2 On-Site Testing: Moving Decision Making from the Lab to the Field

Transcript

Hello! Welcome to the second talk of this week that is ‘On Site Testing Moving Decision Making from Lab to the Field’. So, this is a very important talk in a context that detection and identification usually very critical for containment of a particular pathogen and to protect the growers from severe losses. But usually the process takes longer time when it comes from the field to the lab conditions and then decision making is done on the lab basis. If the decision making system is changed from the context that the decision making could be obtained in the field condition then certainly it is going to save time and help the decision making process.

So ‘On-site testing’ basically is a term that is often used to describe two distinct activities, Firstly detection that is the initial locating of the pest or pathogen infected sample which is in most instances is performed visually. The second is activity is identification, and it is usually achieved by sending suspected samples to a laboratory. So, providing technological solutions to enable more rapid decision making is a must nowadays. It is not necessarily just inspection services who benefit from these techniques, they can be deployed throughout the farm to fork to limit losses caused by pathogens. How best to deploy detection methods is however a matter for policy makers and other stakeholders. Deploying simplified detection and identification methods remotely helps to speed up inspection as well as facilitates trade. So, that is why on-site testing is gaining popularity and devices have been developed to make decision making process in the field itself.

Performing diagnostics is part of a decision making process to prevent or limit spread of pathogens. The faster the decisions are made more effective the action may be. Traditionally once a potential disease has been located, samples are sent to a laboratory for testing that causes delay in the decision making process. Furthermore, If pathogens go unnoticed at pre[1]symptomatic infection stages, this failure of visual observation can lead to its spreading unchecked until it has built up to such a level that it can be seen. So, decision making at field level is very significant. These issues have led the drive to develop technological solutions that would fulfil two complementary roles.

Firstly, putting tools into the hands of those on the front line to enable rapid identification of pathogens and it would prevent delays.

Secondly, developing detection tools that guide those on the front line to the site of the problem, at the pre-symptomatic infection. So these tools are very vital for development for technological solutions at the on-site testing. These tools when linked together enable a more efficient detection and diagnosis process enabling faster deployment of control measures. Definitely, faster deployment of control measures would lead to least losses by the growers. Methods based on latex agglutination have been performed for plant diseases since in the early 1980s. For example Ani Biotech has developed a potato virus test kits by Ani Biotech on the basis of latex agglutination method. Since then more refined methods have been developed to enable rapid identification. Early test kits based on latex agglutination on glass or plastic slides, required:a large number of temperature labile reagents, had multiple steps in which reagents were added sequentially, and the interpretation of the result was often subjective, requiring a fair amount of training and experience to the reproduce.

Some of these second generation kits (e.g. Alert kits by Neogen) also incorporated chemical substrates, effectively recreating laboratory ELISA methodology, yet performed rapidly on a solid support. This provided advantages in terms of both usability and interpretation of results which were no longer subjective and easily interpreted by non-specialists even in the field situation. So, this is a technological advancement that is how it is helping on the on-site testing or decision making at the field level.

The most significant innovation came in the late 1990s with the application of homogeneous test kit formats developed and exploited. The Lateral Flow Device (LFD) format was exploited initially in the phytodiagnostics arena for the detection of potato viruses for use in seed certification systems and proved to be a considerable improvement over previous formats. The underlying chemistry in an LFD is effectively the same as a latex agglutination kit, the accumulation of antibody coated latex or may be colloidal gold particles caused by the presence of the antibody target. The key difference however is that the binding occurs during the capillary flow of sample and reagents along a membrane, but not in the solution as it was in the earlier cases.

Testing based on LFD technology remains the simplest and most rapid option for field use where specific binding reagents for the targets of interest are available. The only significant drawbacks to LFD approaches to field detection are the availability of reagents with a specificity appropriate for the application and the inherent lack of amplification that limits sensitivity. For simple pathogens such as viruses and to some degree bacteria and fungi, antisera or monoclonal antibodies with a useful level of specificity are often available, but for more complex targets this is often not the case. So, LFD approaches has certain limitations as we have seen that. It is good for of certain simple pathogens like fungi, bacteria and viruses but if it is a complex disease then definitely these LFD’s are not sufficient help the diagnosticians under field condition.

In-Field Identification

So, In-Field Identification where applications of LFD’s technologies are used very quickly they can collect the plant tissues and they can get the tissue macerated and the tissue extract can be then subjected to the LFD device and within few minutes the presence or absence of the specific bands can help us to identify that whether the plant is affected by the organism being tested is there or not. Similarly, there are other devices which require no electricity rather a movable or portable water bath or a heat block can be used to go for amplification test and then subject to its analysis and this again help us to take decision in the field itself but without coming to the laboratory. For greater sensitivity and specificity, molecular biology methods such as PCR is used to amplify target nucleic acids. However, implementation of these methods on-site has been a challenge. A number of companies have produced fieldable real-time PCR equipment. Whilst portable real-time PCR has been evaluated extensively there are a couple of significant drawbacks to it implementation. Firstly, in PCR methods, extraction of nucleic acid generally requires reasonably elaborate extraction methods to avoid co-purification of compounds which inhibit the enzymes. Secondly, whilst rugged, portable and in some cases battery powered equipment is available it remains expensive, largely due to the need for careful temperature regulation and sensitive detectors that are required for its detection. To solve both of these problems subsequent research has been focused on evaluation of isothermal amplification chemistries, which could eliminate both the problems associated with the normal PCR reactions.

Isothermal amplifications are methods in which the amplification reaction is incubated at a single temperature. This gives advantages in terms of simplicity over PCR, since the reactions do not need to be cycled accurately between temperatures, thus water-baths, dry-blocks or incubators can be used to incubate reactions. Plant pathogen assays by the Loop mediated AMPlification (LAMP) method is the most widely adopted method to date under field situation.

Finding Pathogens – to finding a pathogens require a sense of having a plant infected by a pathogen which can be spelled out as: Sniffing Pathogen Infection. Then one must see the infection from a distance and one must use Surveillance Tools for detection and diagnosis of the possible occurrence of plant pathogens. So these small equipments that are coming up that are replacing some high sensitive equipments like PCR and these are helping the investigators to take a decision at the on-site level without taking back the samples to the lab for its verification. It is only in certain cases where on-site testing is not confirmatory only then the samples are then brought back to the laboratory for further analysis. So, this is how one can see that how decision making processes has been now moved from the lab to field. Based on development of these technologies like LFDs and isothermal amplifications processes. With this we have come to an end of the topic On-site testing and decision making from lab to field. In the next we will be talking about virtual diagnostics networks and we will see how it s is helping any investigators or growers to take a decision for plant disease management.

Thank You.