assay+based+on+unique+markers+derived+from+genotyping+by+sequencing+data+for+rapid+in+planta+diagnosis+of+Panama+disease+caused+by+tropical+race+4+in+banana%5BTitle%5D)+AND+%22Plant+Pathol%22%5BJournal%5D)">A loop-mediated isothermal amplification (LAMP) assay based on unique markers derived from genotyping by sequencing data for rapid in planta diagnosis of Panama disease caused by tropical race 4 in banana.</a> Plant Pathol. 68 (9), 1682-1693 (2019).</li>
<li>Martzy, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Challenges+and+perspectives+in+the+application+of+isothermal+DNA+amplification+methods+for+food+and+water+analysis%5BTitle%5D)+AND+%22Anal+Bioanal+Chem%22%5BJournal%5D)">Challenges and perspectives in the application of isothermal DNA amplification methods for food and water analysis.</a> Anal Bioanal Chem. 411, 1695-1702 (2019).</li>
<li>Ghouneimy, A., Mahas, A., Marsic, T., Aman, R., Mahfouz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CRISPR-based+diagnostics%3A+Challenges+and+potential+solutions+toward+point-of-care+applications%5BTitle%5D)+AND+%22ACS+Synth+Biol%22%5BJournal%5D)">CRISPR-based diagnostics: Challenges and potential solutions toward point-of-care applications.</a> ACS Synth Biol. 12 (1), 1-16 (2023).</li>
<li>Chen, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CRISPR-Cas12a+target+binding+unleashes+indiscriminate+single-stranded+DNase+activity%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity.</a> Science. 360 (6387), 436-439 (2018).</li>
<li>Gootenberg, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nucleic+acid+detection+with+CRISPR-Cas13a%2FC2c2%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Nucleic acid detection with CRISPR-Cas13a/C2c2.</a> Science. 356 (6336), 438-442 (2017).</li>
<li>Chen, Y., Mei, Y., Jiang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Universal+and+high-fidelity+DNA+single+nucleotide+polymorphism+detection+based+on+a+CRISPR%2FCas12a+biochip%5BTitle%5D)+AND+%22Chem+Sci%22%5BJournal%5D)">Universal and high-fidelity DNA single nucleotide polymorphism detection based on a CRISPR/Cas12a biochip.</a> Chem Sci. 12 (12), 4455-4462 (2021).</li>
<li>Shin, K., Kwon, S. H., Lee, S. C., Moon, Y. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sensitive+and+rapid+detection+of+citrus+scab+using+an+RPA-CRISPR%2FCas12a+system+combined+with+a+lateral+flow+assay%5BTitle%5D)+AND+%22Plants+%28Basel%29%22%5BJournal%5D)">Sensitive and rapid detection of citrus scab using an RPA-CRISPR/Cas12a system combined with a lateral flow assay.</a> Plants (Basel). 10 (10), 2132(2021).</li>
<li>Zhang, Y. M., Zhang, Y., Xie, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+CRISPR%2FCas12a-based+DNA+detection+for+fast+pathogen+diagnosis+and+GMO+test+in+rice%5BTitle%5D)+AND+%22Mol+Breed%22%5BJournal%5D)">Evaluation of CRISPR/Cas12a-based DNA detection for fast pathogen diagnosis and GMO test in rice.</a> Mol Breed. 40 (1), 11(2020).</li>
<li>Wang, Q., Qin, M., Coleman, J. J., Shang, W., Hu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+and+sensitive+detection+of+Verticillium+dahliae+from+complex+samples+using+CRISPR%2FCas12a+technology+combined+with+RPA%5BTitle%5D)+AND+%22Plant+Dis%22%5BJournal%5D)">Rapid and sensitive detection of Verticillium dahliae from complex samples using CRISPR/Cas12a technology combined with RPA.</a> Plant Dis. 107 (6), 1664-1669 (2023).</li>
<li>Sun, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+and+sensitive+detection+of+two+fungal+pathogens+in+soybeans+using+the+recombinase+polymerase+amplification%2FCRISPR-Cas12a+method+for+potential+on-site+disease+diagnosis%5BTitle%5D)+AND+%22Pest+Manag+Sci%22%5BJournal%5D)">Rapid and sensitive detection of two fungal pathogens in soybeans using the recombinase polymerase amplification/CRISPR-Cas12a method for potential on-site disease diagnosis.</a> Pest Manag Sci. 80 (3), 1168-1181 (2024).</li>
<li>Chen, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+frontline%2C+rapid%2C+nucleic+acid-based+Fusarium+circinatum+detection+system+using+CRISPR%2FCas12a+combined+with+recombinase+polymerase+amplification%5BTitle%5D)+AND+%22Plant+Dis%22%5BJournal%5D)">A frontline, rapid, nucleic acid-based Fusarium circinatum detection system using CRISPR/Cas12a combined with recombinase polymerase amplification.</a> Plant Dis. 107 (6), 1902-1910 (2023).</li>
<li>Mu, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CRISPR-Cas12a-based+diagnostics+of+wheat+fungal+diseases%5BTitle%5D)+AND+%22J+Agric+Food+Chem%22%5BJournal%5D)">CRISPR-Cas12a-based diagnostics of wheat fungal diseases.</a> J Agric Food Chem. 70 (23), 7240-7247 (2022).</li>
<li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visual+detection+of+Fusarium+temperatum+by+using+CRISPR-Cas12a+empowered+LAMP+assay+coupled+with+AuNPs-based+colorimetric+reaction%5BTitle%5D)+AND+%22LWT%22%5BJournal%5D)">Visual detection of Fusarium temperatum by using CRISPR-Cas12a empowered LAMP assay coupled with AuNPs-based colorimetric reaction.</a> LWT. 185, 115190(2023).</li>
<li>Liang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+an+RPA-based+CRISPR%2FCas12a+assay+in+combination+with+a+lateral+flow+strip+for+rapid+detection+of+toxigenic+Fusarium+verticillioides+in+maize%5BTitle%5D)+AND+%22Food+Control%22%5BJournal%5D)">Development of an RPA-based CRISPR/Cas12a assay in combination with a lateral flow strip for rapid detection of toxigenic Fusarium verticillioides in maize.</a> Food Control. 157, 110172(2024).</li>
<li>Matthews, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+combined+recombinase+polymerase+amplification+CRISPR%2FCas12a+assay+for+detection+of+Fusarium+oxysporum+f.+sp.+cubense+tropical+race+4%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">A combined recombinase polymerase amplification CRISPR/Cas12a assay for detection of Fusarium oxysporum f. sp. cubense tropical race 4.</a> Sci Rep. 15 (1), 2436(2025).</li>
<li><a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22RNA+Clean+%26+Concentrator-5%3A+Instruction+Manual+Version+3.1.3%22">RNA Clean & Concentrator-5: Instruction Manual Version 3.1.3</a>. , Zymo Research Corporation. Irvine, CA. (2023).</li>
<li><a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Genie+III+user+manual%2C+instrument+software+version+v3.18.2%22">Genie III user manual, instrument software version v3.18.2</a>. , Optigene. Horsham, U.K. (2021).</li>
<li>Li, S. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CRISPR-Cas12a-assisted+nucleic+acid+detection%5BTitle%5D)+AND+%22Cell+Discov%22%5BJournal%5D)">CRISPR-Cas12a-assisted nucleic acid detection.</a> Cell Discov. 4, 1-4 (2018).</li>
<li>Magdama, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparative+analysis+uncovers+the+limitations+of+current+molecular+detection+methods+for+Fusarium+oxysporum+f.+sp.+cubense+race+4+strains%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Comparative analysis uncovers the limitations of current molecular detection methods for Fusarium oxysporum f. sp. cubense race 4 strains.</a> PLoS One. 14 (9), e0222727(2019).</li>
<li>Mckillop, C. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aMckillop%2C+C+%22Independent+review+blames+unreliable+test%2C+not+Biosecurity+Queensland%2C+for+false+Panama+diagnosis%22">Independent review blames unreliable test, not Biosecurity Queensland, for false Panama diagnosis</a>. , ABC News, Australia. (2016).</li>
<li>Mckillop, C., Craig, Z. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aMckillop%2C+C+%22Quarantine+lifted+on+Mareeba+banana+farm+suspected+of+having+Panama+disease%22">Quarantine lifted on Mareeba banana farm suspected of having Panama disease</a>. , ABC News, Australia. (2015).</li>
<li>Wang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cas12aVDet%3A+A+CRISPR%2FCas12a-based+platform+for+rapid+and+visual+nucleic+acid+detection%5BTitle%5D)+AND+%22Anal+Chem%22%5BJournal%5D)">Cas12aVDet: A CRISPR/Cas12a-based platform for rapid and visual nucleic acid detection.</a> Anal Chem. 91 (19), 12156-12161 (2019).</li>
<li>Fozouni, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Amplification-free+detection+of+SARS-CoV-2+with+CRISPR-Cas13a+and+mobile+phone+microscopy%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Amplification-free detection of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy.</a> Cell. 184 (2), 323-333 (2021).</li>
<li>Ding, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ultrasensitive+and+visual+detection+of+SARS-CoV-2+using+all-in-one+dual+CRISPR-Cas12a+assay%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Ultrasensitive and visual detection of SARS-CoV-2 using all-in-one dual CRISPR-Cas12a assay.</a> Nat Commun. 11 (1), 4711(2020).</li>
<li>Parida, M., Sannarangaiah, S., Dash, P. K., Rao, P. V., Morita, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Loop-mediated+isothermal+amplification+%28LAMP%29%3A+A+new+generation+of+innovative+gene+amplification+technique%3B+perspectives+in+clinical+diagnosis+of+infectious+diseases%5BTitle%5D)+AND+%22Rev+Med+Virol%22%5BJournal%5D)">Loop-mediated isothermal amplification (LAMP): A new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases.</a> Rev Med Virol. 18 (6), 407-421 (2008).</li>
<li>Zeng, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CRISPR-Cas12a-driven+MXene-PEDOT%3APSS+piezoresistive+wireless+biosensor%5BTitle%5D)+AND+%22Nano+Energy%22%5BJournal%5D)">CRISPR-Cas12a-driven MXene-PEDOT:PSS piezoresistive wireless biosensor.</a> Nano Energy. 82, 105711(2021).</li>
<li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+and+sensitive+detection+of+toxigenic+Fusarium+asiaticum+integrating+recombinase+polymerase+amplification%2C+CRISPR%2FCas12a%2C+and+lateral+flow+techniques%5BTitle%5D)+AND+%22Int+J+Mol+Sci%22%5BJournal%5D)">Rapid and sensitive detection of toxigenic Fusarium asiaticum integrating recombinase polymerase amplification, CRISPR/Cas12a, and lateral flow techniques.</a> Int J Mol Sci. 24 (18), 14134(2023).</li>
<li>Manzo-Sánchez, G., Orozco-Santos, M., Martínez-Bolaños, L., Garrido-Ramírez, E., Canto-Canche, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diseases+of+quarantine+and+economic+importance+in+banana+tree+%28Musa+sp.%29+in+M%C3%A9xico%5BTitle%5D)+AND+%22Rev+Mex+Fitopatol%22%5BJournal%5D)">Diseases of quarantine and economic importance in banana tree (Musa sp.) in México.</a> Rev Mex Fitopatol. 32 (2), 89-107 (2014).</li>
<li>Blomme, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bacterial+diseases+of+bananas+and+enset%3A+Current+state+of+knowledge+and+integrated+approaches+toward+sustainable+management%5BTitle%5D)+AND+%22Front+Plant+Sci%22%5BJournal%5D)">Bacterial diseases of bananas and enset: Current state of knowledge and integrated approaches toward sustainable management.</a> Front Plant Sci. 8, 1290(2017).</li>
</ol>

The authors declare no competing financial or intellectual interests.

Regular surveillance is an important strategy for effective prevention and containment efforts of Foc TR4 and other important invasive pests. Detection protocols that can be applied in-field, and rapidly and accurately identify pathogens, can fast-track decision-making and limit costs associated with surveillance efforts. To this end, the protocol presented here combined a simplified DNA extraction, conducted directly on sampled plant material, which was subsequently included in a one-tube assay combining RPA and Cas12a ...

Fusarium wilt, caused by the soilborne fungal pathogen Fusarium oxysporum f. sp. cubense (Foc), is globally regarded as one of the most devastating transboundary plant pathogens in history1. Once introduced into a new area, Foc remains invisible until infected plants develop symptoms. This can take months, allowing the fungus to establish and spread unknowingly. Once established, it is almost impossible to eradicate economically due to the production of resilient chlamydospores and the perennial nature of banana production. The importance of prevention and early detection, thus, is paramount in protecting vulnerable growers1. The current epidemic of the disease, caused by Foc tropical race 4 (TR4), requires detection by regular surveillance to identify symptomatic banana plants in high-risk areas. Abiotic constraints such as water stress and certain nutrient deficiencies produce external yellowing, which is similar to symptoms produced by Foc TR4 infection. Additionally, symptoms resulting from infection by different Foc races cannot be distinguished. Laboratory-based purification and subsequent phenotypic and molecular-based detection are, therefore, necessary for reliable Foc TR4 detection2. Various molecular assays are available for Foc TR4 detection, including PCR, quantitative (q) PCR, and loop-mediated isothermal amplification (LAMP) assays3,4,5,6,7. Most of these protocols, however, still require expensive and specialized skills and equipment, which must be conducted in laboratory settings. Recombinase polymerase amplification (RPA) can be performed isothermally and, therefore, eliminates the need for such equipment and can be considered for in-field detection protocols8.
Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have increasingly been utilized for improved molecular diagnostics9. These systems are employed as they are inexpensive, simple, and do not require the use of special instrumentation, and can therefore be considered for detection in-field. CRISPR-based diagnostics typically involve the isothermal amplification of a target sequence, followed by target recognition via CRISPR-Cas proteins and the collateral cleavage of a DNA or RNA fluorescent reporter dye to indicate the presence of the target10,11. In CRISPR-Cas12a systems, specifically, CRISPR RNA (crRNA) guides Cas12a to recognize and cleave nucleic acid targets. This crRNA can be designed to target a specific DNA or RNA region of interest through hybridization to a complementary sequence. Once bound, it results in Cas12a cleaving the target with single-nucleotide specificity, followed by collateral cleavage of reporter molecules12. Recently, CRISPR-Cas12a systems were employed to detect various economically important fungal plant pathogens13,14,15,16,17,18,19,20.
In this protocol, the development of a single-tube detection technique is described, combining a simplified DNA extraction, recombinase polymerase amplification (RPA), and CRISPR/Cas12a technology (RPA-Cas12a) for the detection of Foc TR4, which was visualized both on a portable fluorometer and with the naked eye under an inexpensive light-emitting diode (LED) blue light transilluminator21. The method can be further optimized for in-field detection to improve prevention and containment strategies. A similar approach can also be utilized to design Cas12a-based detection assays for other plant pathogens.

<table><tbody><tr><td>EnGen Lba Cas12a (Cfp1) Protein</td><td>New England Biolabs</td><td>M0653T</td><td></td></tr><tr><td>EnGen Lba Cas12a Diluent</td><td>New England Biolabs</td><td>B0653A</td><td></td></tr><tr><td>Eppendorf PCR Tubes</td><td>Merck</td><td>EP0030124332</td><td></td></tr><tr><td>Eppendorf Safe-Lock Micro Test Tubes</td><td>Merck</td><td>EP0030121708</td><td></td></tr><tr><td>Extraction Bags Universal</td><td>BIOREBA</td><td>430100</td><td></td></tr><tr><td>GeneRuler 100 bp DNA Ladder</td><td>Thermo Fischer Scientific</td><td>SM0241 / SM0242</td><td></td></tr><tr><td>Genie Centrifuge Mini-6KO</td><td>OptiGene</td><td>OP-FUGE</td><td></td></tr><tr><td>Genie III</td><td>OptiGene</td><td>https://www.optigene.co.uk/instruments/genie-iii/&amp;nbsp;</td><td></td></tr><tr><td>Genie Strip Holder</td><td>OptiGene</td><td>GBLOCK-01 / GBLOCK-02 / GBLOCK-03</td><td></td></tr><tr><td>Genie Strips</td><td>OptiGene</td><td>OP-0008-50 / OP-0008-500</td><td></td></tr><tr><td>HiScribe T7 High Yield RNA Synthesis Kit</td><td>New England Biolabs</td><td>E2040S</td><td></td></tr><tr><td>NanoDrop ND-1000 UV/Vis Spectrophotometer</td><td>Thermo Fischer Scientific</td><td>https://caeonline.com/buy/spectrometers/nanodrop-nd-1000/293669253</td><td></td></tr><tr><td>NEBuffer 3</td><td>New England Biolabs</td><td>B7003S</td><td></td></tr><tr><td>NEBuffer r2.1</td><td>New England Biolabs</td><td>B6002S</td><td></td></tr><tr><td>Polyvinylpyrrolidone (PVP) powder</td><td>Sigma-Aldrich</td><td>9003-39-8</td><td></td></tr><tr><td>RNA Clean and Concentrator-5 Kit</td><td>Zymo Research</td><td>R1013</td><td></td></tr><tr><td>RNA Loading Dye</td><td>Thermo Fischer Scientific</td><td>R0641</td><td></td></tr><tr><td>SmartBlue Blue Light Transilluminator</td><td>Accuris Instruments</td><td>E4000-E</td><td></td></tr><tr><td>Sodium hydroxide (NaOH) pellets</td><td>Merck, EMPARTA</td><td>K277</td><td></td></tr><tr><td>Tris (hydroxylmethyl) aminomethane</td><td>Merck</td><td>77-86-1</td><td></td></tr><tr><td>TwistAmp Liquid Basic Kit</td><td>TwistDx</td><td>TALQBAS01</td><td></td></tr></tbody></table>

combined recombinase polymerase amplification crispr/cas12a assay for detecting fusarium oxysporum f. sp. cubense tropical race 4

Combined Recombinase Polymerase Amplification CRISPR/Cas12a Assay for Detecting Fusarium oxysporum f. sp. cubense Tropical Race 4

Regular and accurate surveillance stands central to the efficient management of plant diseases. It can indicate which course of action is most appropriate, and whether prevention, eradication, or no action is required. Surveillance based on symptomology in host plants alone is often not reliable due to similarities in the symptoms caused by biotic and abiotic stresses. Laboratory-based molecular methods such as polymerase chain reaction (PCR) and quantitative (q)PCR are the most commonly and reliably used for plant pathogen detection, but rely on expensive equipment and skilled operators. Here, we describe a protocol combining a simplified DNA extraction, recombinase polymerase amplification (RPA), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a (RPA-Cas12a) for the detection of the invasive pathogen, Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4). The technique provides a simple single-tube detection alternative that is analytically robust with improved specificity compared to available molecular detection assays and negates the need for expensive and sophisticated laboratory equipment.

Successful detection of Foc TR4 from infected plant material using simplified DNA extraction followed by the combined RPA-Cas12a assay when conducted in the Genie III portable fluorometer is illustrated in Figure 4A. Positive samples show visible fluorescence (Lane 6-8), while negative samples show low endpoint fluorescence (Lane 1-5). When sample tubes were viewed under LED blue light, the Foc TR4-infected samples also showed visible fluorescence (Figure 4B). I...

In this protocol, a combined recombinase polymerase amplification CRISPR/Cas12a assay is illustrated for the detection of the invasive plant pathogen, Fusarium oxysporum f. sp. cubense tropical race 4.

Regular and accurate surveillance stands central to the efficient management of plant diseases. It can indicate which course of action is most ...

We are interested in the rapid and accurate detection of the Fusarium wilt of banana pathogen, known as Foc TR4. The combined use of recombinase polymerase amplification and CRISPR negates the use of expensive machines and improves the accuracy of current assays. To begin, transfer 250 milligrams of each piece of symptomatic pseudostem into a separate extraction bag.Add two milliliters of 0.5 molar sodium hydroxide polyvinylpyrrolidone solution into each extraction bag containing plant material. Using a manual grinder, such as a pestle, grind the infected vessels thoroughly inside the extraction bag. Then, add 195 microliters of 100 millimolar Tris-HCl buffer at pH eight into a 1.5 milliliter tube.Pipette five microliters of the plant extract from the extraction bag into the 1.5 milliliter tube containing Tris-HCl buffer to dilute the extract 40-fold. Vortex the tube for 15 to 30 seconds and store it at minus 20 degrees Celsius until further use. Using the RNA transcription kit, add the given components to a 0.2 milliliter PCR tube.Incubate the prepared tube at 37 degrees Celsius in a water bath or PCR machine for at least four hours. Now, use a spectrophotometer to quantify the transcribed RNA. Dilute the RNA to a concentration of 250 nanograms per microliter in a final volume of 40 microliters using nuclease-free water.Then, treat the RNA sample with DNase I provided in the RNA cleaning kit following the manufacturer's instructions. After that, clean the RNA sample using the RNA cleaning kit. Use a NanoDrop device to determine the quantity and quality of the cleaned CRISPR RNA.Then, dilute the RNA to 100 nanograms per microliter with nuclease-free water. Now, heat the CRISPR RNA mixed with RNA loading dye to 70 degrees Celsius for 10 minutes in a water bath, and run the sample on a 1.5%agarose gel to visualize the CRISPR RNA through electrophoresis. Prepare 20 microliters of Cas12a reaction mixture in a separate 0.5 milliliter tube using the given components.Next, prepare 9.5 microliters of RPA reaction mixture in a separate 0.5 milliliter tube with the components shown here. Place an eight-tube strip into a tube rack set on ice, and transfer the 20 microliters of Cas12a reaction mixture into the base of each tube in the strip. Then, transfer the 9.5 microliters of RPA reaction mixture into the lid of each tube in the strip.Add 0.5 microliters of 280 nanomolar magnesium acetate to the RPA mix in the lid, and mix by pipetting up and down. Next, add 0.5 microliters of deionized water to the lid of the non-template control tube, and close it carefully to prevent the RPA and Cas12a reaction mixtures from mixing. Now, add 0.5 microliters of template DNA to the 10 microliters of RPA reaction mixture containing magnesium acetate in the tube strip lid.Mix the contents by pipetting up and down. Carefully close all tube strip lids to ensure that the RPA and Cas12a reaction mixtures do not mix. Afterward, switch on the portable fluorimeter and create a run profile for the RPA Cas12a assay.Open the saved run profile, and place the tube strip inside the fluorimeter to incubate for 30 minutes at 37 degrees Celsius. After 30 minutes, remove the tube strip from the fluorimeter, invert the tube six times to mix the RPA and Cas12a reactions, and briefly spin the strip down in a mini centrifuge for five seconds. Then, return the tube strip to the fluorimeter and incubate for another 30 minutes at 37 degrees Celsius using the saved run profile.Interpret the average relative fluorescence units measured on the portable fluorimeter. Finally, visualize the reaction tubes under LED blue light at an excitation wavelength of 465 nanometers using a blue light transilluminator, and capture images of the fluorescent signal using a phone camera. Positive samples produced visible fluorescence in the Genie III portable fluorimeter following RPA Cas12a detection of Foc TR4 DNA, while negative samples yielded low endpoint fluorescence.When viewed under LED blue light, the sample tubes containing DNA from Foc TR4 infected plant material displayed visible fluorescence, while tubes with DNA from uninfected tissue did not show any fluorescence. We have developed the first assay that employs CRISPR technology for the detection of Foc TR4. Once optimized, the assay offers faster, more accurate detection on inexpensive machines than existing commercial assays.Our further work will explore a smartphone-linked fluorescent detector to be combined with the assay.

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Learn a rapid CRISPR/Cas12a detection assay for Fusarium oxysporum f. sp. cubense tropical race 4 using recombinase polymerase amplification. This method enables accurate, cost-effective pathogen detection in banana crops.

Combined Recombinase Polymerase Amplification CRISPR/Cas12a Assay for Detecting Fusarium oxysporum f. sp. cubense Tropical Race 4 (Video) | JoVE Journal

Combined Recombinase Polymerase Amplification CRISPR/Cas12a Assay for Detecting Fusarium oxysporum f. sp. cubense Tropical Race 4

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