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

Huang long bing, abbreviated as HLB. It's a devastating citrus disease worldwide. In the most citrus-producing countries, such as China and the USA, HLB is caused by the phloem-restricted bacteria Candidatus Liberibacter asiaticus, abbreviated as CLas.

Early detection of CLas were important for growers facilitating early intervention and preventing disease spread. So here we developed a new method for the rapid and portable HLB diagnosis, combining with the recombinase polymerase amplification and CRISPR-Cas12a system. We called it CLas-DETECTR.

The sensitivity of our platform is much higher than PCR. Furthermore, it shows similar result with qPCR when the leaf samples was used. Compare with the conventional CLas detections, our method can be finished in 90 minutes, and works in an isothermal condition.

The result also can be visualized through a handheld fluorescent detection device in the field. The CLas-DETECTR contains four steps:solution preparation, citrus total DNA isolation, isothermal DNA amplification, and the results of visualization. The schematic of the CLas-DETECTR assay is illustrated here.

Buffer A, Solution B, and Solution C are prepared as described in the protocol. To save time and make this method suitable for field CLas detection, the E.coli polyethylene glycol-based approach were used to obtain crude plant extracts for DNA amplification. Citrus leaves are used in the tool.

Punch the few pieces of leaf discs from leaf. Put leaf discs into a 1.5 milliliter Eppendorf tube. Then, add 200 microliter of Buffer A into the tube.

Grind the leaf discs until smooth with a plastic rod manually. Leave the tube undisturbed for 10 minutes. The supernatant is subsequently applied to DNA amplification.

For isothermal DNA amplification, add one microliter of supernatant from step 2.3 and one microliter of magnesium acetate into Solution B and mix well. In the lab, incubate them in a simple incubator at 37 degree for 15 minutes. To visualize the results, add 10 microliter of the mixture from step 3.2 into Solution C and mix well.

Incubate them in the 37 degree incubator for 60 minutes. Wear a goggle, and observe the green fluorescence signal through a handheld fluorescent detection device. To test the specificity of CLas-DETECTR, first pure colonies of Agrobacterium tumefaciens GV3101, Xanthomonas citri subsp.

citri which is the pathogen of the citrus canker and Burkholderia stabilis strain 1440 isolated in our lab were used to extract their genomic DNA. Then this three bacteria genomic DNA and the CLas-positive Newhall leaves genomic DNA were subjected to the CLas-DETECTR test. To test the sensitivity of CLas-DETECTR, a series of CLas DNA fragment dilutions were detected using PCR, CLas-DETECTR, and qPCR as described in the protocol After the specificity and the sensitivity test, the CLas-DETECTR method was used to detect the presence of the CLas in field.

Leaf samples collected from Newhall sweet orange tree grown in the germplasm resource nursery on the campus of the Gannan Normal University, Jiangxi, China. qPCR, a convincing CLas detection method usually used in the lab was also applied. The leaf samples from HLB-infected and HLB-uninfected Newhall tree, in which the presence of the CLas was confirmed in our lab, were subjected to the CLas-DETECTR test.

The green fluorescence signal was captured in the HLB-infected sample, but not in the HLB-uninfected and negative control. We determined the specificity of CLas-DETECTR using nucleic acids extracted from other bacteria. In the CLas-DETECTR assay, only gDNA extracted from CLas-positive Newhall leaves demonstrated green fluorescent signals.

gDNA extracted from Agrobacterium tumefaciens GV3101, Xanthomonas citri subsp. citri and Burkholderia stabilis strain 1440 did not, which means the CLas-DETECTR can detect CLas specifically. We also tested the sensitivity of CLas-DETECTR using a series of CLas DNA dilutions, listed in the protocol.

PCR can detect 201 copies per megaliter. On the other hand, CLas-DETECTR can detect 2.01 copies per megaliter, suggesting its availability as a sensitive field diagnostic. qPCR can detect 0.201 copies per megaliter and the Ct value is higher than 36.

So CLas-DETECTR is two orders of magnitude more sensitive than traditional PCR and one order of a magnitude less sensitive than qPCR when diluted CLas specific amplicons was used. Finally, reexamined the feasibility of the CLas-DETECTR for field samples, and compared its sensitivity with qPCR. In figures, we see.

We can see that the Ct value is higher than 36 when the CLas concentration is less than 0.2 copies per megaliter, which is a very low concentration. Among the 15 samples, the Ct value of the samples 1, 2, 3, 4, 8, 10, 13 and 14 detected by qPCR was less than 36. An apparent green fluorescence signals was observed.

On the other hand, when the Ct value of the samples 6, 7, 9, 12, and 15 detected by qPCR was undetermined, no green fluorescent signals was observed. In addition, weak green fluorescent signals was observed when the CT value of sample 5 and 11 detected by qPCR was higher than 36. The results will suggest that our platform is a rapid, robust and sensitive tool for HLB diagnosis in the field.