Adapting Molecular Diagnostics to Field Conditions

Enhancing food security by providing effective control of infectious diseases in livestock requires major investment in developing diagnostic technologies of sufficient sensitivity and precision to enable veterinary authorities to accurately identify animal carriers of disease and to carry out appropriate measures for containing an outbreak. Diagnosis typically requires the identification of the infective organism, and this may be possible at point-of-care in certain instances where the disease agent can be identified microscopically from the various body fluids. However, in many cases, this method is not applicable and diagnosis requires laboratory backup to provide the information. Gene amplification methods provide a way of doing this without resorting to culture of viral or bacterial pathogens or collecting large amounts of biological samples.

Adapting Molecular Diagnostics to Field Conditions These methods, based on the Polymerase Chain Reaction (PCR) are highly sensitive and specific, but incur a delay in transferring materials, analysing and providing the results. In some instances, this delay can be counterproductive in establishing an efficient disease control programme. For instance, in the Foot and Mouth Disease outbreak in the UK in 2001, delays in obtaining diagnosis meant that nearly 25% of farms declared infected on clinical grounds were actually free from disease. Being able to improve this decision-making gap would be an advantage, especially in the case of transboundary diseases, including Highly Pathogenic Avian Influenza, (HPAI) where suspected disease may occur in remote areas, away from the laboratory and where speedy diagnosis of a dangerous potential zoonosis is of paramount importance. Loop mediated isothermal amplification (LAMP) provides a means for applying molecular technologies to the pen-side as it does not require thermal cycling and the resulting colour change can be seen without the need for equipment (see Figure).

Recently, Mr Oliver Scheiber, a consultant at APU, investigated the potential of applying LAMP technologies to the diagnosis of Avian Influenza (AI), and Peste des Petit Ruminants (PPR). The work involved looking at technical modifications of the LAMP process based on the strand-displacing polymerase Bst, to make it sufficiently robust for field conditions; these involved testing alternative polymerases, using PCR enhancers, optimizing reaction temperatures and testing lyophilized master mixes. The Avian influenza RT-LAMP was based on a published method to which various modifications were applied in order to evaluate potential improvements in its functionality.

Originally the test results were read using a turbidimeter, but such equipment is not routinely used in most laboratories. Hence, the first step was to change the method to a fluorometric one, by adding EVA-Green dye to the reaction mixture as an indicator. The amount of pyrophosphate precipitate that develops during the reaction was minimized by lowering the magnesium concentration, thereby solving the problems encountered in reading the fluorescence signal. Interestingly, lower magnesium concentrations had the added effect of making signal development faster. The dNTP and the primer concentration were then optimised, leading to lower reagent usage and faster amplifications.

An alternative strand-displacing polymerase (Bsm), supplied by Fermentas was evaluated for use in the LAMP and was found to be comparable with Bst. Betaine is an agent that has been used successfully for increasing yield and specificity of PCR products by reducing the formation of secondary structure caused by GC-rich regions. Sugar polymers can also enhance the activity of certain enzymes, so its use in LAMP was evaluated. Betaine actually inhibited AI-RT-LAMP when using Bst, whereas Bsm needed a certain concentration of betaine to work. The disaccharide could replace Betaine in LAMP for both Bst and Bsm reducing the costs of the mastermix.

Adapting Molecular Diagnostics to Field Conditions Isothermal conditions used for LAMP are usually between 60 to 65°C, with an optimum of 62.5°C. However, in our tests performed between 55 to 65°C it was found that optimum temperatures for both Bsm and Bst was below 60°C and gave faster results than published positive results for AI-RT-LAMP (optimum around 57°C).

Applying the same reagents and concentrations a PPR-LAMP was performed on vaccine c-DNA using primers designed by Dr. Gang Li, Chinese Academy of Agricultural Sciences. It was found that the reaction worked extremely similar to the AI test. The results obtained with the AI LAMP give promise that this procedure can be further developed into a rapid and quality assured diagnostic system. The EVA green based platform will help to better evaluate these tests with established laboratory equipment in our counterpart laboratories for the fitness for purpose and their future field application.