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Multiplex Point-of-Care Diagnostic Platform Development Essay

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Updated: Nov 4th, 2020


Diverse citrus products are popular among the population due to their excellent qualities. They are healthy and rich in vitamins that is why the citrus industry has always been profitable. However, there are some dangerous factors faced by this industry. Greening is among the major threats which can negatively impact the citrus industry through decreasing product quality and quantity and thus increasing their price due to the necessity to plan time- and cost-consuming strategies to fight citrus diseases. Greening first was discovered and described in the early 1900s in China, as huanglongbing (HLB, meaning “yellow shoot disease”) and spread all over the world since then (1).

It has affected citrus industries in China, the United States, and other significant citrus manufactures. In contrast to HLB, Citrus Tristeza Virus (CTV) is an endemic virus that is persistent in Australia. At present, the most efficient strategy is its early detection and eradication. Thus, there is a need to develop efficient methods to detect and eliminate pathogens dangerous for citrus crops. The aim of this study is to develop bionanosensors that are cheap, specific, sensitive, easy-to-use, and can be used to detect pathogens before the development of symptoms. The following literature review will provide theoretical grounds for further research.

Importance of Citrus Industry: in Australia and Globally

Citrus is among the world’s core fruit crops. They are popular due to their diversity, availability, and value for human diets (2). Liu, Heying, and Tanumihardjo (2) single out oranges, lemons, limes, grapefruit, and tangerines as the most popular “citrus fruits with commercial importance.” Citrus fruits are cultivated in more than 140 countries. However, the majority of the crop is gathered in tropical and subtropical areas (Figure 1).

Global production of citrus fruit has grown rapidly at the beginning of the 20th century from about “30 million metric tons in the late 1960s” to “a total estimate of over 105 million metric tons between 2000 and 2004” (2). As of 2009, China, Brazil, the U.S.A., India, Mexico, and Spain were considered leaders in citrus production.

The world’s major producing regions for citrus fruits
Figure 1. The world’s major producing regions for citrus fruits (highlighted in orange) (2).

Australia is not the biggest citrus produces, but it is considered to be the place of origin of citrus fruit together with New Caledonia and New Guinea (2). Australian citrus production is dated back to the year 1787 when “the English First Fleet set sail under instructions to introduce plants and seeds for sustainable horticulture”(3). At present, the citrus industry is a meaningful part of the Australian economy with about 1,900 growers cultivating more than 28,000 hectares of citrus (3).

According to Australian Citrus (3), the main areas for citrus growing are Riverland, South Australia; Murray Valley, Victoria, and New South Wales; Riverina, New South Wales, and the Central Burnett region in Queensland (Figure 2). The national strategic plan of citrus industry development puts market and market development as a priority (4). The industry is in the process of change introducing new varieties of fruits, an extension of export markets, and providing more regional support to farers (4).

Australian citrus production regions
Figure 2. Australian citrus production regions (3).

Constrains of Citrus Industry

Despite widespread and profitability, the citrus industry is developing some constraints. One of the problems is connected with abiotic and biotic stress. Another one is connected with the first and is conditioned by the high costs of dealing with stresses and harm of those interventions to the environment.

Biotic and antibiotic stress and the role of the current project in dealing with the disease

Gong and Liu (5). provide the review of genetic transformation and genes for resistance to stresses influencing the citrus industry. These stresses, both biotic and abiotic, include “drought, extreme temperature, salinity, citrus canker, citrus tristeza virus, and Huanglongbing (or citrus greening)” (5). The authors state that phytohormone ethylene can be applied to regulate various aspects of plant growth and development, and cope with environmental stresses (5).

In its turn, “control of ethylene production will modify stress tolerance” (5). Furthermore, the generation of transgenic plants with improved biotic stress tolerance is needed. Many efforts have been concentrated on creating transgenic plants able to resist CTV and canker (5). Coat protein genes were applied in this process. The authors define stress molecules – polyamines – found in living organisms and responsible for diverse processes of metabolism and development, biotic, and abiotic stress among them (5).

High costs and harm to the environment

The development of stress-resistant citrus plants is, evidently, a need of time. However, this process is tine and cost-consuming. Still, it is necessary because other ways of fighting citrus plant disease can be harmful to the environment, and no actions will lead to economic losses. Thus, according to Gong and Liu (5)., citrus canker is the cause of the substantial decrease in crops in citrus-growing countries. One of the ways to deal with cancer is the use of chemicals such as copper bactericides (5). Nevertheless, it is dangerous for the environment and threatens the safety of food consumption.

Global Citrus Market

The global citrus market includes world markets of citrus trade all over the world. The market is divided in accordance with the type of fruit. Thus, in oranges production, Brazil has leading positions with almost 5 tons of oranges produced (6). For tangerine and mandarin crops, “global production for 2016/17 is forecast at 28.5 million metric tons, down slightly from last year with a smaller crop in China more than offsetting larger crops in the European Union, Morocco, and Turkey”(6). European Union is a leader in the production of lemon and lime because of favorable weather conditions and a simultaneous decrease in production in the United States and Argentina (6).

Threats of citrus import and export

Although citrus trade is a significant sector for economies of countries which are leading producers of citrus fruits, import and export have some threats to be considered. The major danger of export and import is the transfer of endemic plant diseases (1). Such citrus disease as greening, for example, affects plants in many countries. However, the Australian citrus industry is not much influenced by this dangerous agent.

Consequently, active import and export can bring diseases not characteristic of this or that region and thus negatively influence the citrus industry in any country. Thus, efficient diagnostic methods are necessary to timely diagnose citrus crops and prevent its spread.

Control of the Existing and New Pathogens

At present, the major means of control over postharvest diseases of citrus fruit are synthetic fungicides such as “imazalil (IZ)., thiabendazole (TBZ)., sodium ortho-phenyl phenate (SOPP)., fludioxonil (FLU)., pyrimethanil or different mixtures of these compounds.” (7). However, these methods are banned in many countries. Talib, Boubaker, Boudyach, and Aoumar (7). suggest alternative environment-friendly methods for controlling postharvest diseases of citrus fruits. For example, they introduce such biological approaches as “use of antagonistic micro-organisms; application of naturally derived bioactive compounds; and induction of natural resistance.”(7).

Another method suggested by Talib, Boubaker, Boudyach, and Aoumar (7). is the application of natural plant-derived compounds. Youseff, Abd-Elsalam, Hussein, Tanzania, and Ippolito (8). recommend organic and inorganic salts as postharvest alternative control means of citrus. However, the authors agree that the prevention of citrus fruit diseases can be more efficient (8). Thus, “regular monitoring of the disease appearance and weather conditions, improved postharvest handling, control agents, timely use of effective fungicides, transport, and storage conditions” can be useful not only for monitoring of the existing pathogens but also for timely detection and elimination of new ones (8).

Methods of Pathogen Detection

The most widely accepted methods used for pathogen detection are symptom analysis, culture methods, serological methods, and PCR based molecular detection (9). Symptom-based analysis requires specialized training and is mostly too late to control aggressive pathogens. Culturing methods need specialized training and equipment, and take a longer time to produce results. Serological methods such as dipstick type assays have recently become available for some plant pathogens. Nevertheless, their limit of detection (LOD). is only 1:1000 and there are problems with detection when pathogen mutates (10).

PCR based assays are more sensitive with LOD of 10-9 but they need specialized training and equipment. The current study is aimed at the development of bionanosensors that are cheap, specific, sensitive, easy-to-use, and can be used to detect pathogens before the development of symptoms. The LOD for such nanosensor is up to femtogram.

Lau presents a multiplex point-of-care diagnostic in the thesis (9). The principle of the RPA/SERS assay is illustrated in Figure 3.

Schematic illustration of RPA/SERS multiplex assay.
Figure 3. Schematic illustration of RPA/SERS multiplex assay. (A). Three steps method of RPA/SERS which involved RPA and 2 times hybridization steps. (B). Single-step method of RPA/SERS which performed RPA and hybridization in a single tube (9).

On the whole, the first step was to extract total genomic DNA from plant tissue with the help of a modified Solid Phase Reversible Immobilization (SPRI). method (9). After that, RPA was used to amplify peculiar genomic regions for every pathogen. The created primers or RPA products would include “a biotin handle on one end and a 5′ overhang sequence of 10 nt on the opposite end, which functions as a barcode for hybridizing to SERS nanotags” (9). Amplification was followed by the capture of biotin/RPA/SERS products by streptavidin magnetic beads (9). The amplification and hybridization of SERS nanotags were finally optimized “to occur simultaneously in a single-tube to enable a faster, simpler assay” (9).

The efficiency of Multiplex Point-of-Care Diagnostic Platform

A multiplex point-of-care diagnostic platform aimed at the detection of plant pathogens has been the topic of recent research. Thus, Lau, Wang, Wee, Botella, and Trau (11) provide a field demonstration of a multiplexed point-of-care diagnostic platform. Recombinase polymerase amplification (RPA).12 is an illustration of an isothermal technique (11). It has recently observed some novice diagnostic applications.

RPA, if compared to PCR, is based on enzymes, “at a single low temperature, to separate dsDNA, assist in primer/target recognition and primer extension”(11). The benefits of RPA application comprise highly effective amplification and low constant operating temperature (11). Moreover, RPA “is highly sensitive with a detection limit as low as 6.25 fg of genomic DNA input with specificity >95%”(11). Also, POC-compatible readouts can be used with RPA to empower field applications. Still, these approaches, although useful, are not applicable to multiplex RPA assays (11).

Babu, Washburn, Miller, Poduch, Sarigul, Knox, et al. provide research dedicated to a rapid assay for detection of Rose rosette virus using reverse transcription-recombinase polymerase amplification using multiple gene targets (12). This example of RPA use is proof of its efficiency in various cultures. Thus, RT-RPA analysis of the field samples demonstrated a positive result with a majority of the samples (12).

Daher mentions several factors that have an impact on the effectiveness and robustness of amplification on a multiplex point-of-care diagnostic platform which should be considered. First of all, there is the necessity of reduction of “evaporation problems, liquid volume, incubation temperature, the concentration of bound and unbound primers, and the size of the solution and surface reaction products must be determined”(10).


On the whole, current literature on the issue of citrus fruits pathogen detection is diverse. However, there is no much research on the problem of applying the multiplex point-of-care diagnostic platform to detect HLB and CTV. Rodrigues (13) defines the primary trends in the citrus industry which determine its development and will influence the direction of the research in the field. Thus, continuing globalization of industry will stimulate the growth of problems with citrus plant diseases and, consequently, production cost.

Secondly, the production technology observes a tendency to complication. Rodrigues (13) also predicts the decrease in the number of citrus manufacturers and an increase in the size of their production. Moreover, consumers’ demand for safe, traceable, and eco-friendly products will have a great impact on the manufactures (13).

Thus, there is a vital need for the development of an efficient detection strategy that will be able to identify citrus pathogens already in the conditions of the nursery. Otherwise, more and more farms can become infected by HLB and CTV which can kill the citrus plant within a couple of years. The multiplex point-of-care diagnostic platform can become a tool to identify HLB and CTV at early stages and thus save time and costs which will be needed in case the infected plants are moved to farms.


  1. National Research Council, Division on Earth and Life Studies, Board on Agriculture and Natural Resources, Committee on the Strategic Planning for the Florida Citrus Industry: Addressing Citrus Greening Disease (Huanglongbing). Strategic planning for the Florida citrus industry: addressing the citrus greening disease. Washington: The National Academies Press; 2010.
  2. Liu YQ, Heying E, Tanumilhardjo SA. . Comprehensive Reviews in Food Science and Food Safety. 2012. Web.
  3. Citrus Australia (AU). Our industry: Australian Citrus. 2017. Web.
  4. Vos R. Australian Citrus Industry: industry development needs assessment & recommendations; 2009.
  5. Gong XQ, Liu JH. . PCTOC. 2013. Web.
  6. United States Department of Agriculture. . 2017. Web.
  7. Talib I, Boubaker H, Boudyach EH, Aoumar AAB. . JAppMicrobiol. 2014. Web.
  8. Youseff K, Abd-Elsalam KA, Hussein A, Sanzani SM, Ippolito A. . AgrBiol Sci. 2017. Web.
  9. Han Yih Lau PhD. Development of point-of-care and multiplex diagnostic methods for the detection of plant pathogens [doctoral thesis]. [Quensland (AU).]: University of Queensland; 2016.
  10. Rana Daher PhD. Recombinase polymerase amplification technology: assessment for nucleic acid-based point-of-care diagnostics [doctoral thesis]. [Quebek (CA).]: University Laval; 2015.
  11. Lau HY, Wang Y, Wee EJH, Botella JR, Trau, M. Field demonstration of a multiplexed point-of-care diagnostic platform for plant pathogens. AnalChem. 2016; 88:8074-8081.
  12. Babu B, Washburn BK, Miller SH, Poduch K, Sarigul T, Knox GW, et al. A rapid assay for detection of Rose rosette virus using reverse transcription-recombinase polymerase amplification using multiple gene targets. JVirologMeth. 2017;240:78-84.
  13. Rodrigues JLA. Global citrus industry trends – Opportunities and challenges. 2017. Web.
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