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Highly Sensitive and Rapid Detection of Citrus Huanglongbing Pathogen (‘Candidatus Liberibacter asiaticus’) Using Cas12a-Based Methods

    Affiliations
    Authors and Affiliations
    • Matthew S. Wheatley1
    • Yong-Ping Duan2
    • Yinong Yang1 †
    1. 1Department of Plant Pathology and Environmental Microbiology and Huck Institute of the Life Sciences, The Pennsylvania State University, University Park, PA 16802
    2. 2USDA/ARS U.S. Horticultural Research Laboratory, Fort Pierce, FL 34945
    Published Online:6 Dec 2021https://doi.org/10.1094/PHYTO-09-20-0443-R

    Abstract

    Citrus huanglongbing (HLB) or greening is one of the most devastating diseases of citrus worldwide. Sensitive detection of its causal agent, ‘Candidatus Liberibacter asiaticus’ (CLas), is critical for early diagnosis and successful management of HLB. However, current nucleic acid–based detection methods are often insufficient for the early detection of CLas from asymptomatic tissue and unsuitable for high-throughput and field-deployable diagnosis of HLB. Here we report the development of the Cas12a-based DNA endonuclease-targeted CRISPR trans reporter (DETECTR) assay for highly specific and sensitive detection of CLas nucleic acids from infected samples. The DETECTR assay, which targets the five-copy nrdB gene specific to CLas, couples isothermal amplification with Cas12a transcleavage of a fluorescent reporter oligonucleotide and enables detection of CLas nucleic acids at the attomolar level. The DETECTR assay was capable of specifically detecting the presence of CLas across different infected citrus, periwinkle, and psyllid samples and shown to be compatible with lateral flow assay technology for potential field-deployable diagnosis. The improvements in detection sensitivity and flexibility of the DETECTR technology position the assay as a potentially suitable tool for early detection of CLas in infected regions.

    Citrus is one of the most economically important fruit crops worldwide, valued at roughly US$17 billion from the sales of fresh fruit and juices (Arredondo Valdés et al. 2016). In recent years, extensive economic loss in citrus production has resulted from the citrus greening disease or huanglongbing (HLB), caused by phloem-limited, fastidious, gram-negative bacteria in Candidatus Liberibacter spp. (Jagoueix et al. 1994). This genus includes three HLB-associated species, ‘Candidatus Liberibacter africanus’, ‘Candidatus Liberibacter americanus’, and Candidatus Liberibacter asiaticus’ (CLas), with CLas being the most devastating and broadly distributed species worldwide (Song et al. 2017). CLas is transmitted via natural insect vectors such as citrus psyllids or through the grafting of infected tissues. To manage CLas, it is critical to use early detection technology for disease diagnosis and quarantine of infected crops and insect vectors to minimize crop loss and prevent transmission into disease-free citrus growing regions (Iftikhar et al. 2016). So far various technologies such as electron microscopy, serology, DNA probes, enzyme-linked immunosorbent assay, conventional PCR, quantitative PCR (qPCR), and canine olfactory detection have been used to diagnose and confirm infection (Arredondo Valdés et al. 2016; Gottwald et al. 2020; Ghosh et al. 2021; Zhong et al. 2018). However, it is still challenging to rapidly and reliably detect HLB at early infection stage with these techniques because of the very low titer of CLas within infected plants. Therefore, it is imperative to significantly improve early detection technologies for sensitive, accurate, and high-throughput diagnosis of CLas infection.

    Recently, the clustered regularly interspersed short palindromic repeats/CRISPR associated (CRISPR/Cas) system has been adapted as a molecular diagnostic tool outside its conventional genome editing applications (Wheatley and Yang 2021). A number of CRISPR/Cas-based detection strategies such as DETECTR, HOLMES, HOLMES version 2, SHERLOCK, and SHERLOCK version 2 have been demonstrated as point-of-care diagnostic tools for highly sensitive and specific detection of pathogen nucleic acids in biomedical samples (Chen et al. 2018; Gootenberg et al. 2017, 2018; Li et al. 2019). These CRISPR/Cas-based diagnostic methods rely on the direction of Cas12a or Cas13a by CRISPR RNA (crRNA) to target nucleic acids and the subsequent cleavage of fluorescent reporter oligonucleotides. When coupled with isothermal amplification methods such as recombinase polymerase amplification (RPA), CRISPR/Cas-based detection strategies resulted in rapid (1 to 2 h) and robust detection of nucleic acids (RNA or DNA) at the attomolar (10−18) (Chen et al. 2018; Gootenberg et al. 2017; Li et al. 2019) and zeptomolar (10−21) (Gootenberg et al. 2018) sensitivity with single-nucleotide specificity. Most recently, Cas12a-based methods have been used to detect plant RNA viruses or viroids and Magnoporthe oryzae in rice and wheat (Aman et al. 2020; Jiao et al. 2021; Kang et al. 2021; Zhang et al. 2020). Therefore, the adoption of CRISPR/Cas-based detection for phytopathogen nucleic acid should enable the rapid and sensitive diagnosis of various other plant diseases including HLB.

    In this study, we used Lachnospiraceae bacterium (Lba) Cas12a, also known as Cpf1, for highly specific and sensitive detection of CLas nucleic acids. Cas12a is a class 2 type V Cas nuclease that is capable of cleaving both single-stranded DNA (ssDNA) and double-stranded DNA and has recently been used for detection of pathogen DNAs (Chen et al. 2018; Zetsche et al. 2015). Cas12a binds to DNA sequences complementary to the crRNA spacer, adjacent to a T-rich protospacer adjacent motif (PAM, (T)TTN), to generate double-strand breaks in DNA (Fig. 1A). Upon recognition of the target sequence, the RuvC nuclease domain will create a staggered double-strand break in the target and nontarget strands, generating an overhang on the nontarget strand (Zetsche et al. 2015). The cleavage of target DNA triggers the collateral nuclease activity of Cas12a, which is capable of indiscriminate transcleavage of ssDNA substrates (Chen et al. 2018; Li et al. 2019) such as the fluorescent reporter oligonucleotide for the Cas12a-enabled detection or DNA endonuclease-targeted CRISPR trans reporter (DETECTR). By targeting the five-copy nrdB gene specific to CLas (Zheng et al. 2016), here we developed a highly sensitive and specific DETECTR assay for detection of CLas nucleic acids in various citrus, periwinkle, and insect samples.

    Fig. 1.

    Fig. 1. Diagram of ‘Candidatus Liberibacter asiaticus’ (CLas) DNA endonuclease-targeted Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) trans reporter (DETECTR) assay. A, Cas12a (Cpf1) shown with spacer (red) of CRISPR RNA (crRNA) (brown) bound to target double-stranded DNA (dsDNA) (black) proximal to protospacer adjacent motif (PAM) (blue). The RuvC nuclease domain becomes active upon correct base-pairing of spacer and target sequence, leading to the cleavage of both strands of the dsDNA target (scissors) and the generation of a 5′ staggered 5-bp overhang. After activation of Cas12a, the RuvC domain remains active with ssDNase activity (arrow). B, Diagram of CLas nrdB and conserved five-copy number region with recombinase polymerase amplification (RPA) primer location (purple), nrdB spacer (red), and PAM (blue). C, nrdB amplicon for CLas DETECTR assay with primer location, protospacer, and PAM. D, Schematic of CLas DETECTR assay, which includes DNA amplification by RPA and subsequent Cas12a detection based on trans-cleavage of ssDNA reporters.

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    MATERIALS AND METHODS

    Nucleic acid preparation and in vitro cleavage assay.

    We extracted total genomic DNAs from CLas-infected plant or psyllid tissues by using the cetyltrimethylammonium bromide/column purification method as described previously (Zhou et al. 2011). The nrdB target DNA fragment was amplified by PCR from the CLas-containing genomic DNA with a pair of specific primers (nrdB RPA F1, CAT GCG AGA TGA ATC ACT GCA TCT CAA and nrdB RPA R3, ACC GAT TTG GTG ACA ACG ATT GGC G; also see Table 1) and cloned into the pGEM T-Easy vector system (Promega, WI). To synthesize the crRNA targeting nrdB, we generated the partially double-stranded DNA template by annealing the T7 promoter sequence (T7 in vitro transcription [IVT] F: TAA TAC GAC TCA CTA TAG) with the nrdB crRNA oligonucleotide (nrdB IVT: TCG AAA TCG CCT ATG CAC ATA TCT ACA CTT AGT AGA AAT TAC TAT AGT GAG TCG TAT TA). IVT by T7 RNA polymerase was performed with the HiScribe T7 High Yield RNA Synthesis Kit (NEB, MA) based on the manufacturer’s instruction. The resulting crRNA was purified with the RNA Clean & Concentrator kit (ZYMO Research, CA). In vitro cleavage assay was performed at 37°C for 1 h in 20 µl of 1× NEB 2.1 reaction buffer with 50 nM nrdB target amplicon, 50 nM En Gen Lba Cas12a (NEB), and 50 nM nrdB crRNA. The reaction products were treated with proteinase K at room temperature for 10 min before being run on agarose gel for analysis. In addition, two fluorescent reporter oligonucleotides (5′-56-FAMN/TTA TT/3IABkFQ/-3′ and 5′-56-FAMN/TTA TT/3Bio/-3′) were synthesized by IDT (Coralville, IA). 56-FAMN/TTA TT/3IABkFQ/ is conjugated with fluorescein (FAM) and fluorescence quencher (FQ) for the fluorescence microplate reader, and FAMN/TTA TT/3Bio contains both FAM and biotin modifications for lateral flow assay.

    TABLE 1. List of oligonucleotide primers used in this study

    Cas12a detection of CLas target nrdB.

    Cas12a detection of nrdB target plasmid was performed with preincubated Cas12a detection components consisting of 500 nM EnGen Lba Cas12a (NEB, MA), 625 nM in vitro transcribed crRNA, and 500 nM custom fluorescent reporter (56-FAMN/TTA TT/3IABkFQ) at 37°C for 30 min before the addition of target DNA. Collateral detection was initiated by adding a series of diluted plasmid DNAs in 20 μl of NEB 2.1 buffer to a final concentration of 50 nM Lba Cas12a, 62.5 nM crRNA, and 50 nM 56-FAMN/TTA TT/3IABkFQ. Reactions were incubated at 37°C in the Synergy H1 BioTEK microplate reader for 60 or 120 min time courses with fluorescence detection (excitation 495 nm, emission 535 nm) occurring every 2 min.

    CLas nrdB DETECTR assay.

    The DETECTR assay combines RPA with a TwistAmp nfo assay kit (TwistDx, UK) with the Cas12a detection as described in a recent study (Zhang et al. 2020). The RPA assay was performed in a 20-μl reaction (TwistDx Liquid Basic) consisting of 50 ng of genomic DNA, 0.48 μM forward and reverse primers, RPA enzyme mixes, and reaction buffer. The reaction was initiated with the addition of 14 nM magnesium acetate and incubated at 37°C for 10 min. Subsequent Cas12a detection was performed at 37°C for 1 h in a Synergy H1 BioTEK microplate reader after 2 μl of RPA reaction product was added into the preincubated Cas12a components. The detection values from the CLas-infected samples were normalized to the maximum mean fluorescent signal observed with the crRNA targeting the five-copy number nrdB target. A one-way ANOVA and Dunnett’s posttest were used to determine the positive value cutoff (P ≤ 0.05) for the identification of CLas from infected samples.

    qPCR assay of CLas nrdB.

    qPCR was performed with TempAssure 0.1-ml PCR tube strips (USA Scientific) with optical caps in a real-time thermal cycler (Bio-Rad CFX96). Each tube contains a 20-μl reaction mixture, which includes the GoTaq qPCR Master Mix (Promega), a series of diluted nrdB plasmid DNAs (identical to the ones used in the Cas12a DETECTR assay), and the same primer pair (F1 and R3) used in the RPA assay. Thermal cycling reactions consisted of 2 min at 95°C followed by 40 cycles of 15 s at 95°C and 1 min at 60°C.

    Lateral flow assay.

    The CLas nrdB DETECTR reaction was performed as described previously with the RPA reaction products and Cas12a detection components containing the 5′-56-FAMN/TTA TT/3Bio/-3′ fluorescent reporter. Lateral flow assay was carried out according to the HybriDetect protocol (Milenia Biotec, Germany). The HybriDetect assay buffer (100 μl) was added to the DETECTR reaction and incubated at room temperature for 5 min. A lateral flow strip was dipped into the reaction mix for 5 to 10 min for visual detection.

    RESULTS

    Optimization of RPA for specific amplification of CLas nrdB target.

    To use this technology for specific and sensitive detection of CLas, we chose to target the nrdB gene, which contains five copies of a conserved region within the CLas genome that has previously been reported to provide robust detection of CLas via qPCR from symptomatic tissues (Zheng et al. 2016). Specific pairs of primers for RPA were designed to amplify the nrdB fragment across all five copies. To enable Cas12a-based detection, a crRNA specific to nrdB target site was designed and synthesized via IVT. Figure 1B and C depict the conserved region of the nrdB gene and the location of the crRNA spacer, PAM, and primers used in this study. A schematic of the CLas DETECTR assay that combines isothermal amplification (RPA) with Cas12-based detection is shown in Figure 1D.

    To ensure optimal amplification of the nrdB target from the genomic DNA, specific primer combinations were tested and evaluated in the RPA assay with a CLas-positive sweet orange DNA sample (SO1) and a CLas-free citrus DNA sample. Of 11 RPA primer pairs screened, three showed high specificity and amplicon yield (Fig. 2A). The RPA F1/RPA R3 primer combination was chosen for subsequent RPA of the nrdB target based on the best amplicon yield and specificity observed.

    Fig. 2.

    Fig. 2. Evaluation of recombinase polymerase amplification (RPA) primers and in vitro DNA cleavage efficiency. A, RPA testing of primer combinations for nrdB target amplification from ‘Candidatus Liberibacter asiaticus’ (CLas)-positive citrus genomic DNA. Each primer combination was used to amplify the nrdB target from SO1, a CLas-positive sweet orange DNA sample. The RPA reaction proceeded for 10 min at 37°C. After column purification, the DNA concentration was measured before being run on 1.5% Tris-acetate-EDTA agarose gel. Lane 1, RPA F1/RPA R1; Lane 2, RPA F1/RPA R2; Lane 3, RPA F1/RPA R3. Molecular weight size marker was included in the form of a 100-bp DNA ladder. B, In vitro cleavage of nrdB target fragment (amplified with the RPA F1/RPA R3 primer pair) by Cas12a and nrdB crRNA. D, digested by Cas12a and nrdB CRISPR RNA, UD, undigested.

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    Cas12a detection of nrdB target and assay sensitivity of DETECTR versus qPCR.

    Before proceeding with Cas12a detection, in vitro cleavage of nrdB target DNA was tested with the nrdB crRNA and Lba Cas12a nuclease. As shown in Figure 2B, the 247-bp target DNA was specifically and efficiently cleaved to produce two predicted fragments. To test the sensitivity and specificity of Cas12a-based detection, the nrdB plasmid was diluted at various molar concentrations and incubated with the Cas12a detection components for 2 h at 37°C. A nontarget plasmid was used as a negative control. A Synergy H1 fluorescent microplate reader was used to report the accumulation of fluorescent signal production created by the activation of Cas12a and recognition of the nrdB target within a 384-well fluorescent microplate. As shown in Figure 3, Lba Cas12a was capable of detecting the DNA target reliably, down to 1 nM nrB plasmid. To determine the sensitivity of the CLas DETECTR assay, which combines Cas12a detection with RPA, the nrdB plasmid was further titrated to various molar concentrations (mM to aM) and used as a standard. A comparison between Cas12a detection alone and the DETECTR assay of the nrdB plasmid is shown in Figure 4A. For the DETECTR assay series, a 10-min RPA was performed before the 1-h collateral detection via Cas12a for all samples, whereas the Cas12a-only series omitted the initial RPA portion. By itself, Cas12a was capable of successful detection at the nanomolar level. When coupled with the RPA, the CLas DETECTR assay reached attomolar sensitivity. With the same primers and nrdB plasmid dilution series, however, real-time qPCR was capable of detecting the nrdB DNA only at 1.0 or at most 0.1 fM levels (Fig. 4B), which is 100 or possibly 1,000 times less sensitive than the Cas12a-based DETECTR assay.

    Fig. 3.

    Fig. 3. Cas12a-based detection of target DNA versus nontarget plasmid. Cas12a detection of nrdB plasmid at known concentrations compared to 100 nM nontarget plasmid (NC). Error bars represent mean ± SD when n = 3 replicates.

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    Fig. 4.

    Fig. 4. Detection of ‘Candidatus Liberibacter asiaticus’ (CLas) nrdB DNA with Cas12a alone, DNA endonuclease-targeted CRISPR trans reporter (DETECTR), and quantitative PCR (qPCR). A, Sensitivity of DETECTR versus Cas12a without recombinase polymerase amplification. Note that the CLas DETECTR assay enables robust detection of CLas nucleic acids at attomolar sensitivity. Error bars represent mean ± SD when n = 3 replicates. B, Sensitivity of real-time qPCR for nrdB DNA detection. Error bars represent mean ± SD when n = 3 replicates. qPCR experiments were performed three times with similar results. NC, negative control; ND, not detected.

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    Specific detection of CLas from infected samples via the DETECTR assay.

    After evaluating its sensitivity and specificity, we used the CLas DETECTR assay to detect and confirm the presence of CLas nucleic acids in the infected citrus, periwinkle, and psyllid samples collected from Florida. A total of 28 DNA samples from various species (sweet orange, pumelo, grapefruit, periwinkle, and psyllid) were tested along with a negative control (uninfected ‘Duncan’ grapefruit sample). As expected, the CLas DETECTR assay confirmed that all 26 samples were positive for CLas compared with the negative control (Fig. 5). Interestingly, two grapefruit DNA samples (GFTN1 and GFTN2) were previously tested by qPCR to be CLas negative. However, they showed weak positivity of CLas in the DETECTR assay.

    Fig. 5.

    Fig. 5. Detection of ‘Candidatus Liberibacter asiaticus’ (CLas)-infected samples via DNA endonuclease-targeted CRISPR trans reporter (DETECTR) assay. Detection of CLas was performed on 28 DNA samples from sweet orange (orange), pumelo (green), grapefruit (yellow), periwinkle (purple), psyllid (gray). The grapefruit samples GFTN1 and GFTN2 were potentially infected but showed negative for CLas based on quantitative PCR detection. Genomic DNAs extracted from uninfected grapefruit cultivar Duncan (NC1) and CLas-free psyllids (NC2) were used as negative controls. Error bars indicate the mean of the highest fluorescence value over 1 h ± SD when n = 3 replicates. A two-tailed t test was done to provide P values of infected samples compared with control, where *P ≤ 0.01 and **P ≤ 0.001.

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    Lateral flow compatibility for the CLas DETECTR assay.

    In addition to fluorescence detection with a microplate reader, we used lateral flow strips (HybriDetect, Milenia Biotec) for a visual and semiquantitative DETECTR assay that will probably facilitate in-field detection of CLas. Two samples from each plant or insect species, along with various 1,000-fold dilutions of the standard, were run on lateral flow strips to determine the presence of CLas (Fig. 6). We modified the CLas DETECTR assay for the lateral flow strip assay by using the ssDNA reporter oligonucleotide that contained 5′-FAM and 3′-biotin modifications, enabling the reaction product to migrate to the test band on an immunostrip after cleavage of the ssDNA reporter oligonucleotide by the activated Cas12a. All infected samples tested showed positive detection of CLas on the lateral flow strips (Fig. 6). The use of diluted samples from the nrdB plasmid standard indicated the attomolar sensitivity for the lateral flow assay.

    Fig. 6.

    Fig. 6. Lateral flow reading from ‘Candidatus Liberibacter asiaticus’ DNA endonuclease-targeted CRISPR trans reporter assay. Lateral flow strips were incubated for 10 min in the hybridization product before the photo was taken. Both serial dilution of the nrdB plasmid (100 aM, 10 aM, 1 aM, and negative control 0) and genomic DNAs from the infected samples were tested with the lateral flow assay. The lateral flow strip is comprised of three segments, the sample application pad, the streptavidin band (control), and the antibody capture band (sample). Following sample application, the intact oligonucleotide reporter will be bound by streptavidin band. Anti-fluorescein antibodies labeled with gold nanoparticles will bind to the fluorescein end of the reporter resulting in the generation of a dark purple color. When the oligonucleotide reporter is cleaved in the presence of target DNA due to collateral activity, the gold nanoparticle labeled antibodies continue to flow over to the antibody capture band (sample), forming a dark purple color at the second location. GFT, grapefruit; NC1, negative control 1 (uninfected grapefruit DNA); NC2, negative control 2 (uninfected psyllid DNA); PM, pumelo; PSY, psyllid; PW, periwinkle; SO, sweet orange.

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    DISCUSSION

    This study reports the development and validation of Cas12a-based diagnostics for sensitive and specific detection of CLas by targeting the conserved region of its nrdB gene. Previously, the nrdB gene was used to detect CLas nucleic acids via qPCR (Zheng et al. 2016) and was reported to increase CLas detection sensitivity threefold compared with the 16S rRNA gene (Fujikawa and Iwanami 2012) because of the higher copy number of the conserved nrdB region and was twice as stable as the intragenic prophage sequences (Morgan et al. 2012). Following this premise for increased sensitivity, we designed specific primers and a crRNA based on the nrdB region for highly sensitive and specific detection of CLas nucleic acids. In combination with RPA, the Cas12a-based DETECTR assay achieved attomolar sensitivity for detection of CLas DNAs (Fig. 4A). By contrast, we were able to achieve only 1.0 or at most 0.1 fM sensitivity for the nrdB DNA detection in our real-time qPCR experiments (Fig. 4B). Therefore, the Cas12a DETECTR assay is 100 or even 1,000 times more sensitive than the qPCR for detecting CLas DNA. Other techniques that boast early detection capabilities of CLas use specialized instrumentation for volatile organic compounds analysis along with in situ stains to visualize leaves and locations of concentrated CLas for direct confirmation or to increase the sensitivity of nucleic acid diagnosis approaches (Pandey and Wang 2019; Arredondo Valdés et al. 2016). These techniques show great promise, but they may have limited feasibility for rapid and high-throughput diagnosis of CLas infection. Although further testing and optimization are needed, the Cas12a-based DETECTR assay will lead to more sensitive and rapid detection of CLas nucleic acid and probably facilitate in-field diagnosis of HLB.

    Other techniques of isothermal amplification such as loop-mediated isothermal amplification (LAMP), strand displacement amplification, and nicking enzyme amplification reaction can be used as a substitute for RPA in CRISPR/Cas-based detection of pathogen nucleic acids (Keremane et al. 2015; Li et al. 2019; Rigano et al. 2014), which are also commonly used with lateral flow technology. The HOLMES version 2 platform uses LAMP before Cas12b-based detection (Li et al. 2019). Once activated, Cas12b also exhibits general ssDNase activity, which could pose problems for ssDNA amplicon intermediates during LAMP that could be destroyed. However, Cas12b used in HOLMES version 2 exhibits a higher rate of transactivation, or reporter degradation, as compared with Cas12a (Li et al. 2019; Teng et al. 2019), which could result in a shorter assay with higher sensitivity, in turn producing a faster diagnostic result.

    The DETECTR assay can truly shine when used to detect pathogens that exist in low titers such as CLas, phytoplasmas, and some viruses, where qPCR and quantitative real-time PCR results can be inconclusive because of their limit in detection. However, other pathogens that are more abundant may also benefit from the Cas12a-based detection that omit the initial RPA step. Because of the sufficient sensitivity of LbaCas12a, as shown by Chen et al. (2018) (10 pM) and this study (1 nM), Cas12a detection alone may be suitable for detection of many pathogens based on the recognition of nucleic acid sequences from total DNAs of infected or asymptomatic tissues. Omitting the RPA portion of the reaction reduces the cost, as shown by Gootenberg et al. (2018). Recently, another sensitive Cas nuclease–based detection system, CRISPR-Chip, has been reported to detect target genes without amplification down to 1.7 fM via immobilized dCas9 on a graphene transistor (Hajian et al. 2019). These methods may hold promise in rapid detection by omitting amplification of target nucleic acids.

    Currently, Cas12a-based detection methods such as DETECTR are limited in multiplexing capabilities. Because ssDNase activity is nonspecific after the activation of Cas12a, it would not be possible to distinguish which reporter was degraded between Cas12 homologs used in the same reaction. SHERLOCK version 2, a CRISPR/Cas-based detection platform that uses both Cas12a and Cas13a, is capable of multiplexed pathogen detection because of the dinucleotide preferences in the ssRNase activity of Cas13a (Gootenberg et al. 2017). Each ssRNA reporter for Cas13a contained its own specific fluorophore that was detectable in a microplate reader, indicating which nuclease became activated based on the fluorescent readings. Only a single Cas12a can be used in this reaction because of the nonspecific nature of the ssDNase activity after Cas12a activation. SHERLOCK version 2 has demonstrated the ability to simultaneously detect four different pathogens in a single reaction, creating a platform for highly sensitive and rapid multiplexed pathogen detection in point-of-care diagnostics (Chen et al. 2018; Gootenberg et al. 2018; Li et al. 2019).

    Together these results show that CRISPR/Cas-based detection strategies can improve on current HLB diagnostics by providing specific and highly sensitive detection of CLas nucleic acids. The CLas DETECTR assay can detect the nrdB target down to the attomolar level in both microplate reader and lateral flow strip assays. In addition, we have shown that the CLas DETECTR assay has the potential to be used in field diagnostics to rapidly detect CLas from infected samples by using lateral flow strips or portable endpoint fluorometers. The improvement in detection sensitivity and ease of use compared with traditional methods such as qPCR position the CLas DETECTR assay as a promising tool for HLB diagnostics in CLas-infected regions.

    The author(s) intend to file a U.S. patent application.

    LITERATURE CITED

    Funding: This work was supported by the USDA National Institute of Food and Agriculture and Hatch Appropriations under project PEN04659 and accession 1016432.

    The author(s) intend to file a U.S. patent application.