Name: On-site DNA Detection of Trypanosomatid Parasites and Nosema ceranae Through Alkaline Lysis Coupled to RPA/CRISPR/Cas12a System (Video) | JoVE Journal
Uploaded: 2025-07-18T14:50:00.000+00:00
Duration: 7 min 46 s
Description: Learn rapid DNA detection of bee pathogens using alkaline lysis with RPA and CRISPR/Cas12a. This field-adaptable method detects Lotmaria passim and Nosema ceranae with improved sensitivity over qPCR.

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5 minutes

This work was supported by the grant &#34;New molecular tools for rapid monitoring of honeybee diseases in apiaries in the province of Granada&#34; (GOPG-GR-23-0002) under the call Aid to operational groups of the European Innovation Association (AEI) in agricultural productivity and sustainability of the Andalusian government. We also acknowledge the support by the Spanish Programme for Knowledge Generation and Scientific and Technological Strengthening of the R+D+I System, grant PID2021-126938OB-I00 funded by MCIN/AEI/10.13039/501100011033 and by &#34;ERDF/EU&#34;. We strongly appreciated the help and resources provided by beekeepers of the Asociaci&#243;n de Peque&#241;os Apicultores de Granada (APAG) and Cooperativas Agroalimentarias de Granada (FAECA). The study is part of the PhD thesis of D. Pedro Garc&#237;a Olmedo. Doctoral Program in Fundamental and Systems Biology (University of Granada, UGR).

All procedures were conducted in accordance with institutional and national guidelines for working with invertebrates and using the lowest number of bees needed for proof of concept. Honeybees were transported in specialized hive boxes with ventilation and appropriate temperature and stored in the lab at -20&#8239;&#176;C until analysis.
1. HotSHOT alkaline cell lysis
<ol>
	<li>Solution preparation
	<ol>
		<li>HotSHOT Lysis buffer-preparation of 100 mL
		<ol>
			<li>Weigh 99.993 mg of NaOH and 8.32 mg of EDTA, add 90 mL of distilled water, and mix. Adjust the pH to 12 and the volume to 100 mL; autoclave.</li>
		</ol>
		</li>
		<li>40 mM Tris-HCl, pH 5
		<ol>
			<li>To 484.544 mg of Tris, add 90 mL of deionized water, adjust the pH to 5 with HCl, and bring the volume to 100 mL.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Lysis protocol
	<ol>
		<li>Dissect the abdomen of the bee using a sterile scalpel and transfer the dissected tissue to a 1.5 mL microcentrifuge tube. 
		NOTE: For pathogen detection, only the abdomens were used, and the remaining body parts were discarded.</li>
		<li>Add 400 &#181;L of HotSHOT buffer, macerate the tissue using a disposable pestle, and further homogenize it by vortexing. 
		NOTE: The pestle can be reused if decontaminated by incubating the material overnight in 0.1 N NaOH solution, then rinsed with double deionized water, followed by one autoclave cycle and subsequent drying in an incubator.</li>
		<li>Incubate the homogenate at 95&#8239;&#176;C for 10 min to lyse the cells. After incubation, immediately place the tube on ice to cool.</li>
		<li>To neutralize the reaction, add an equal volume (400 &#181;L) of 40 mM Tris-HCl, resulting in a final concentration of 20 mM Tris-HCl in the solution. 
		NOTE: This lysis procedure is illustrated in Figure 2.</li>
	</ol>
	</li>
</ol>
2. Isothermal amplification by RPA
NOTE: The design of the primers was made with the help of the eprimer3 software tool (https://www.bioinformatics.nl/cgi-bin/emboss/eprimer3), although other primer designing tools could be used.&#160;
<ol>
	<li>Preparation of working solutions
	<ol>
		<li>RPA primer solutions
		<ol>
			<li>Prepare stock solutions at a concentration of 100 &#181;M according to the manufacturer&#39;s instructions. For example, reconstitute 10.0 nmol of lyophilized primer in 100 &#181;L of Diethyl Pyrocarbonate (DEPC)-treated water and store at -20&#8239;&#176;C. Prepare working solutions at a 10 &#181;M concentration by diluting the stock 1:10; mix 90 &#181;L of DEPC-treated water with 10 &#181;L of the 100 &#181;M stock solution in a 0.2 mL tube. Store these solutions at -20&#8239;&#176;C.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>RPA protocol
	<ol>
		<li>Prepare the master mix according to the number of reactions to be performed. Include both a negative control (substituting ARCL with DEPC-treated water) and a positive control using pathogen DNA (in this example, DNA from cell culture L. passim promastigotes and bees positive for Nosema spp. (spores detected by microscopy)) (Table 1). 
		NOTE: Always prepare the RPA master mix on ice and use filter tips to avoid contamination.</li>
		<li>Transfer an aliquot of 46.5 &#181;L of the master mix into each 0.2 mL tube.</li>
		<li>Add magnesium acetate (MgOAc) and template DNA to the caps of the tubes, following the volumes specified in Table 2. Briefly centrifuge the tubes to mix the contents and initiate the RPA reaction. 
		NOTE: For better reproducibility, briefly vortex and centrifuge all components before use. Positive and negative controls must be included in each batch to validate the reaction.</li>
		<li>Incubate the samples at 39&#8239;&#176;C for 40 min for the RPA amplification reaction. 
		NOTE: After incubation, RPA reactions can be directly used for downstream detection (e.g., CRISPR/Cas12a-based assays), described in section 3.</li>
	</ol>
	</li>
</ol>
3. Pathogen DNA detection by CRISPR/Cas12a
NOTE: Detection after RPA can be performed in different ways. In this protocol, we will detail detection by fluorescence (FAM-TTTTTTTT-MGB) and immunochromatographic strips (Biotin-TTTTTTTT-FITC).
Perform all procedures in a clean, DNA-free area (e.g., dedicated PCR space). Clean work surfaces and pipettes with 0.5% bleach, followed by 70% ethanol. Always use filter tips and wear gloves. Handle DNA or amplified material in a separate area from the reagent preparation zone. Thaw all the reagents on ice and maintain cold conditions throughout preparation.
<ol>
	<li>Preparation of working solutions
	<ol>
		<li>crRNA 
		NOTE: The designing of Nosema ceranae and Lotmaria passim crRNA was performed using the Eukaryotic Pathogen CRISPR guide RNA/DNA Design Tool (EuPaGDT)&#160;http://grna.ctegd.uga.edu/.&#160;For customized crRNA design, other software such as GPP sgRNA Designer (https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design) or CHOPCHOP (https://chopchop.cbu.uib.no/) may be used.&#160;
		<ol>
			<li>To prepare a 100&#8239;&#181;M crRNA stock solution, reconstitute 10&#8239;nmol of each lyophilized crRNA with 100&#8239;&#181;L of DEPC-treated water and store at -20&#8239;&#176;C. To prepare a 1&#8239;&#181;M working solution, perform a 1:100 dilution by mixing 1&#8239;&#181;L of the 100&#8239;&#181;M stock solution with 99&#8239;&#181;L of DEPC-treated water in a 0.2&#8239;mL tube (Figure 3). 
			NOTE: Both solutions should be stored at -20 &#176;C.</li>
		</ol>
		</li>
		<li>Cas12a preparation 
		NOTE: Cas12a can be provided at concentrations of either 1&#8239;&#181;M or 100&#8239;&#181;M.
		<ol>
			<li>If supplied at 100&#8239;&#181;M, dilute it to a 1&#8239;&#181;M working solution before use by mixing 1&#8239;&#181;L of the 100&#8239;&#181;M Cas12a enzyme with 99&#8239;&#181;L of the diluent provided in the kit (1:100 dilution, Figure 3). 
			NOTE: The Cas12a enzyme concentration can be adjusted depending on the sample type. As the enzyme is stored in a glycerol-based diluent, it is important to ensure that the volume of enzyme added does not exceed 20% of the total reaction volume to avoid potential inhibitory effects.</li>
		</ol>
		</li>
		<li>Preparation of FAM and Biotin probe solutions 
		NOTE: The FAM probe is provided in lyophilized form.
		<ol>
			<li>To prepare a 100&#8239;&#181;M stock solution, resuspend the probe by adding 1&#8239;&#181;L of DEPC-treated water per 1&#8239;nmol of probe (e.g., for 102&#8239;nmol, add 102&#8239;&#181;L of DEPC-treated water). Store the stock solution at -20&#8239;&#176;C. For use in the detection mastermix, dilute the stock 1:10 (90 &#181;L of DEPC-treated water with 10 &#181;L of 100 &#181;M FAM) to obtain a 10&#8239;&#181;M working solution (Figure 3).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>CRISPR/Cas12a detection protocol
	<ol>
		<li>Fluorescence-based detection (FAM probe)
		<ol>
			<li>In a 1.5 mL microcentrifuge tube, prepare the reaction mix as described in Table 3, excluding the amplicons.</li>
			<li>Mix thoroughly using a vortex and distribute the mixture into PCR tubes.</li>
			<li>Then, add 4&#8239;&#181;L of RPA amplicon (described in step 2.2) to each tube.</li>
			<li>Incubate the reactions at 37&#8239;&#176;C for 120 min in a thermocycler with fluorescence reading capability, recording fluorescence every minute.</li>
			<li>Optional: At the end of the incubation, transfer the reactions to 0.2&#8239;mL tubes and visualize using a gel documentation system for imaging.</li>
		</ol>
		</li>
		<li>Detection using a Biotin probe in a lateral flow test
		<ol>
			<li>In a 1.5 mL microcentrifuge tube, add the components shown in Table 4.</li>
			<li>Make the mix as described in section 3.2.1, mix, and distribute it into a PCR plate. Finally, add 4 &#181;L of amplicon to the respective wells.</li>
			<li>Incubate for 120 min in a thermocycler (without fluorescence reading) or thermal block at 37 &#176;C.</li>
			<li>Transfer the samples to 0.2 mL tubes and perform the detection with immunostrips. To do this, add 50 &#181;L of running buffer and 10 &#181;L of the reaction mixture into each tube and insert the strip directly into the tube. Incubate for 15 min at room temperature and read the results after 10 min of immersion, as illustrated in Figure 3.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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<li>Hao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Application+of+a+lyophilized+CRISPR%2FCas12a+and+RPA+assay+for+rapid+detection+of+Actinobacillus+pleuropneumoniae%5BTitle%5D)+AND+%22Microchem+J%22%5BJournal%5D)">Application of a lyophilized CRISPR/Cas12a and RPA assay for rapid detection of Actinobacillus pleuropneumoniae.</a> Microchem J. 206, 111443(2024).</li>
<li>Lin, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+universal+all-in-one+RPA-Cas12a+strategy+with+de+novo+autodesigner+and+its+application+in+on-site+ultrasensitive+detection+of+DNA+and+RNA+viruses%5BTitle%5D)+AND+%22Biosens+Bioelectron%22%5BJournal%5D)">A universal all-in-one RPA-Cas12a strategy with de novo autodesigner and its application in on-site ultrasensitive detection of DNA and RNA viruses.</a> Biosens Bioelectron. 239, 115609(2023).</li>
</ol>

The authors have no conflicts of interest to disclose.

In this study, we developed and validated a practical molecular diagnostic protocol for detecting honeybee pathogens. The workflow integrates amplification-ready cell lysis, isothermal amplification using RPA, and highly specific detection via CRISPR/Cas12a. Among the chemical DNA extraction methods evaluated, the HotSHOT alkaline lysis protocol offers significant advantages in cost, speed, and simplicity. It efficiently produces amplification-ready lysates without the need for commercial kits. This method has proven reliable in various applications, including pathogen detection in honeybees14,15,16,17,18. The high-temperature alkaline conditions disrupt cell membranes and hydrogen bonding between DNA strands, releasing single-stranded DNA suitable for downstream amplification. Although potential limitations include the presence of PCR inhibitors or suboptimal neutralization, alkaline lysis has been shown to work effectively in diverse organisms such as archaea19, bacteria20, and fungi14, and is compatible with PCR, qPCR, and isothermal methods like LAMP21 and RPA12.
In our hands, the RPA/CRISPR/Cas12a system showed strong specificity and satisfactory sensitivity. The use of crRNAs targeting conserved regions in each pathogen allowed for accurate identification with no cross-reactivity to host DNA or unrelated microorganisms. Previous applications of this system in honeybees have achieved detection limits of approximately 6.5 &#215; 102 and 6.2 &#215; 101 copies/&#181;L for Deformed Wing Virus types A and B8,12. However, these assays have not been optimized for in-field conditions with rapid RNA and/or DNA extraction. In this work, we adapted the method specifically for DNA pathogens --&#160;N.&#160;ceranae and L.&#160;passim -- enabling field-compatible detection.
Our results show that RPA/CRISPR/Cas12a outperformed conventional PCR in identifying positive samples (16 vs. 12), likely due to the improved signal using the CRISPR/Cas12a collateral cleavage activity, which remains effective even in crude lysates. The improvement in sensitivity over PCR-based methods has been previously reported in different pathogen models such as Yersinia enterocolitica22, Diaporthe aspalathi23, or Vibrio vulnificus24. Furthermore, quantitative analysis revealed an equivalent limit of detection of RPA/CRISPR/Cas12a and qPCR under laboratory conditions (~96 parasite copies/&#181;L). This implies a good performance of the method with comparable specificity to TaqMan qPCR, due to the added sequence recognition provided by the crRNA. Furthermore, the assay requires only minimal equipment, does not depend on a thermocycler or fluorescence reader, and can be visualized using lateral flow strips as previously developed for other pathogens25,26. This simplifies interpretation and makes the test highly accessible for field technicians and beekeepers.
The current protocol yields robust results, but further improvements are possible. Adapting the assay to target other DNA-based bee pathogens would only require redesigning RPA primers and crRNAs. For RNA viruses, the protocol can be expanded to include a reverse transcription step (RT-RPA) as previously demonstrated for several viral infectious diseases26,27,28, including DWV-A and DWV-B in honeybees15. Additionally, the total assay time -- currently around 180 minutes -- can likely be reduced through protocol optimization for decreasing the timing of each step. Another alternative would be one-pot RPA/CRISPR/Cas12a that combines all RPA/CRISPR/Cas12a reagents in a single reaction. This all-in-one methodology has been successfully applied for the detection of a wide range of viral and bacterial pathogens25,26,29. Besides and despite that ssDNA probes have been extended and popularized for Cas12a detection, some studies have reported the capacity of Cas12a of trans-cleavage double-stranded DNA (dsDNA) probes that may be useful to be tested due to the stability of dsDNA30. This would lead to a time-to-result under 30 minutes, further enhancing field applicability. Lyophilizing RPA and CRISPR reagents would also improve reagent stability and eliminate the need for a cold chain, increasing suitability for remote or low-resource environments. The successful performance of RPA/CRISPR/Cas12a using lyophilized reagents has been successfully determined for the detection of Actinobacillus pleuropneumonia31 and respiratory infectious viruses28,32, including SARS-Cov226.
In summary, this study presents a fully in-field, cost-effective, and portable molecular diagnostic platform for honeybee pathogen detection. This methodology, as it stands, would last 180 min approximately, but its total timing could still be subjected to further improvements. While we focused on L. passim and Nosema spp. as proof-of-concept targets, the system is modular and adaptable to a wide range of DNA-based bee pathogens (i.e., Paenibacillus larvae or Melisococcus plutonious) as well as for monitoring commensal symbionts of interest. This approach expands access to molecular diagnostics beyond traditional laboratory settings and supports early intervention strategies in apiculture. More broadly, our results demonstrate the viability of CRISPR-based technologies in field-ready diagnostic applications.

<a href="https://app.jove.com/t/6641">This corrects</a> the article <a href="https://dx.doi.org/10.3791/68871">10.3791/68874</a>

An erratum was issued for:&#160;<a href="https://www.jove.com/t/68874">On-site DNA Detection of Trypanosomatid Parasites and Nosema ceranae Through Alkaline Lysis Coupled to RPA/CRISPR/Cas12a System</a>. The Authors&#160;section was&#160;updated.

Erratum: On-site DNA Detection of Trypanosomatid Parasites and Nosema ceranae Through Alkaline Lysis Coupled to RPA/CRISPR/Cas12a System

Bees are key species implicated in the pollination of plants and are essential for plant reproduction, biodiversity, and food security1. Their pollination services benefit a wide range of crops with high economic value, contributing an estimated $235&#8211;577 billion annually to global agriculture. However, the worldwide decline in bee populations has raised serious concerns due to its potential consequences for ecological stability and crop productivity, which have resulted in an estimated loss of $4.8&#8211;16.3 billion in heavily indebted poor countries2. The mass of bee disappearance involves multiple interactions between several factors: pesticide application, habitat loss, climate change, and infections by pathogens, including viruses, bacteria, fungi, and protists.
Among these microorganisms, Nosema spp., a microsporidian fungus that disturbs the functioning of the gut, and trypanosomatid parasites &#8212;&#160;particularly Lotmaria passim &#8212;&#160;are widely distributed in honeybee colonies worldwide3,4. Recent findings show that L. passim infections, even when asymptomatic, can downregulate immune-related genes and alter energy metabolism in honeybees, potentially compromising their resilience under additional stress5. Similarly, Nosema spp. infections reduce bee longevity, suppress immune function, and disrupt the colony&#39;s foraging dynamics, leading to reduced honey production and increased colony mortality. Moreover, coinfections involving both Nosema spp. and trypanosomatids may have synergistic effects, exacerbating physiological stress and weakening host defenses. These findings underscore the importance of monitoring multiple pathogens simultaneously.
Addressing these threats requires the development of rapid and accurate diagnostic tools, which in turn depend on a deeper understanding of the biology, transmission, and survival strategies of these pathogens6. The detection of these pathogens through traditional methods, including light microscopy and molecular amplification, remains reliable but faces certain limitations. Examples of qPCR, RTqPCR (requires cycling at different temperatures), and isothermal methods, such as Loop Mediated Amplification (LAMP, operates at constant temperature), have been developed for fungal, viral, bacterial, or protozoan parasites6,7,8,9,10. However, the implementation of these methods needs specialized equipment, together with trained personnel and well-equipped laboratory facilities, which restricts their practicality for field applications and large-scale monitoring. To address these issues, new molecular techniques have been developed &#8212;&#160;the amplification of DNA targets with recombinase polymerase amplification (RPA), together with CRISPR/Cas12a-based detection &#8212;&#160;a fast, specific, and sensitive approach. The method works isothermally and produces visual results that make it suitable for on-site or point-of-care diagnostics, according to Shao et al.11 and Xiao et al.12.
RPA&#160;is a simple and rapid DNA amplification method that operates at a constant&#160;temperature, typically between 37&#8239;&#176;C and 42&#8239;&#176;C. It relies on recombinase enzymes, single-stranded DNA-binding proteins, and a strand-displacing polymerase to amplify DNA without the need for thermal cycling. RPA is especially suitable for on-site testing and field diagnostics due to its speed, minimal equipment requirements, and compatibility with crude samples11,12,13. To enhance specificity, the RPA product can be detected using the Cas12a endonuclease, which also functions within the same temperature range. Cas12a binds to a CRISPR guide RNA (gRNA), a short RNA that guides Cas12a towards the amplified target sequence and binds to the complementary sequence in the DNA. Upon binding, Cas12a becomes activated and exhibits collateral (trans) cleavage activity, specifically against single-stranded DNA (ssDNA). This enables the use of ssDNA reporter molecules labeled with fluorophores or biotin, which can generate fluorescence or visual signals, including lateral flow strip readouts. Together, RPA and CRISPR/Cas12a form a powerful molecular diagnostic platform that is rapid, sensitive, and suitable for use in field or resource-limited settings12.
To further enhance the practicality of this molecular diagnostic approach, we also implemented a simplified amplification-ready cell lysis (ARCL) method based on sodium hydroxide (NaOH) lysis of bee abdomens11,14, eliminating the need for extensive sample&#160;preparation and laboratory reagents. This rapid nucleic-acid extraction technique, combined with the isothermal amplification and CRISPR/Cas12a detection system, enables a truly field-deployable workflow that significantly reduces both time and cost. Importantly, the assay maintains the quality, high sensitivity, and specificity comparable to qPCR, the current gold standard for pathogen detection in bees.
Here, we successfully applied this methodology for the detection of Nosema ceranae and L. passim in honey bees (Figure 1). We compared this method to standard qPCR assays and validated its performance using naturally infected samples. Our findings show that this approach is a practical and reliable alternative for field-based monitoring of honeybee health, with strong potential for large-scale surveillance and early intervention efforts in beekeeping.

<table><tbody><tr><td>0.2 mL DNAse- and RNAse-free Eppendorf tubes</td><td>Axygen</td><td>MTC-02-C</td><td></td></tr><tr><td>1.5 mL DNAse- and RNAse-free Eppendorf tubes</td><td>Axygen</td><td>MTC-150-C</td><td></td></tr><tr><td>10 &amp;mu;L low retention filtered tips</td><td>LabClinics</td><td>5-063-P5-0</td><td></td></tr><tr><td>100 &amp;mu;L low retention filtered tips</td><td>LabClinics</td><td>5-120-P5-0</td><td></td></tr><tr><td>1000 &amp;mu;L low retention filtered tips</td><td>LabClinics</td><td>5-203-P4-0</td><td></td></tr><tr><td>BH-200 Series Stuart Dry Block Heater</td><td>Cole-Parmer</td><td>EW-36610-00</td><td></td></tr><tr><td>DEPC-treated water</td><td>Ambicon</td><td>AM9906</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>798681</td><td></td></tr><tr><td>EnGen Lba Cas12a (Cpf1)</td><td>New England Biolabs</td><td>M0653T</td><td></td></tr><tr><td>Sigma-Aldrich</td><td>Sigma-Aldrich</td><td>64-17-5</td><td></td></tr><tr><td>HCl</td><td>Sigma-Aldrich</td><td>7647-01-0</td><td></td></tr><tr><td>Microtube pestles</td><td>Fisherbrand</td><td>12141364</td><td></td></tr><tr><td>Milenia GenLine HybriDetect</td><td>Milenia</td><td>MGHD 1</td><td></td></tr><tr><td>NaOH</td><td>Sigma-Aldrich</td><td>221465</td><td></td></tr><tr><td>RPA kit TwistAmp Liquid Basic</td><td>TwistAmp</td><td>&amp;nbsp;TALQBAS01</td><td></td></tr><tr><td>Scalpel blades</td><td>Swann-Morton</td><td>201</td><td></td></tr><tr><td>Single Chanel Pipete PIPETMAN P1000L Micropipette Metal eject</td><td>Gilson</td><td>F144059M</td><td></td></tr><tr><td>Single Chanel Pipete PIPETMAN P100L Micropipette Metal eject</td><td>Gilson</td><td>F144057M</td><td></td></tr><tr><td>Single Chanel Pipete PIPETMAN P10L Micropipette Metal eject</td><td>Gilson</td><td>F144055M</td><td></td></tr><tr><td>Sodium hypochlorite</td><td></td><td></td><td></td></tr><tr><td>Touch Real-Time PCR Detection System</td><td>BioRad</td><td>CFX96</td><td></td></tr><tr><td>Tris-HCl</td><td>Sigma-Aldrich</td><td>T1503</td><td></td></tr><tr><td>TwistAmp Liquid Basic</td><td>Twist</td><td>TALQBAS01</td><td></td></tr></tbody></table>

on-site dna detection of trypanosomatid parasites and nosema ceranae through alkaline lysis coupled to rpa/crispr/cas12a system

On-site DNA Detection of Trypanosomatid Parasites and Nosema ceranae Through Alkaline Lysis Coupled to RPA/CRISPR/Cas12a System

Early on-site detection of microscopic pathogens is essential for the effective management and health of honeybee colonies. Currently, the gold standard for molecular detection of bee pathogens is qPCR or RT-qPCR. Here, we present a rapid, sensitive, and cost-effective alternative for field-deployable DNA pathogen detection. This method combines direct, amplification-ready cell lysis of worker bee abdomens with recombinase polymerase amplification (RPA), CRISPR/Cas12a-mediated trans-cleavage of reporter probes, and detection via lateral flow assays. We successfully validated this protocol in honeybees infected with Nosema ceranae and Lotmaria passim. The described protocol can be performed using a simple heat block or at room temperature and is potentially applicable to any DNA-based pathogen or gut microbiota of the honeybee. The entire process takes approximately 180 min and achieves a sensitivity comparable to qPCR, detecting as few as 96 parasite copies/&#181;L. In conclusion, this approach offers a promising tool for reliable and rapid field diagnosis of honeybee infections without the need for complex laboratory equipment, making it ideal for on-site colony surveillance.

Here we have described a fast on-site method using alkaline lysis of worker bee abdomens coupled to RPA amplification and CRISPR/Cas12a-based detection by fluorescence and/or lateral flow approaches. The schema of the full workflow is represented in Figure 3, and the schema of the HotSHOT alkaline lysis of worker bee abdomens is in Figure 4. Representative results of the RPA/CRISPR/Cas12a assay are shown in Figure 5. To detect L. passim and N. ceranae, a thermocycler was used to quantify the relative fluorescence units (RFUs) using a FAM-labeled probe (Figure 4A). The RPA reactions were performed using 10 ng of DNA, and the fluorescence data were plotted in a graph (Figure 4B). At the final stage of the assay, samples in conical tubes were photographed using a gel imaging system (Figure 4C). For detection with a biotin-labeled probe, the samples were incubated in a thermoblock (Figure 4A); the detection solution was prepared according to the manufacturer's instructions and applied onto a lateral flow strip (Figure 4B, C). The method resulted in a successful detection of both honeybee pathogens.
To determine the efficiency of RPA reactions, we compared this method with standard qPCR using DNA purified from honeybees using the referenced kit (see the Table of Materials). A total of 32 bee samples were tested with primers targeting the β-tubulin gene of L. passim for both methods and then detected by CRISPR/Cas12a trans-cleavage of fluorescent probes. Notably, as a result, 12 samples were found to be positive by qPCR, while RPA/CRISPR/Cas12a detected L. passim in 16 samples (Figure 5A). This indicates that RPA reaction performance is more sensitive than PCR, as the gold-standard DNA amplification method.
Likewise, we determined the detection limit (LOD) for qPCR and RPA coupled to CRISPR/Cas12a detection for L. passim DNA as the pathogen model using serial dilutions of L. passim DNA ranging from 66 ng to 6 fg. qPCR detected as little as 6 pg, corresponding to approximately 96 parasites (Figure 5B). In comparison, the LOD for RPA corresponded to 96 parasites (Figure 5C, D).
<img alt="DNA detection process diagram; bee sample, alkaline lysis, RPA, CRISPR-Cas12, fluorescence results." class="xfigimg" src="/files/ftp_upload/68874/68874fig1.jpg" data-altupdatedat="1762509930000" data-alt="Figure 1"> 
Figure 1: General workflow of the HotSHOT alkaline lysis coupled with RPA amplification and Cas12 detection methods in honeybees. Abbreviations: RPA = recombinase polymerase amplification; CRISPR = Clustered Regularly Interspaced Short Palindromic Repeats; Cas = CRISPR-associated protein. <a href="https://www.jove.com/files/ftp_upload/68874/68874fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Honeybee dissection sequence: sample prep, HotShot, 95°C incubation, neutralization process." class="xfigimg" src="/files/ftp_upload/68874/68874fig2.jpg" data-altupdatedat="1762509930000" data-alt="Figure 2"> 
Figure 2: Processing of amplification-ready cell lysis with HotSHOT buffer. (A) Cut the honeybee abdomen with a scalpel and put it in a centrifuge tube; (B) with a pestle, make a maceration with 400 µL of HotShot; (C) incubate on a thermoblock at 95 °C for 10 min; and (D) neutralize with 400 µL of 40 mM Tris-HCl. <a href="https://www.jove.com/files/ftp_upload/68874/68874fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="crRNA, Cas12a enzyme, and probes preparation; diagram of master mix detection process and solutions." class="xfigimg" src="/files/ftp_upload/68874/68874fig3.jpg" data-altupdatedat="1762509930000" data-alt="Figure 3"> 
Figure 3: General workflow for the preparation of Cas12a enzyme and FAM- and Biotin-labeled reporter probe solutions. Abbreviations: DEPC-water = Diethyl Pyrocarbonate-treated water; crRNA= RNA CRISPR; Cas12 = type of CRISPR-associated protein. <a href="https://www.jove.com/files/ftp_upload/68874/68874fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="FAM vs. Biotin detection; PCR setup, amplification graph, lateral flow assay for β-tubulin analysis." class="xfigimg" src="/files/ftp_upload/68874/68874fig4.jpg" data-altupdatedat="1762509930000" data-alt="Figure 4"> 
Figure 4: Detection of Lotmaria passim and Nosema ceranae. by the CRISPR/Cas12a method with FAM and biotin probes. For FAM detection, (A) samples are being incubated in a thermocycler; (B) the relative fluorescence units (RFU´s) of L. passim and N.ceranae; (C) visualization of the same samples under a photodocumentation system. In biotin tests, (a) a sample incubating in a thermo block; (b) samples were added in an immunostrip from the kit (see the Table of Materials); (c) the results after incubation. Abbreviations: LP= Lotmaria passim; NC= Nosema ceranae; NTC= no template control; RFU: relative fluorescence units <a href="https://www.jove.com/files/ftp_upload/68874/68874fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Detection limits of PCR vs. RPA; includes qPCR and RPA graphs, plus RPA detection results." class="xfigimg" src="/files/ftp_upload/68874/68874fig5.jpg" data-altupdatedat="1762509930000" data-alt="Figure 5"> 
Figure 5: Comparison between qPCR and RPA, and detection limit of Lotmaria passim. (A) Detection of L. passim in honeybee samples using conventional qPCR and RPA. (B) qPCR detection limit of L. passim using SsoFast EvaGreen. (C) Detection limit of L. passim using CRISPR/Cas12a with a FAM-labeled probe; (D) visualization of the same CRISPR/Cas12a detection (from C) under a gel imaging system (top row), and corresponding lateral flow results using CRISPR/Cas12a with a biotin-labeled probe (bottom row). Abbreviations: RPA = recombinase polymerase amplification; qPCR= quantitative polymerase chain reaction. <a href="https://www.jove.com/files/ftp_upload/68874/68874fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Final Concentration</td>
			<td>10 µL</td>
			<td>50 µL</td>
		</tr>
		<tr>
			<td>2x Reaction Buffer</td>
			<td>1x</td>
			<td>5 µL</td>
			<td>25 µL</td>
		</tr>
		<tr>
			<td>dNTPs (10 mM)</td>
			<td>1.8 mM</td>
			<td>1.8 µL</td>
			<td>9 µL</td>
		</tr>
		<tr>
			<td>10x Basic E-Mix</td>
			<td>1x</td>
			<td>1 µL</td>
			<td>5 µL</td>
		</tr>
		<tr>
			<td>Forward Primer (10 µM)</td>
			<td>480 nM</td>
			<td>0.5 µL</td>
			<td>2.4 µL</td>
		</tr>
		<tr>
			<td>Reverse Primer (10 µM)</td>
			<td>480 nM</td>
			<td>0.5 µL</td>
			<td>2.4 µL</td>
		</tr>
		<tr>
			<td>20x Core Reaction Mix</td>
			<td>1x</td>
			<td>0.5 µL</td>
			<td>2.5 µL</td>
		</tr>
		<tr>
			<td>DEPC Water</td>
			<td>--</td>
			<td>0.7 µL</td>
			<td>3.7 µL</td>
		</tr>
	</tbody>
</table>
Table 1: Mastermix components for RPA with final volumes of 10 µL and 50 µL, and corresponding working concentrations:
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Final concentration</td>
			<td>10 µL reaction</td>
			<td>50 µL reaction</td>
		</tr>
		<tr>
			<td>280 mM MgOAc</td>
			<td>1.8 mM</td>
			<td>0.5 µL</td>
			<td>2.5 µL</td>
		</tr>
		<tr>
			<td>DNA</td>
			<td>10-0.1 ng</td>
			<td>1 µL</td>
			<td>2 µL</td>
		</tr>
	</tbody>
</table>
Table 2: Volume of reagents to be added per sample in a reaction of RPA
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>[stock]</td>
			<td>[final]</td>
			<td>1x (µL)</td>
		</tr>
		<tr>
			<td>Cas12a enzyme</td>
			<td>1 µM</td>
			<td>50 nM</td>
			<td>2.5</td>
		</tr>
		<tr>
			<td>cRNA</td>
			<td>1 µM</td>
			<td>100 nM</td>
			<td>5</td>
		</tr>
		<tr>
			<td>FAM probe</td>
			<td>10 µM</td>
			<td>250 nM</td>
			<td>1.25</td>
		</tr>
		<tr>
			<td>NEB buffer 10x</td>
			<td>10x</td>
			<td>1x</td>
			<td>5</td>
		</tr>
		<tr>
			<td>RPA amplicon</td>
			<td>-</td>
			<td>-</td>
			<td>4</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>-</td>
			<td>-</td>
			<td>32.25</td>
		</tr>
		<tr>
			<td>Total</td>
			<td></td>
			<td></td>
			<td>50</td>
		</tr>
	</tbody>
</table>
Table 3: Concentrations of reagents to make a mix of FAM detection by the method CRISPR/Cas 12a. 
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>[stock]</td>
			<td>[final]</td>
			<td>1x (µL)</td>
		</tr>
		<tr>
			<td>Cas12a enzyme</td>
			<td>1 µM</td>
			<td>50 nM</td>
			<td>2.5</td>
		</tr>
		<tr>
			<td>cRNA</td>
			<td>1 µM</td>
			<td>100 nM</td>
			<td>5</td>
		</tr>
		<tr>
			<td>Biotin probe</td>
			<td>10 µM</td>
			<td>500 nM</td>
			<td>2.5</td>
		</tr>
		<tr>
			<td>NEB buffer 10x</td>
			<td>10X</td>
			<td>1X</td>
			<td>5</td>
		</tr>
		<tr>
			<td>RPA amplicon</td>
			<td>-</td>
			<td>-</td>
			<td>4</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>-</td>
			<td>-</td>
			<td>31</td>
		</tr>
		<tr>
			<td>Total</td>
			<td></td>
			<td></td>
			<td>50</td>
		</tr>
	</tbody>
</table>
Table 4: Concentrations of reagents to make a mix of biotin detection by the CRISPR/Cas 12a method.

Here, we describe a simple and quick procedure for detecting DNA from bee pathogens, such as Lotmaria passim and Nosema ceranae, using an amplification-ready cell lysis, recombinase polymerase amplification, and CRISPR/Cas12a assays.

Early on-site detection of microscopic pathogens is essential for the effective management and health of honeybee colonies. Currently, the gold standard ...

Our main research interest focus on understanding the mechanism of transmission lifecycles, as well as improving diagnostic methods of the main bee pathogens, including trypanosomatid parasites. The advent of new hydrothermal nucleic acid techniques, which enable exponential amplification of nucleic acids at constant temperatures is making possible the detection of pathogens without the need for complex infrastructures or lab equipment. In our field of molecular target identification and selection for pathogen detection, we use isothermal RPA combined with CRISPR-Cas12a for rapid, sensitive, and portable molecular diagnostics.Current challenges include optimizing a site's sensitivity and specificity, minimizing false positive, simplifying sample preparation, and developing roles, deployable, diagnostic, adaptable to diverse pathogen and conditions. We have addressed the need for a fast, simple, and field adaptable method to detect honeybee pathogens, overcoming limitations of traditional molecular diagnostics. To begin, obtain an anesthetized honeybee.With a sterile scalpel, dissect the abdomen of the bee. Transfer the dissected tissue into a 1.5 milliliter microcentrifuge tube. Add 400 microliters of HotSHOT buffer to the tube, then use a disposable pestle to macerate the tissue thoroughly.Vortex the tube to further homogenize the tissue. Next, incubate the tube in a heating block set to 95 degrees Celsius for 10 minutes to lyse the cells. After incubation, transfer the tube immediately onto ice to cool.Neutralize the reaction with 400 microliters of 40 millimolar tris-HCL, resulting in a final concentration of 20 millimolar tris-HCL in the solution. For isothermal amplification by RPA, first prepare the master mix according to the number of reactions required. Use diethyl pyrocarbonate-treated water instead of amplification-ready cell lysis for the negative control and pathogen DNA for the positive control.Transfer 46.5 microliters of the master mix into 0.2 milliliter PCR tubes. Add magnesium acetate and template DNA to the caps of each PCR tube according to specified volumes. Briefly centrifuge the tubes to mix and initiate the RPA reaction.Then incubate the tubes in a thermocycler at 39 degrees Celsius for 40 minutes to carry out the RPA amplification reaction. Alternatively, incubate the samples using DNA-free Eppendorf tubes in a thermal block. First, prepare a 100 micromolar CRISPR RNA stock solution.Reconstitute 10 nanomoles of lyophilized CRISPR RNA in 100 microliters of diethyl pyrocarbonate-treated water. To prepare a one micromolar working solution, mix one microliter of the 100 micromolar stock with 99 microliters of diethyl pyrocarbonate-treated water in a 0.2 milliliter tube. To prepare the Cas12a enzyme solution, dilute the 100 micromolar solution to a one micromolar working solution by mixing one microliter of the enzyme with 99 microliters of the diluent provided in the kit.Now, prepare a 100 micromolar stock solution of the FAM probe by resuspending it in one microliter of diethyl pyrocarbonate-treated water per one nanomole of probe. For the 10 micromolar working solution, mix 90 microliters of DEPC-treated water with 10 microliters of the 100 micromolar stock. Next, prepare the CRISPR reaction mix, excluding the amplicons in a 1.5 milliliter microcentrifuge tube.Vortex the suspension to mix well, then distribute it into PCR tubes. Now, pipette four microliters of RPA amplicon into each tube. Place the tubes in a thermocycler set to 37 degrees Celsius and incubate for 120 minutes with fluorescence recorded every minute.Alternatively, incubate the samples using DNA-free Eppendorf tubes in a thermal block. At the end of the incubation, transfer the reactions into 0.2 milliliter tubes for visualization using a gel documentation system. To detect biotin probe in a lateral flow test, prepare the flow test components in a 1.5 milliliter tube.After vortexing the solution, distribute it into a PCR plate. Then add four microliters of amplicon to the respective wells. Incubate the PCR tubes at 37 degrees Celsius for 120 minutes using a thermocycler or thermal block without fluorescence reading.Then transfer the samples to 0.2 milliliter tubes. Then add 50 microliters of running buffer and 10 microliters of the reaction mixture to each tube. Insert ImmunoStrips and incubate for 15 minutes at room temperature.Read results after 10 minutes of immersion. Recombinase polymerase amplification detected Lotmaria passim in 16 out of 32 honeybee samples, whereas quantitative polymerase chain reaction identified only 12 positives, demonstrating higher sensitivity of the RPA method. The detection limit of quantitative polymerase chain reaction was approximately 9.6 parasites as shown by the amplification curve with the lowest visible signal.The detection limit of the recombinase polymerase amplification coupled to CRISPR-Cas12a matched that of QPCR, detecting as low as six picograms corresponding to 96 parasites.

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Research

JoVE Journal

Biology

The Trypanosoma cruzi agent of Chagas disease produces long-lasting asymptomatic infections that abruptly develop into clinically recognized pathology. The following research protocol describes a short-run family-based epidemiological study to unravel the T. cruzi infection transmitted sexually from parent to progeny.

Sexual Transmission of American Trypanosomes from Males and Females to Naive Mates

Fine Needle Aspiration is a technique, whereby cells are obtained from a lesion or organ using a thin needle. Aspirated material is smeared, stained and examined under a microscope for diagnosis or used for molecular biology, cytometry or in vitro analysis. It is cheap, simple, quick and causes minimal trauma.

Detection of Extravascular Trypanosoma Parasites by Fine Needle Aspiration

This article details a protocol for rapid identification of indels induced by CRISPR/Cas9 and selection of mutant lines in the mosquito Aedes aegypti using high-resolution melt analysis.

Indel Detection following CRISPR/Cas9 Mutagenesis using High-resolution Melt Analysis in the Mosquito Aedes aegypti

The present work describes the steps for producing ready-to-use qPCR for T. cruzi DNA detection that can be pre-loaded on the reaction vessel and stored in the refrigerator for several months.

Ready-To-Use qPCR for Detection of DNA from Trypanosoma cruzi or Other Pathogenic Organisms

In this work, a rapid, sensitive, and portable detection method for Candidatus Liberibacter asiaticus based on recombinase polymerase amplification combined with CRISPR-Cas12a was developed.

Field-Deployable Candidatus Liberibacter asiaticus Detection Using Recombinase Polymerase Amplification Combined with CRISPR-Cas12a

We present a protocol that combines recombinase polymerase amplification with a CRISPR/Cas12a system for trace detection of DNA viruses and builds portable smartphone microscopy with an artificial intelligence-assisted classification for point-of-care DNA virus detection.

DNA Virus Detection System Based on RPA-CRISPR/Cas12a-SPM and Deep Learning

Here, we present the step-by-step preparation of premixed, lyophilized recombinase-based isothermal amplification and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based reactions, which can be used for the detection of nucleic acid biomarkers of infectious disease pathogens or other genetic markers of interest.

Point-of-care CRISPR-based Diagnostics with Premixed and Freeze-dried Reagents

To facilitate the rapid and precise detection of Acinetobacter baumannii, we present a protocol that employs Recombinase Polymerase Amplification (RPA) in conjunction with LbaCas12a endonuclease for identifying A. baumannii infections.

Rapid and Specific Detection of Acinetobacter baumannii Infections Using a Recombinase Polymerase Amplification/Cas12a-based System

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.

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

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

Learn rapid DNA detection of bee pathogens using alkaline lysis with RPA and CRISPR/Cas12a. This field-adaptable method detects Lotmaria passim and Nosema ceranae with improved sensitivity over qPCR.

On-site DNA Detection of Trypanosomatid Parasites and Nosema ceranae Through Alkaline Lysis Coupled to RPA/CRISPR/Cas12a System

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