Detection Of Citrus Tristeza Virus Research Articles

Identification of an Isolate of Citrus Tristeza Virus by Nanopore Sequencing in Korea and Development of a CRISPR/Cas12a-based Assay for Rapid Visual Detection of the Virus

Citrus tristeza virus (CTV) is a highly destructive viral pathogen posing a significant threat to citrus crops worldwide. The disease management and crop protection strategies necessitate the development of rapid and accurate detection methods. In this study, we employed Oxford Nanopore sequencing (ONT) to detect CTV in Citrus unshiu samples. Subsequently, we developed a specific and sensitive detection assay combining CRISPR/Cas12a with reverse transcription-recombinase polymerase amplification. The CRISPR-Cas12a assay exhibited exceptional specificity for CTV, surpassing conventional RT-PCR by at least 10-fold in sensitivity. Remarkably, the developed assay detected CTV in field samples, with zero false negatives. This diagnostic approach is user-friendly, cost-effective, and offers tremendous potential for rapid on-site detection of CTV. Therefore, the CRISPR-Cas12a assay plays a significant role in managing and preserving citrus trees that are free from viruses in the industry.

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Simple template-based reverse transcription-recombinase polymerase amplification assay for routine diagnosis of citrus tristeza virus.

This study reports a simple template-based reverse transcription-polymerase amplification assay (ST-RT-RPA) for detection of citrus tristeza virus (CTV) from crude plant extract lysed in NaOH:EDTA (1:1) without the need of tedious RNA isolation. The developed assay showed versatility in its usage as amplification can be performed at wide temperature range (14°C to 42°C) and incubation time (4 to 32min), although the best conditions were 38°C for 30min. The developed ST-RT-RPA assay could detect the CTV up to 10-8 dilution of crude plant extract of NaOH:EDTA and up to 0.01 fg µl-1 of RNA of CTV-infected plant tissues and 0.001 ag µl-1 of plasmid DNA containing viral insert, thus exhibiting sufficient sensitivity. ST-RT-RPA assay showed high specificity without any cross-reaction with other citrus pathogens (Indian citrus ringspot virus, citrus yellow mosaic virus, citrus yellow vein clearing virus, and Candidatus Liberibacter asiaticus) and was more sensitive in detection of CTV infection in field samples as compared to standard reverse transcription-polymerase chain reaction (RT-PCR) with later showing false negative in 7.92% of samples tested after 1 week of sampling. The developed ST-RT-RPA assay used minimally processed crude plant extract as template, tolerant to sample degradation in transit and storage, while it can be easily performed at wide temperatures and could be adopted in resource-poor setup.

Reproducibility and Sensitivity of High-Throughput Sequencing (HTS)-Based Detection of Citrus Tristeza Virus and Three Citrus Viroids.

The credibility of a pathogen detection assay is measured using specific parameters including repeatability, specificity, sensitivity, and reproducibility. The use of high-throughput sequencing (HTS) as a routine detection assay for viruses and viroids in citrus was previously evaluated and, in this study, the reproducibility and sensitivity of the HTS assay were assessed. To evaluate the reproducibility of HTS, the same plants assayed in a previous study were sampled again, one year later, and assessed in triplicate using the same analyses to construct the virome profile. The sensitivity of the HTS assay was compared to routinely used RT-PCR assays in a time course experiment, to compensate for natural pathogen accumulation in plants over time. The HTS pipeline applied in this study produced reproducible and comparable results to standard RT-PCR assays for the detection of CTV and three viroid species in citrus. Even though the limit of detection of HTS can be influenced by pathogen concentration, sample processing method and sequencing depth, detection with HTS was found to be either equivalent or more sensitive than RT-PCR in this study.

A new single-chain variable fragment (scFv) antibody provides sensitive and specific detection of citrus tristeza virus

Citrus tristeza virus (CTV) is the most economically important virus disease of citrus worldwide. To develop a specific serological assay for CTV, a Tomlinson phage display antibody library of single chain variable fragments (scFv) was screened with a recombinant CTV coat protein (CTV-CP) heterologously expressed in Escherichia coli. The phage clones were checked by ELISA to identify clones with high specificity for CTV-CP. Eight clones were strongly reactive with CTV-CP. Nucleotide sequencing of these clones revealed that all of them contained the same sequence. Thus, the phage-displayed scFv antibody was termed scFvF10. Evaluation of scFvF10 binding to CTV-CP by plate-trapped antigen ELISA (PTA-ELISA) and immunoblotting, showed that it was specific and allowed sensitive detection of CTV-CP. Homology-based molecular modeling and docking analysis confirmed that the interaction between CTV-CP and scFvF10, with a binding energy of -738 kj mol-1, occurred mainly by 12 intermolecular hydrogen bonds. Moreover, triple-antibody sandwich (TAS)-ELISA using scFvF10 as second antibody showed high sensitivity in the detection of CTV infected samples. The CTV detection limit of scFvF10 by PTA-ELISA and TAS-ELISA were 0.05 and 0.01 μg CP/mL, respectively. Our results with different diagnostic assays demonstrated that scFvF10 has the potential to be used as an efficient tool for CTV-infected plant diagnosis.

Development of a real-time RT-PCR method for the detection of Citrus tristeza virus (CTV) and its implication in studying virus distribution in planta.

Tristeza is an economically important disease of the citrus caused by Citrus tristeza virus(CTV) of genusClosterovirus and familyClosteroviridae. The disease has causedtremendous losses to citrus industry worldwide by killing millions of trees, reducing the productivity and total production. Enormous efforts have been made in many countries to prevent the viral spread and the losses caused by the disease. To understand the reason behind this scenario, studies on virus distribution and tropism in the citrus plants are needed. Different diagnostic methods are available for early CTV detection but none of them is employed for in planta virus distribution study. In this study, a TaqMan RT-PCR-based method to detect and quantify CTV in different tissues of infected Mosambi plants (Citrus sinensis) has been standardized. The assay was very sensitive with the pathogen detection limit of > 0.0595fg of in vitro-transcribed CTV-RNA. The assay was implemented for virus distribution study and absolute CTV titer quantification in samples taken from Tristeza-infected trees. The highest virus load was observed in the midribs of the symptomatic leaf (4.1 × 107-1.4 × 108/100mg) and the lowest in partial dead twigs (1 × 103-1.7 × 104/100mg), and shoot tip (2.3 × 103-4.5 × 103/100mg). Interestingly, during the peak summer months, the highest CTV load was observed in the feeder roots (3 × 107-1.1 × 108/100mg) than in the midribs of symptomatic leaf. The viral titer was highest in symptomatic leaf midrib followed by asymptomatic leaf midrib, feeder roots, twig bark, symptomatic leaf lamella, and asymptomatic leaf lamella. Overall, high CTV titer was primarily observed in the phloem containing tissues and low CTV titer in the other tissues. The information would help in selecting tissues with higher virus titer in disease surveillance that have implication in Tristeza management in citrus.

Detection and Molecular Characterization of 'Candidatus Liberibacter asiaticus' and Citrus Tristeza Virus Associated with Citrus Decline in Bhutan.

Citrus, mainly mandarin (Citrus reticulata Blanco), is an economically important fruit crop in Bhutan. Despite having favorable agroclimatic conditions for citrus cultivation, the early decline of fruit-bearing orchards coupled with low crop productivity is a major concern among citrus growers. During a recent survey, an association of 'Candidatus Liberibacter asiaticus' (citrus greening) and citrus tristeza virus (CTV), either singly or as mixed infections in declined citrus trees, was recorded in all four major citrus-growing districts (Tsirang, Dagana, Zhemgang, and Sarpang). Using PCR-based diagnosis, a higher incidence of citrus greening (27.45%) and tristeza (70.58%) was observed in symptomatic field samples. Detection and characterization of 'Ca. L. asiaticus' was performed based on the 16S ribosomal DNA, prophage gene, 50S ribosomal rplA-rplJ gene, and tandem repeats of the CLIBASIA_01645 locus. Similarly, the coat protein, p23, and p18 genes were used as genetic markers for the detection and characterization of Bhutanese CTV. The 'Ca. L. asiaticus' isolates from Bhutan segregated into classes II and III based on the CLIBASIA_01645 locus, analogous to Indian isolates from the northeast region and Term-A based on the CLIBASIA_05610 locus. CTV isolates of Bhutan were observed as closely related to the VT strain, which is considered to be the most devastating. To the best of our knowledge, this is the first study on molecular characterization of 'Ca. L. asiaticus' and CTV isolates and their association with citrus decline in Bhutan.

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

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.

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

Citrus tristeza virus (CTV) is the etiologic agent of the destructive Tristeza disease, a massive impediment for the healthy citrus industry worldwide. Routine indexing of CTV is an essential component for disease surveys and citrus budwood certification for production of disease-free planting material. Therefore, the present study was carried out to develop an efficient serological assay for CTV detection based on the RNA binding protein (CTV-p23), which is translated from a subgenomic RNA (sgRNA) that accumulates at higher levels in CTV-infected plants. CTV-p23 gene was amplified, cloned and polyclonal antibodies were raised against recombinant CTV-p23 protein. The efficacy of the produced polyclonal antibodies was tested by Western blots and ELISA to develop a quick, sensitive and economically affordable CTV detection tool and was used for indexing of large number of plant samples. The evaluation results indicated that the developed CTV-p23 antibodies had an excellent diagnostic agreement with RT-PCR and would be effective for the detection of CTV in field samples. Furthermore, CTV-p23 gene specific primers designed in the present study were found 1000 times more sensitive than the reported coat protein (CTV-p25) gene specific primers for routine CTV diagnosis. In silico characterizations of CTV-p23 protein revealed the presence of key conserved amino acid residues that involved in the regulation of protein stability, suppressor activity and protein expression levels. This would provide precious ground information towards understanding the viral pathogenecity and protein level accumulation for early diagnosis of virus.

Virus-directed synthesis of emitting copper nanoclusters as an approach to simple tracer preparation for the detection of Citrus Tristeza Virus through the fluorescence anisotropy immunoassay

Citrus Tristeza Virus (CTV) was detected by targeting the virus capsid protein, CP25, through a competitive fluorescence anisotropic immunoassay. In this assay, instead of using chromophore labeled tracers, a simple approach in which the target antigen serves both as the main target and tracer was developed. For this, CP25 was expressed in E.coli and the purified recombinant protein was manipulated for copper nanocluster synthesis. The resultant CP25/CuNCs optically showed quantum dot-like behavior and emitted light in a range of 535–715 nm. The CP25/CuNCs could also interact with the anti-CTV antibody and showed polarized emission. In this assay, the CP25 protein competes with the fluorescent CP25/CuNCs used as a tracer for immune-complex formation with the anti-CTV antibody. The results showed that the FA values decreased linearly from 400 pg/mL to 25 ng/mL with an increase in CP25 protein levels and the limit of detection (LOD) was 220 pg/mL. The procedure of this assay can be useful for the detection of low molecular weight proteins or peptides in general, due to the availability of various kinds of metal nanoclusters and synthesis protocols.

Development of a sensitive and reliable reverse transcription-droplet digital polymerase chain reaction (RT-ddPCR) assay for the detection of Citrus tristeza virus

Citrus tristeza virus (CTV) is one of the most important citrus viruses in the world. In this study, we established a reverse-transcription droplet digital polymerase chain reaction (RT-ddPCR) method for the sensitive and accurate quantification of CTV. Quantitative linearity, sensitivity and accuracy of RT-ddPCR were compared to those of reverse-transcription real time PCR (RT-qPCR) by using 10-fold serial dilutions of the CTV RNA transcripts. Both methods showed a high degree of linearity (R2 = 0.991) and quantitative correlation, although RT-ddPCR revealed 100-fold higher sensitivity than RT-qPCR. The detection results for heat-treatment citrus samples also showed that the positive detection rate of RT-ddPCR (73.2%) was higher than that of RT-qPCR (53.6%). In summary, the results indicated that RT-ddPCR may contribute to improved analytical sensitivity and accuracy for CTV detection.

Ultrasensitive immunoassay for detection of Citrus tristeza virus in citrus sample using disposable microfluidic electrochemical device

Tristeza is a disease that affects citrus crops in general, caused by the Citrus tristeza virus (CTV). It is considered an economically important virus diseases in citrus, which is present in the main citrus producing regions all around the world. Early detection of CTV is crucial to avoid any epidemics and substantial economic losses for the citrus growers. Consequently, the development of rapid, accurate, and sensitive methods capable of detecting the virus in the early stages of the disease is highly desired. Based on that, a low-cost and rapid magneto-immunoassay methodology to detect the capsid protein from CTV (CP-CTV) was proposed. For this, magnetic beads were decorated with antibodies anti-CP-CTV and horseradish peroxidase enzyme (HRP) and applied for the capture and separation of CP-CTV from the sample solutions. The magnetically captured biomarker was detected using a simple disposable microfluidic electrochemical device (DμFED) constructed by rapid prototyping technique and composed by an array of immunosensors. In DμFED, the electrodes were modified with monoclonal antibody anti-CP-CTV and the detection was carried out using amperometry, based on the hydroquinone/H2O2 catalytic redox reaction due to the presence of HRP label in an immune-sandwich structure. The proposed immunoassay presented excellent linearity with a wide linear range of concentration of 1.95–10.0 × 103 fg mL−1 and ultralow detection limit of 0.3 fg mL−1.The disposable device was successfully applied for the detection of Citrus tristeza virus in healthy and infected plant samples, where it showed good agreements with the comparative method of enzyme-linked immunosorbent assay (ELISA). The developed immunoassay methodology showed a sensitive and selective way in the detection of CTV. Hence, it can be considered as a promising analytical alternative for rapid and low-cost diagnosis of Tristeza disease in citrus.

Rapid and Sensitive Detection of Citrus tristeza virus Using Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Assay.

Loop-mediated isothermal amplification (LAMP) is one recently developed gene amplification technique that emerges as a simple and quick diagnostic tool for early detection of nucleic acid targets. The LAMP technique works on the principle of strand displacement activity of Bst polymerase. It contains a set of four specially designed primers, which recognizes six different regions on the target nucleotide sequence. In the LAMP reaction, amplification is carried out in an isothermal conditions (60-65°C) using simple and inexpensive device like water bath or dry bath. Additional benefits of LAMP technique are that final results can be seen directly with naked eyes by adding intercalating dye SYBR Green I in the reaction tube. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is one of the novel techniques used for detection of RNA targets. The technology has been successfully applied for rapid and sensitive detection of Citrus tristeza virus (CTV) by using four oligo-primers, targeting a conserved coat protein gene (CPG) of an Indian CTV isolate. The result of assay is visible in naked eyes easily in the presence of SYBR Green I (100×) or on 1.5% agarose gel electrophoresis. CTV-RT-LAMP could be used away from plant pathology laboratories even in remote location.

Tissue-Print and Squash Capture Real-Time RT-PCR Method for Direct Detection of Citrus tristeza virus (CTV) in Plant or Vector Tissues.

Direct systems to process samples allow high-throughput testing or identification of Citrus tristeza virus (CTV) by the sensitive real-time reverse transcription coupled to polymerase chain reaction (RT-PCR) neither with extract preparation nor nucleic acid purification. Immobilized CTV targets are amplified from fresh sections of plant tissues or squashes of fresh or already caught vectors onto paper, nitrocellulose, or positively charged nylon membranes. The printed or squashed support can be stored or mailed at room temperature. These validated user-friendly methods are recommended by IPPC-FAO standard for CTV diagnosis, detection, and identification. The methods are safe, not under current quarantine regulations because they do not involve any risk of introduction of exotic CTV isolates or vectors and are discrete and useful for epidemiological studies or screening for large-scale analyses. In this chapter, tissue-printing and squashing capture methods for direct sample preparation without extract preparation or nucleic acid extraction and purification were coupled with validated real-time RT-PCR detection protocols based on TaqMan chemistry for CTV detection.

Detection of Citrus tristeza virus and Coinfecting Viroids.

Citrus can host a number of important vector- and graft-transmissible pathogens which cause severe diseases. Citrus disease management and clean stock programs require pathogen detection systems which must be economical and sensitive to maintain a healthy citrus industry. Rapid diagnostic tests for simultaneous detection of major graft-transmissible disease agents enable reduction of cost and time. The genetic and biological features of viruses and viroids can vary according to the strains/variants, with severe and mild strains described within the same species. The use of diagnostic tests that can allow to selectively discriminate severe strain(s) is a powerful tool to intercept the most harmful strains and to reduce the need for biological indexing. Moreover a combination of these detection methods will facilitate the studies on the interactions between CTV and viroids, a research topic only partially explored so far.

PCR cuantitativa para la detección del virus de la tristeza de los cítricos en Colombia

Se aplicó la técnica de reacción en cadena de la polimerasa en tiempo real (qRT-PCR), usando SYBR Green para la detección específica del virus de la tristeza de los cítricos (CTV) en Colombia. Una pareja de iniciadores, diseñados a partir de secuencias conservadas en los marcos abiertos de lectura (ORF’s) 1b y 2, permitió amplificar el ARN genómico (ARNg), y un producto de 186 pb fue obtenido sin la presencia de dímeros o inespecificidad. El análisis de la curva de fusión mostró un pico entre 81 oC y 83 oC, con un coeficiente de correlación de 0,998 y una eficiencia de 99,1 %. La curva estándar se desarrolló a partir de una amplificación del producto de 186 pb y permitió realizar un análisis cuantitativo de las muestras, con un rango de detección de 1x108 hasta 1x103 copias de RNAg y con valores de coeficiente de variación bajos. La acumulación de CTV fue más alta en tejido foliar y en frutos que en corteza; entretanto, las diferencias demostradas entre varias especies de cítricos susceptibles a la infección fueron mínimas. Los resultados de este trabajo muestran que la mayor concentración de virus se encontró en el tercio superior de las plantas analizadas, seguida por el tercio bajo y, por último, el tercio medio. La qRT-PCR es un método específico y sensible de interés práctico en los procesos de detección de las enfermedades virales presentes en el cultivo de los cítricos y otros cultivos de interés comercial.

High incidence of citrus tristeza virus in mandarin (Citrus reticulata) in North-East states of India.

Khasi mandarin is one of the most remunerative crop in North Eastern Hills region of India playing a very critical role in the socio-economic upliftment of the people. The orchards of this region were found to express the typical symptoms of citrus tristeza virus (CTV). An extensive survey of the Khasi mandarin orchard for six North Eastern states of India namely Arunachal Pradesh, Meghalaya, Assam, Nagaland, Sikkim and Tripura, was carried out to establish the identity of these viruses using ELISA and PCR/RT-PCR techniques. Out of 300 Khasi mandarin tree samples collected from the six states, 172 were found to be positive for CTV infection by DAS-ELISA indicating 57.33% overall CTV disease incidence. Results revealed presence of CTV in all the surveyed states showing a maximum incidence of 66.00% in Arunachal Pradesh followed by 62.00% in Assam, 60.00% in Meghalaya and Nagaland, 54.00% in Sikkim and 42.33% in Tripura. Higher CTV concentration was recorded in the age group>15years (69.09%) followed by 10-15 (57%) and 5-10years (43.33%). However, this study, to the best of our knowledge, is the first report for the detection of CTV in Khasi mandarin from Arunachal Pradesh, Nagaland and Tripura and also the first authentic survey of overall disease incidence of CTV Khasi mandarins from the six major mandarin growing North Eastern states of India.

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

Tristeza is a devastating disease of citrus and reported to be present in almost all countries where it is cultivated as a commercial crop. The etiological agent of this disease is Citrus tristeza virus (CTV), a member of the genus Closterovirus with in the family Closteroviridae. The pathogen is restricted to the phloem tissue of the infected citrus plant and has a monopartite ss (+) RNA genome of ∼20kb size. Till date, there is no effective control measure available for this virus. Management of tristeza depends on destruction of CTV infected field plants, production of virus-free planting material for new orchard establishment and controlling viruliferous aphid vectors responsible for field spread of the pathogen. Availability of rapid diagnostic assay is essential for rapid and efficient detection of the pathogen. In the present investigation, RT-LAMP (reverse transcription-loop mediated isothermal amplification), a highly sensitive, robust and low cost assay has been developed for rapid detection of CTV in infected citrus plant samples. Based on conserved nucleotide sequences available in GenBank and specific to p25 gene (major coat protein gene) of predominant CTV isolates of India, four primer sets (CTV-F3, CTV-B3, CTV-FIP and CTV-BIP) ware designed and custom synthesized. The amplified LAMP products obtained after maintaining isothermal condition of 65°C for 60min duration could be visible easily with necked eyes in presence of SYBR Green I (100X). Subsequently, LAMP products were verified by electrophoresis run in 1.5% agarose gel. The RT-LAMP results obtained with known CTV isolates maintained in screen house of CCRI, Nagpur were validated using field samples and thereafter it was further confirmed by conventional RT-PCR (reveres transcription-polymerase chain reaction) assay. The sensitivity of CTV-RT-LAMP protocol standardized in the present study was 100 times more than conventional one step RT-PCR assay. It also has maximum detection limit up to 0.0001ng RNA in individual reaction mixture. CTV-RT-LAMP assay is a simple, sensitive, rapid and less costly detection technique. This assay could be used for CTV diagnosis in pathology laboratories having limited facility and resources and even by citrus nurseries situated in remote locations. As per our knowledge and available literature, the present study reports first time about the usefulness of RT-LAMP assay for detection of CTV from India.

On-site detection of Citrus tristeza virus (CTV) by lateral flow immunoassay using polyclonal antisera derived from virions produced by a recombinant CTV

Maintenance of virus-free citrus in nurseries and orchards is essential to control the spread of aphid-transmitted Citrus tristeza virus (CTV). To this end, a lateral flow immunoassay (LFIA) was developed to detect CTV that was user-friendly and field-deployable. Polyclonal antisera was generated in rabbit against purified virions from Nicotiana benthamiana inoculated with an infectious recombinant cDNA clone of CTV (rCTV). Affinity purified rCTV IgG (1 mg/ml) and Mouse IgG (0.5 mg/ml) were conjugated with gold nanoparticles and coated onto a glass fiber membrane conjugate pad. Affinity purified rCTV IgG and anti-mouse IgG were used as test line and control line, respectively on the test strip. The LFIA detected CTV within 10 min and was sensitive up to a 1:80 dilution of crude plant sap extract. The LFIA was validated using CTV strains T30, T36, VT and RB from greenhouse and field sources and results were 100% in agreement with Enzyme-Linked Immunosorbent Assay and Reverse Transcription quantitative PCR. The immunostrip readily detected CTV after six months in storage in a sealed bag with silica gel at room temperature indicating good stability. The LFIA test strips offered a cost-effective tool to detect CTV in the field or nursery by non-skilled personnel without laboratory equipment.

Label-free electrochemical immunosensor for direct detection of Citrus tristeza virus using modified gold electrode

This paper presents the first label-free and sensitive electrochemical immunosensor for efficient and rapid detection of Citrus tristeza virus (CTV). The specific antibody against coat protein (CP) of CTV was successfully immobilized on 11-mercaptoundecanoic acid (MUA) and 3-mercapto propionic acid (MPA) modified gold electrode via carbodiimide coupling reaction using N-(3-dimethylaminopropyl)-N′-ethyl carbodiimide hydrochloride (EDC) and N-HydroxySuccinamide (NHS). Electrochemical characterizations of the immunosensor (anti-CTV/MUA-MPA/Au electrode) were carried out by cyclic voltammetry and electrochemical impedance spectroscopy. Electrochemical detection was performed by differential pulse voltammetry in [Fe(CN)6]3−/4− solution. The decrease in the [Fe(CN)6]3−/4− current upon increasing antigen concentrations on the immunosensor surface was determined in the range of 1nM to 5μM. The developed immunosensor provided a much lower limit of detection (LOD=0.27nM) than ELISA and other reported immunoassay techniques for CTV detection. The specificity of the immunosensor was also examined and no significant interference from other analytes in the real sample was observed. The proposed immunosensor is simple, relatively inexpensive, and shows good sensitivity for this application.