A subscription to JoVE is required to view this content. Sign in or start your free trial.
* These authors contributed equally
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.
The early detection of Candidatus Liberibacter asiaticus (CLas) by citrus growers facilitates early intervention and prevents the spread of disease. A simple method for rapid and portable Huanglongbing (HLB) diagnosis is presented here that combines recombinase polymerase amplification and a fluorescent reporter utilizing the nuclease activity of the clustered regularly interspaced short palindromic repeats/CRISPR-associated 12a (CRISPR-Cas12a) system. The sensitivity of this technique is much higher than PCR. Furthermore, this method showed similar results to qPCR when leaf samples were used. Compared with conventional CLas detection methods, the detection method presented here can be completed in 90 min and works in an isothermal condition that does not require the use of PCR machines. In addition, the results can be visualized through a handheld fluorescent detection device in the field.
Huanglongbing (HLB) is one of the most problematic citrus diseases worldwide1. HLB is caused by the phloem-colonizing and fastidious bacteria Candidatus Liberibacter spp., including Candidatus Liberibacter asiaticus (CLas), Ca. L. africanus, and Ca. L. americanus2. The most prevalent HLB-associated species in China and the USA is CLas, which is transmitted by Asian citrus psyllids (Diaphorina citri) or through grafting3. After being infected by CLas, citrus trees demonstrate growth decline until death2. The common symptoms of citrus leaves infected with CLas are blotchy mottle, green islands (small circular dark green dots), raised corky veins on thicker and leathery leaves, and nonuniform yellowing shoots2. In addition, fruits infected with CLas appear small and lopsided2.
Since no citrus variety is resistant to HLB and there is no therapeutic cure for HLB, the prevention of HLB requires the quarantine and isolation of CLas-positive citrus trees2,3. Therefore, early detection is critical for monitoring and quarantine to prevent the spread of CLas and minimize economic losses3. In addition, sensitive CLas detection is needed due to the low titer of CLas in plants during the early stage of infection3. In China, CLas detection is usually conducted by certain certified test centers. However, the detection process usually takes at least 1 week, and the detection fee is expensive. Therefore, to help monitor the HLB incidence
Various technologies have been applied to diagnose HLB4,5,6,7,8,9. Polymerase chain reaction (PCR) and quantitative PCR (qPCR) are the most used tools for CLas detection due to their high sensitivity and specificity4,5. However, those technologies rely heavily on expensive instruments and highly skilled personnel. In addition, several isothermal amplification methods, such as loop-mediated isothermal amplification (LAMP), have been developed as attractive alternatives to conventional PCR methods due to their simplicity, rapidity, and low cost8,9,10. However, it is challenging to apply them to accurately detect CLas due to the non-specific amplification signals, which may cause false-positive results.
RNA-guided CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) endonuclease-based nucleic acid detection has been developed as a next-generation molecular diagnostics technology owing to its high sensitivity, specificity, and reliability11,12,13,14. These CRISPR/Cas diagnostics technologies rely on the collateral nuclease activity of Cas proteins to cleave single-stranded DNA (ssDNA) modified with a fluorescent reporter and a fluorescence quencher at each end of the oligonucleotides, as well as a fluorescence detection device to capture the released fluorescent reporter11,12. The nuclease activity of several Cas effectors activated by the CRISPR RNA (crRNA) target duplex can indiscriminately cleave the surrounding non-target ssDNA11. CRISPR-Cas12a (also called Cpf 1), a class 2 type V-A CRISPR/Cas system, demonstrates several advantages compared with Cas9, such as a lower mismatch tolerance and greater specificity13. The Cas12a/crRNA system has been applied for the sensitive and specific detection of the nucleic acids of human pathogens and phytopathogens14,15,16,17,18. Therefore, utilizing the Cas12a/crRNA system should enable the accurate and sensitive detection of the nucleic acid of CLas.
Cas12a alone is not theoretically sensitive enough to detect low levels of nucleic acids. Therefore, to improve its detection sensitivity, CRISPR-Cas12a detection is typically combined with an isothermal amplification step14,15. Recombinase polymerase amplification (RPA) enables sensitive and rapid isothermal DNA amplification in a temperature range from 37 °C to 42 °C19.
A detection platform called DETECTR (DNA Endonuclease Targeted CRISPR Trans Reporter) that combines the DNase activity of Cas12a with RPA and a fluorescence readout has been recently devised12 and has been shown to detect nucleic acid with higher sensitivity20. Furthermore, the fluorescence signal emitted from the positive samples can be observed through a handheld fluorescence detection device in the field.
Since we amplified DNA with RPA, designed crRNA targeting the five-copy nrdB (ribonucleotide reductase β- subunit) gene specific to CLas21, and employed the DNase activity of the Cas12a protein, we called this CLas detection method CLas-DETECTR. Compared with existing CLas detection methods, CLas-DETECTR is fast, accurate, sensitive, and deployable.
1. Construction of the CLas-DETECTR
NOTE: The construction of CLas-DETECTR is a four-step process: solution preparation, citrus total DNA isolation, isothermal DNA amplification, and result visualization. The schematic of the CLas-DETECTR assay is illustrated in Figure 1A.
2. Specificity test
NOTE: To test the specificity of CLas-DETECTR, the rhizosphere bacterium Agrobacterium tumefaciens GV3101, Xanthomonas citri subsp. citri (Xcc), and Burkholderia stabilis strain 1440 isolated in the lab24 were subjected to the CLas-DETECTR test.
NOTE: Candidatus Liberibacter (CLas) cannot be cultured. One can only obtain CLas DNA from the extraction of genomic DNA from citrus tissues infected with CLas. Xcc is the causal agent of another important citrus disease, citrus canker. Burkholderia stabilis strain 1440 is an anti-Xcc bacterium isolated in the lab. Therefore, pure strains for all these three bacteria were used
3. Sensitivity comparison
4. Sample detection
NOTE: After the specificity and sensitivity test, the CLas-DETECTR method was used to detect the presence of CLas in the field leaf samples collected from Newhall sweet orange trees grown in the germplasm resource nursery on the campus of Gannan Normal University, Jiangxi, China. A qPCR was performed to verify the results.
Here, we have described a portable platform, CLas-DETECTR, combing the RPA and CRISPR-Cas12a systems to diagnose HLB in the field. The schema of CLas-DETECTR is illustrated in Figure 1A.
When leaf samples from the HLB-infected and HLB-uninfected Newhall trees (Figure 1B), for which the presence of CLas was confirmed by PCR (Figure 1C), were subjected to the CLas-DETECTR test, a green fluorescence signal w...
This study presents a rapid and portable method to detect CLas named CLas-DETECTR, which combines the RPA and CRISPR-Cas12a systems. The workflow is illustrated in Figure 1. CLas-DETECTR detects CLas with specificity and sensitivity (Figure 2 and Figure 3). Furthermore, using Newhall leaf samples, CLas-DETECTR detects CLas with the same sensitivity as qPCR (Figure 4). Notably, the detec...
The authors declare that they have no competing interests.
This work was financially supported by the National Key R & D Program of China (2021YFD1400805), the Major Science and Technology R & D Program of Jiangxi Province (20194ABC28007), Projects of Jiangxi Education Department (GJJ201449), and the Collaborative Innovation of Modern Agricultural Scientific Research in Jiangxi Province (JXXTCX2015002(3+2)-003).
Name | Company | Catalog Number | Comments | |
AxyPrep DNA Gel Extraction Kit | Corning | 09319KE1 | China | |
Bacterial Genomic DNA Extraction Kit | Solarbio | D1600 | China | |
EnGen LbCas12a | TOLOBIO | 32104-01 | China | |
Ex Taq Version 2.0 plus dye | TaKaRa | RR902A | China | |
Handheld fluorescent detection device | LUYOR | 3415RG | China | |
Hole puncher | Deli | 114 | China | |
Magnesium acetate, MgOAc | TwistDx | TABAS03KIT | UK | |
NaOH | SCR | 10019718 | China | |
NEB buffer 3.1 | NEB | B7203 | USA | |
PCR strip tubes | LABSELECT | PST-0208-FT-C | China | |
PEG 200 | Sigma | P3015 | USA | |
PrimeSTAR Max DNA Polymeras | TaKaRa | R045A | China | |
Quick-Load Purple 1 kb Plus DNA Ladder | New England Biolabs | N0550S | USA | |
TB Green Premix Ex Taq II (Tli RNaseH Plus) | TaKaRa | RR820B | China | |
TwistAmp Basic | TwistDx | TABAS03KIT | UK |
Request permission to reuse the text or figures of this JoVE article
Request PermissionExplore More Articles
This article has been published
Video Coming Soon
Copyright © 2025 MyJoVE Corporation. All rights reserved