key: cord-0014267-3pkb2hl2
authors: Ghosh, Dilip Kumar; Kokane, Sunil B.; Gowda, Siddarame
title: Development of a reverse transcription recombinase polymerase based isothermal amplification coupled with lateral flow immunochromatographic assay (CTV-RT-RPA-LFICA) for rapid detection of Citrus tristeza virus
date: 2020-11-26
journal: Sci Rep
DOI: 10.1038/s41598-020-77692-w
sha: ab80f364f630a7878546cfd194eb6f0ec7d665d9
doc_id: 14267
cord_uid: 3pkb2hl2

Tristeza is a highly destructive disease of citrus caused by the phloem-limited, flexuous filamentous Citrus tristeza virus (CTV) in the genus Closterovirus and the family Closteroviridae. It has been a major constraint for higher productivity and has destroyed millions of citrus trees globally. CTV is graft transmissible and spread through use of virus infected nursery plants. Therefore, virus detection by using specific and reliable diagnostic tools is very important to mitigate disease outbreaks. Currently, the standard molecular techniques for CTV detection include RT-PCR and RT-qPCR. These diagnostic methods are highly sensitive but time consuming, labor intensive and require sophisticated expensive instruments, thus not suitable for point-of-care use. In the present study, we report the development of a rapid, sensitive, robust, reliable, and highly specific reverse transcription-RPA technique coupled with a lateral flow immunochromatographic assay (CTV-RT-RPA-LFICA). RT-RPA technique was standardized to amplify the coat protein gene of CTV (CTV-p25) and detect double labeled amplicons on a sandwich immunoassay by designing specific labeled primer pair and probe combinations. The optimally performing primer set (CTRPA-F1/CTRPA-R9-Btn) and the corresponding TwistAmp nfo probe (CTRPA-Probe) was optimized for temperature and reaction time using purified cDNA and viral RNA as template. The sensitivity of the developed assay was compared with other detection techniques using in vitro-transcribed RNA. The efficacy and specificity of the assay was evaluated using CTV positive controls, healthy samples, field grown citrus plants of unknown status, and other virus and bacterial pathogens that infect citrus plants. The RT-RPA-LFICA was able to detect ≤ 141 fg of RNA when cDNA used as a template. The assay detected ≤ 0.23 ng/µl of CTV RNA when directly used as template without cross-reactivity with other citrus pathogens. Best results were achieved at the isothermal temperature of 40 °C within 15–20 min. The study demonstrated that RT-RPA-LFICA has potential to become an improved detection technique for end users in bud-wood certification and quarantine programs and a promising platform for rapid point-of-care diagnostics for citrus farmers and small nurseries in low resource settings.

| (2020) 10:20593 | https://doi.org/10.1038/s41598-020-77692-w www.nature.com/scientificreports/ most destructive epidemics occurred in Argentina, California, Brazil, Florida, Spain, Israel, and Venezuela 4 . Phloem-limited long flexuous filamentous virions of CTV (2000 × 11 nm) contained single stranded plus sense RNA of approximately 19.3 kb and organized into 12 ORFs (with UTRs at the 5′ and 3′ termini) that potentially encode at least 19 proteins [3] [4] [5] [6] . Different aphid species, Aphis gossypii Glover, Aphis (Toxoptera) citricidus Kirkaldy and Aphis spiraecola Patch act as vectors and transmit CTV from infected to healthy plants in citrus groves in a semi-persistent manner 3, 7 . Consequently, reduced production and fruit quality and increase in disease severity result in citrus decline. The symptoms of Tristeza (stem pitting, vein clearing, vein flecking, stunting, slow decline, and quick decline) often were mistaken with other diseases and nutrient deficiency 8 . CTV, similar to another major citrus pathogen 'Candidatus Liberibacter spp. ' (causal agent of citrus greening/HLB), is graft transmissible and spread to other areas by propagation of infected buds. Therefore, dispersal of this pathogen could be reduced by use of healthy propagation material. Indexing of mother-plants using specific and reliable molecular detection techniques in the nursery would be an important step to prevent its spread 9 .

In addition to biological indexing, many serological and molecular techniques have been employed for the detection of CTV such as, enzyme-linked immunosorbent assays (ELISA), dot immunobinding assays (DIBA), RT-PCR, RT-qPCR, electron microscopy, and Loop-mediated isothermal amplification (LAMP) [10] [11] [12] [13] [14] [15] [16] . These methods (excluding LAMP), have certain limitations inherently i.e. time consuming, labor intensive, require sophisticated expensive equipments, specific technical expertise and are not able to be used at point-of-care. Some disadvantages of LAMP include need for high temperature (65 °C), more amplification time, susceptible to carryover contaminations, and complex in primers design [17] [18] [19] [20] . During development of any new detection method, concerns must account for sensitivity, specificity, simplicity, cost, robustness and rapidity 21 . The recombinase polymerase amplification (RPA) is a nucleic acid amplification technique that depends on the extension of primers induced by the recombination process and could be performed at isothermal temperature (37-42 °C) within 15-25 min 19, 22 . RPA overcomes the limitations of many current methods as it does not need expensive instruments, provides rapid and reliable results under low-resource conditions and therefore one of the most promising new molecular diagnostic technologies 21 . The specific combination of enzymes involved in an isothermal RPA reaction are a recombinase, a strand displacing DNA polymerase and a single strand binding protein (SSB). The principle of the RPA technology mainly relies on the ATP dependent recombinase enzyme, which forms a nucleoprotein filament by binding the primers and scanning for the homologous double stranded DNA (dsDNA) sequence of the template to facilitate strand exchange at cognate sites. The SSB protein stabilizes the resulting D loop structure to perform the extension at the 3ˈ end of the invading primer using the complementary strand as a template by DNA polymerase I and accomplishes exponential amplification by cyclic repetition [23] [24] [25] [26] [27] . The amplified end product of the RPA reaction can be detected by different methods including agarose gel electrophoresis, colour-based visual detection, real-time fluorescence analysis with exo or fpg probes, and lateral flow immunochromatographic assays (LFICA). The LFICA-based detection system requires a Twist Amp nfo probe labeled with an antigenic molecule such as FAM/FITC/digoxigenin at the 5′ termini and labeled reverse primers with an antigenic biotin molecule at the 5′ termini. The amplified amplicons using these Twist Amp nfo probe and biotin labeled reverse primers can be detected on 'sandwich immunochromatographic' assays such as, the PCRD nucleic acid detector and Milenia GenLine HybriDetect strip (TwistDx Limited, Cambridge, UK) 27, 28 . The different types of RPA-based assays have been reported to detect pathogens in animals [29] [30] [31] , humans 32 37 , Phytophthora spp. 38 and begomoviruses 22 . The aim of the present study was to develop a novel, rapid, robust and highly specific reverse transcription-RPA technique coupled with lateral flow immunochromatographic assay for CTV (working principle is illustrated in Fig. 1 ) and evaluate its efficacy in comparison with RT-PCR and RT-qPCR.

Screening of CTV-RT-RPA-LFICA primers and specificity assessment. Forty different combinations of forward and reverse primers (4 forward and 15 reverse) targeting a portion of the coat protein gene of CTV were analyzed for specificity and cross reactivity using different strains of CTV and other citrus pathogens with primer-BLAST software. During in silico analysis it was observed that all primer sets were unable to find complementary regions against other citrus infecting pathogen except CTV. Among these, 26 combinations of primers were identified as capable of amplifying target CTV RNA by conventional RT-PCR (Fig. 2) . Optimally working primer sets from the initial RPA assays (CTRPAF1/R1, CTRPAF2/R1 and CTRPAF3/R1) were selected for RT-RPA-LFICA. The CTRPA-R9-Btn reverse primer was selected as it showed the minimum complementary energy (ΔG) − 4.41 kcal/mole between reverse primer and probe. The CTRPA-F1/R9-Btn combination was identified as the most optimally performing primer set which consistently amplified a ~ 165 bp specific region of the CTV-p25 gene and was therefore used for further optimization. The CTV positive control and the negative sample used in the present study were further confirmed using conventional RT-PCR. An expected size of ~ 672 bp amplicons with CN150/CN151 primers and ~ 630 bp with P23RBP-F/R primers was observed in CTV infected samples (A1, A2, A3, A4, M1, M2, M3, M4, N1, N2, N3 and N4) and no band was observed in the negative controls (HA1, HA2, HM1, HM2, HN1, HN2). However, amplicons of only six representative samples (A1, A2, M1, M2, N1 and N2) along with positive and negative controls were separated on 1% agarose gel (Fig. 3A,B) . The CTV positive and negative samples were used to evaluate the specificity and efficacy of the RT-RPA primers (CTRPA-F1/CTRPA-R9) by conventional PCR. The expected amplification product of ~ 165 bp was observed in CTV positive samples whereas no amplification was observed in the healthy samples and non-template control (Fig. 3C) . The PCR products amplified by RT-RPA primers were purified by gel elution and sequenced. In silico analyses confirmed sequences were specific for CTV. Optimization of the CTV-RT-RPA-LFIC assay. The CTV-RT-RPA-LFIC assay was optimized using synthesized cDNA and the total RNA isolated from CTV positive citrus plants. A PCRD nucleic acid detector was used to capture and detect CTV specific amplified double labeled products generated by RT-RPA. Upon application of the amplified products on the sample port, the carbon conjugated anti-biotin antibodies react with the Dig/biotin labeled amplicons. The complex of carbon conjugated antibody-Dig/biotin amplicons get captured at the test line (T-lines) and control line (C-line) by manifestation of a coloured visible line. The appearance of both lines occurring simultaneously within 2 min after application of the amplified product was considered as a positive result for CTV whereas development of only the control line indicated negative results (Fig. 4) . It was observed that the sample with higher CTV titer required minimum time (within 60 s) to develop a more intensevisible line (T-lines). It was also observed that RNA templates required more incubation time (20-25 min) than cDNA as initial template. The optimal results of the CTV-RT-RPA-LFIC assay were observed at 40-42 °C sandwich immunochromatographic assay consistently detected target cDNA of CTV up to 10 -5 serial dilution synthesized from 141 ng of in vitro-transcribed RNA as the initial template. A faint band was observed for the 10 -6 serial dilution of target cDNA (corresponding to 3.77 × 10 5 RNA copies). The test (T-line) band intensity approximately correlated with initial template concentration. The detection limit of the CTV-RT-RPA-LFIC assay was ≤ 141 fg of RNA when converted into cDNA and used as a template (3.77 × 10 5 RNA copies) (Fig. 6A ). However, the assay detected ≤ 0.23 ng/µl (6.288 × 10 8 RNA copies) when RNA was used directly as template. The detection limit for conventional RT-PCR was nearly 10 -5 serial dilution (3.77 × 10 6 RNA copies) of target cDNA ( Fig. 6B ) and the detection limit of TaqMan real-time PCR assay was recorded near > 10 -8 serial dilution of target cDNA with Ct value > 33.3 (3.773 × 10 3 RNA copies) (Fig. 7) . The primer pair was modified such that they contained unique nucleotide stretches and did not mix with the template before adding enzyme to avoid any non-specific binding of recombinase enzyme during the reaction. Specificity analysis showed that the developed CTV-RT-RPA-LFIC assay was highly specific to CTV and failed to detect any other non target citrus pathogens ( Fig. 8 ). (Table 1) with the kappa value of 0.926. These results indicate that the developed assay demonstrated an excellent diagnostic agreement with RT-qPCR and would be effective for the detection of CTV in field samples.

CTV is one of the most economically important pathogens of citrus and has destroyed millions of citrus trees worldwide 1 . The virus infection causes reduction in yield and quality of citrus fruits and induced stem pitting and devastating quick-decline symptoms. Citrus is mainly a vegetatively propagated crop and the major pathogens are transmitted through disease-infected buds and propagating planting material. In the field, the horizontal transmission is by aphid vectors in a semi-persistent manner. To prevent outbreaks of the disease, indexing of planting material is a crucial step that requires a specific and reliable virus detection techniques 9 . Numerous techniques have been developed for CTV detection but most of them have limitations viz., time consuming, www.nature.com/scientificreports/ requirement of expensive equipment and trained personnel. RT-PCR and RT-qPCR are the most acceptable techniques for CTV detection but require well setup laboratory, equipment and personel. Therefore, there is need to develop a rapid, robust and reliable on-site detection technique to assist in the certification of virus-free planting material. The RPA approach is an emerging isothermal, low cost, rapid, and point-of-care diagnostic tool 23 . It is a highly sensitive, reliable nucleic acid based method and has become a rapid detection tool for many pathogens including viruses 34 , bacteria 19, 27 , and Phytophthora species 39 . It is also used for detection of RNA viruses without the need for a separate step to synthesize cDNA by RT-RPA 21, 36, 37, 40 . This technology has the potential to be a promising alternative to RT-qPCR 41 . Enzymes (recombinase, SSB and strand displacement DNA polymerase) are required for exponential amplification of the target template 23 . To target the RNA template, extra reverse transcriptase enzyme need to be added to the reaction. Present study is first to report the development of a robust reverse transcription recombinase polymerase-based isothermal amplification technique coupled with lateral flow immunochromatographic assay (CTV-RT-RPA-LFICA) for the rapid detection of CTV.

The specificity of the optimized RT-RPA primers selected to amplify the CTV specific coat protein gene (CTV-p25) demonstrated by comparison with conventional RT-PCR and sequencing of PCR products as cognate gene specific. The analysis of the products of basic RPA by agarose electrophoresis requires an extra chloroform/ isoamyl alcohol purification step to reduce the crowding and complexity of the amplified RPA product on the www.nature.com/scientificreports/ agarose gel for better band visibility compared to non-purified RT-RPA products (Fig. 3D) . The specificity of the RT-RPA primers were also judged by in silico analysis (primer BLAST) that specifically detected the target pathogen and showed no cross reactivity with other major citrus pathogens. Hetero-dimer analysis using the Oligo Analyzer tool was performed between probe and reverse primers to obtain the realistic results on lateral flow immunochromatographic assay. Efforts were also made to obtain a minimum ∆G (free energy of the oligo sequence binding to its complement site) between probe and reverse primer. The ∆G value of − 4.41 kcal/mole for probe and CTRPA-R9 was observed as best to achieve reliable results owing to less complementarity between reverse primer and probe. CTV-RT-RPA-LFICA developed in the present investigation was rapid, needed 20-25 min for amplification and 5 min for visualization. Application of RT-RPA amplified products on the sample port, the test line (T-lines) and control line (C-line) were visible as coloured bands with CTV positive samples simultaneously within 2 min, whereas development of color with only the C-line indicated negative results (Fig. 4) . The assay needs less RT-RPA product (0.5 µl) for end point detection, the visualization process on the PCRD detector, as compared with other techniques. The results also suggested that RNA as an initial template needed more reaction incubation time (20-25 min) compared to cDNA as template (15 min) . Higher pathogen titer in the sample would require much less time (~ 60 s) to develop more intense color band in T-lines. The reaction works at low isothermal temperature of 40 °C in a simple dry bath without expensive thermal cyclers (Fig. 5A) . The reaction also could 42 . The reaction needed a single set of primers, whereas LAMP needs as many as four to six primers 39 . The quick lateral flow immunochromatographic assay optimized using the Twist Amp nfo probe which saved an additional 60 to 80 min compared to RPA where products have to be visualized by agarose gel electrophoresis. The sensitivity of RT-RPA-LFICA was shown to be equal or better compared to conventional RT-PCR, but less sensitive compared to the TaqMan-RT-qPCR (Figs. 6, 7 and Supplementary Fig. S1 ). The ability of RT-RPA-LFICA to detect the CTV using genomic RNA as template with a limit up to 0.23 ng/µl of RNA was demonstrably a strong advantage compared to RT-PCR and TaqMan-qPCR. Specificity analysis showed that the developed CTV-RT-RPA-LFIC assay is highly specific to CTV and did not cross react with any other non-target citrus pathogens (Fig. 8) . The specificity of the assay was further validated by testing seventy-four field grown CTVsuspected samples and compared with RT-PCR and RT-qPCR. These results confirm the robustness and specificity of the newly-designed RT-RPA-LFICA primers-probes and demonstrated excellent diagnostic agreement with RT-qPCR (kappa value = 0.926). The major advantage of this technique is that the result can be easily judged as positive or negative visually and thus a great potential to be used as a point-of-care diagnostic tool and a valuable tool for nursery citrus bud wood certification programs.

Maintenance of CTV infected plants, sample collection and processing. CTV infected (sample code: A1, A2, A3, A4, M1, M2, M3, M4, N1, N2, N3 and N4) and healthy (sample code: HA1, HA2, HM1, HM2, HN1, HN2) plants of different citrus cultivars viz., Acid lime (Citrus aurantiifolia), Mosambi (Citrus sinensis) and Nagpur mandarin (Citrus reticulata) were collected from different geographical regions of India and maintained in an insect-proof screen house at ICAR-CCRI, Nagpur. Four to six leaves were selected from each collected sample and washed with distilled water, wiped with 70% ethanol, blot dried and used for further processing.

the CTV-RT-RPA-LFICA were designed using Twist Amp nfo assay design manual guidelines (www.twist dx.co. uk) to amplify a segment of the coat protein gene of CTV (CTV-p25). Number of coat protein gene sequences of CTV were retrieved from the GenBank and aligned using the software MEGA7. The highly conserved region of coat protein gene (CTV isolate MD; GenBank KY011909.1) was targeted and several sets of primer combinations (4 forward and 15 reverse) were designed and custom synthesized from Integrated DNA Technologies (IDT, Iowa, USA). The standard parameters of RPA primer design were taken into consideration and in silico specificity was considered using primer-BLAST software (www.ncbi.nlm.nih.gov). After initial screening by RT- To confirm the presence of CTV, RT-PCR was conducted using a coat protein gene specific primer pair (CN150/CN151) as previously described by Warghane et al. 43 The PCR products were visualized by 1% agarose gel electrophoresis and the UV GelDoc system (G: Box Syngene). Another primer set, P23RBP-F/R, specific to the RNA binding protein (CTV-P23) gene located at the 3′-terminus and adjacent to the untranslated region of the RNA genome of CTV 44 was designed and custom synthesized (IDT, Iowa, USA). It was also used to validate the RT-RPA technique (Table 3) . PCR amplification was performed using the P23RBP-F/R primer set according to Kokane et al. 45 .

To assess the sensitivity of the developed RT-RPA, an in vitro-transcribed RNA standard was generated by using a MEGAscript T7 transcription kit (Invitrogen). Total RNA was isolated from CTV infected plants and converted into single stranded cDNA using coat protein gene specific reverse primer (RPARNA-P25R2) which contains the RNA polymerase T7 promoter sequence. The cDNA was used as template for PCR amplification of the 700 bp coat protein gene using RPARNA-P25F1/R2 primers ( Table 3 ). The PCR amplified products were checked on 1.5% agarose gel, eluted and sequenced. The sequence validated PCR product (120 ng) containing the T7 RNA polymerase promoter sequence was used as template for in vitro RNA synthesis. The in vitro RNA transcription was performed at 37 °C for 4 h in a mix consisting of 3 mM of each T7 NTPs and 2 µl Enzyme mix in 1 × T7 reaction buffer. After reaction completion, the RNA was treated with 2U TURBO DNase (2U/µl) and recovered by lithium chloride precipitation according to manufacturer's instructions. The copy number of in vitro synthesized RNA was determined using the formula, RNA copy number = Moles of ssRNA × Avogadro's number (6.022 × 10 23 ). The moles of ssRNA was calculated as mass of ssRNA (g)/[(number of ribonucleotides of ssRNA × average molecular weight of a ribonucleotide) + 18.02 g/mol].

TaqMan-qPCR assay. TaqMan-qPCR was used to compare and validate the sensitivity of the CTV-RT-RPA-LFIC assay using the CTV specific primer-probe combination. The coat protein (CTV-p25) gene (Gen-Bank AF260651) specific primers (forward; P25-F and reverse; P25-R) and corresponding probe (labelled with 6-carboxy-fluorescein (FAM) reporter dye at the 5′ terminus, and the Black Hole Quencher (BHQ)-1dye at the 3′ terminus) were custom synthesized at IDT 46 . Plant samples (A1, A2, A3, A4, M1, M2, M3, M4, N1, N2, N3, N4, HA1, HA2, HM1, HM2, HN1, and HN2) were examined by TaqMan-qPCR. Total RNA was converted into cDNA using coat protein gene specific reverse primer (P25R). The cDNA (1 µl) was used as a template for qPCR reactions. The primers and probe concentration were optimized for obtaining highest reporter fluorescence and the lowest Ct (Cycle threshold) value. The TaqMan-qPCR assay was performed using a StepOne Real Time PCR System (Applied Biosystems) in a total of 20 µl reaction volume containing 300 nM each of forward (P25-F) and reverse (P25-R) primers, 250 nM probe (CTV-FAM) with CXR reference dye containing 1x GoTaq qPCR master mix (Promega). The reaction protocol was 95 °C for 2 min, followed by 40 cycles at 95 °C for 15 s, annealing and primer extension for 1 min at 60 °C. All experimental reactions were conducted in triplicate along with non-template controls (NTC) and the data was analyzed using StepOne Software v2.1. To analyze the sensitivity, cDNA was synthesized from 141 ng of in vitro-transcribed RNA. The tenfold serial dilution of cDNA (1, 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 and 10 -10 ) was used for detection limit estimation and validation of RT-RPA.

Primer optimization and screening based on RT-RPA and RT-PCR. The designed sets of primer combinations (4 forward and 15 reverse) were optimized using conventional PCR and RT-RPA. The most optimally performing CTV specific primer sets were used for RT-RPA-LFICA optimization. For screening and vali- Table 3 . Primer and probe sequences used for TaqMan-qPCR assay, conventional RT-PCR and generation of in vitro RNA standard in the present study. 

To activate the reaction, 14 mM magnesium acetate (MgAc) was dispensed into the cap of the tube and spun down to start the reaction. The reaction was incubated in a dry bath (Labtech, USA) at isothermal temperature (40 °C) for 4 min, tubes were vortexed, briefly centrifuged and re-incubated for a further 21 min to achieve optimal amplification. An aliquot of 2.5 μl of SDS (from 20% stock) was added to the final RT-RPA product and heat inactivated at 65 °C for 10 min. Subsequently, the mixture was diluted with three volume of nuclease-free water, extracted with an equal volume of chloroform, and the supernatant was precipitated by chilled isopropanol at -20 °C. The precipitated product was centrifuged, washed with 70% chilled ethanol, air dried, and suspended in 30 μl of nuclease-free water and was analyzed. The in silico hetero-dimer analysis was also performed for primer and probe using IDT-OligoAnalyser tool

and 10 -10 ) synthesized from 141 ng of in vitro-transcribed RNA as an initial template. The cDNA dilutions were used to compare the sensitivity of the assay with RT-qPCR and RT-PCR. The sensitivity of RT-RPA-LFICA was also analyzed based on in vitro synthesized RNA as template. The RNA (235 ng/µl) was tenfold serially diluted to 23.5 ng/µl, 2.35 ng/µl, 0.235 ng/µl, 0.0235 ng/µl, 0.00235 ng/µl, 0.000235 ng/µl and 1 µl of each was used as template. All reactions were performed in triplicate and the non-template reaction mixture was included as negative control. The different samples viz. CTV-unknown samples, CTV positive, and other non-CTV major virus and bacterial pathogens of citrus., Citrus yellow mosaic virus 47,48 , Indian citrus ringspot virus 20 , Candidatus Liberibacter asiaticus 49 , and Candidatus Phytoplasma cynodontis (citrus phytoplasma)

Citrus tristeza virus: a pathogen that changed the course of the citrus industry

The epitope structure of Citrus tristeza virus coat protein mapped by recombinant proteins and monoclonal antibodies

Citrus tristeza virus-host interactions

Plant viral diseases: economic implications

Complete sequence of the Citrus tristeza virus RNA genome

The pathogenicity determinant of Citrus tristeza virus causing the seedling yellows syndrome maps at the 3′-terminal region of the viral genome

Estimation of the number of aphids carrying Citrus tristeza virus that visit adult citrus trees

Citrus tristeza virus p23: a unique protein mediating key virus-host interactions

Development of multiplex polymerase chain reaction assay for simultaneous detection of clostero-, badna-and mandari-viruses along with huanglongbing bacterium in citrus trees

The continuous challenge of Citrus tristeza virus control

Kinetics of accumulation of Citrus tristeza virus RNAs

Comparison of detection methods for Citrus tristeza virus in field trees during months of nonoptimal titer

A real-time RT-PCR assay for detection and absolute quantitation of Citrus tristeza virus in different plant tissues

Molecular cloning, sequencing and phylogenetic analysis of coat protein gene of a biologically distinct Citrus tristeza virus isolate occurring in central India

Characterization of Citrus tristeza virus isolates from Colombia

Development of a simple and rapid reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay for sensitive detection of Citrus tristeza virus

Simultaneous elimination of carryover contamination and detection of DNA with uracil-DNA-glycosylase-supplemented loop-mediated isothermal amplification (UDG-LAMP)

Loop-mediated isothermal amplification (LAMP) based method for rapid and sensitive detection of 'Candidatus Liberibacter asiaticus' in citrus and the psyllid vector

Genome-informed diagnostics for specific and rapid detection of Pectobacterium species using recombinase polymerase amplification coupled with a lateral flow device

A rapid and sensitive reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the detection of Indian citrus ringspot virus

Rapid detection of potyviruses from crude plant extracts

Evaluation of recombinase polymerase amplification for detection of begomoviruses by plant diagnostic clinics

DNA detection using recombination proteins

Recombinase polymerase amplification as a promising tool in hepatitis C virus diagnosis

Rapid detection of Mycobacterium tuberculosis by recombinase polymerase amplification

Nucleic acid amplification using recombinase polymerase: enzymatic approach

Development of a recombinase polymerase based isothermal amplification combined with lateral flow assay (HLB-RPA-LFA) for rapid detection of 'Candidatus Liberibacter asiaticus

Development of a recombinase polymerase amplification combined with lateral-flow dipstick assay for detection of bovine ephemeral fever virus

A new approach for diagnosis of bovine coronavirus using a reverse transcription recombinase polymerase amplification assay

Field-applicable recombinase polymerase amplification assay for rapid detection of Mycoplasma capricolum subsp. capripneumoniae

Development of real-time and lateral flow strip reverse transcription recombinase polymerase amplification assays for rapid detection of peste des petits ruminants virus

Rapid detection of HIV-1 proviral DNA for early infant diagnosis using recombinase polymerase amplification

Non-instrumented incubation of a recombinase polymerase amplification assay for the rapid and sensitive detection of proviral HIV-1 DNA

An isothermal based recombinase polymerase amplification assay for rapid, sensitive and robust indexing of Citrus yellow mosaic virus

Field detection of citrus Huanglongbing associated with 'Candidatus Liberibacter asiaticus' by recombinese polymerase amplification within 15 min

Rapid and sensitive detection of Little cherry virus 2 using isothermal reverse transcription-recombinase polymerase amplification

Rapid diagnostic detection of plum pox virus in Prunus plants by isothermal AmplifyRP (®) using reverse transcription-recombinase polymerase amplification

Development of rapid isothermal amplification assays for detection of Phytophthora spp. in plant tissue

A recombinase polymerase amplification-lateral flow dipstick assay for rapid detection of the quarantine citrus pathogen in China Phytophthora hibernalis

Development of real-time reverse transcription recombinase polymerase amplification (RPA) for rapid detection of peste des petits ruminants virus in clinical samples and its comparison with real-time PCR test

Development of a recombinase polymerase amplification assay for detection of epidemic human noroviruses

Recombinase polymerase amplification for diagnostic applications

Molecular detection and coat protein gene based characterization of Citrus tristeza virus prevalent in Sikkim state of India

Viral-like symptoms induced by the ectopic expression of the p23 gene of Citrus tristeza virus are citrus specific and do not correlate with the pathogenicity of the virus strain

In-silico characterization and RNA-binding protein based polyclonal antibodies production for detection of Citrus tristeza virus

Quantitative detection of Citrus tristeza virus in citrus and aphids by real-time reverse transcription-PCR (TaqMan)

Use of NCM based DNA extraction method for simultaneous detection of citrus mosaic badnavirus and Candidatus Liberibacter asiaticus by duplex PCR

Quantitative distribution of citrus yellow mosaic badnavirus in sweet orange (Citrus sinensis) and its implication in developing disease diagnostics

Genetic diversity of the Indian populations of 'Candidatus Liberibacter asiaticus' based on the tandem repeat variability in a genomic locus

First report of 16SrXIV group phytoplasma associated with witches'-broom disease of acid lime (Citrus aurantifolia) in India

First report of a 'Candidatus Phytoplasma cynodontis'-related strain (group 16SrXIV) associated with Huanglongbing disease on Citrus grandis

The phloem-limited bacterium of greening disease of citrus is a member of the α subdivision of the Proteobacteria

Antimicrobial nano-zinc oxide-2S albumin protein formulation significantly inhibits growth of 'Candidatus Liberibacter asiaticus' in planta

Molecular detection, identification, and sequence analysis of 'Candidatus Liberibacter asiaticus' associated with Huanglongbing disease of citrus in North India

Ultrasensitive detection of phytoplasmas by nested-PCR assays using two universal primer pairs

The data that support the findings of this study are available from the corresponding author upon reasonable request. 

The authors declare no competing interests.

Supplementary information is available for this paper at https ://doi.org/10.1038/s4159 8-020-77692 -w.Correspondence and requests for materials should be addressed to D.K.G.Reprints and permissions information is available at www.nature.com/reprints.Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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