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* 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 cit....
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.
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.......
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 3.1Â | NEB | B7203 | USA | |
PCR strip tubes | LABSELECT | PST-0208-FT-C | China | |
PEG 200Â | 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 |
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