Emerging technologies for the detection of rabies virus: challenges and hopes in the 21st century

Abstract

The diagnosis of rabies is routinely based on clinical and epidemiological information, especially when exposures are reported in rabies-endemic countries. Diagnostic tests using conventional assays that appear to be negative, even when undertaken late in the disease and despite the clinical diagnosis, have a tendency, at times, to be unreliable. These tests are rarely optimal and entirely dependent on the nature and quality of the sample supplied. In the course of the past three decades, the application of molecular biology has aided in the development of tests that result in a more rapid detection of rabies virus. These tests enable viral strain identification from clinical specimens. Currently, there are a number of molecular tests that can be used to complement conventional tests in rabies diagnosis. Indeed the challenges in the 21st century for the development of rabies diagnostics are not of a technical nature; these tests are available now. The challenges in the 21st century for diagnostic test developers are two-fold: firstly, to achieve internationally accepted validation of a test that will then lead to its acceptance by organisations globally. Secondly, the areas of the world where such tests are needed are mainly in developing regions where financial and logistical barriers prevent their implementation. Although developing countries with a poor healthcare infrastructure recognise that molecular-based diagnostic assays will be unaffordable for routine use, the cost/benefit ratio should still be measured. Adoption of rapid and affordable rabies diagnostic tests for use in developing countries highlights the importance of sharing and transferring technology through laboratory twinning between the developed and the developing countries. Importantly for developing countries, the benefit of molecular methods as tools is the capability for a differential diagnosis of human diseases that present with similar clinical symptoms. Antemortem testing for human rabies is now possible using molecular techniques. These barriers are not insurmountable and it is our expectation that if such tests are accepted and implemented where they are most needed, they will provide substantial improvements for rabies diagnosis and surveillance. The advent of molecular biology and new technological initiatives that combine advances in biology with other disciplines will support the development of techniques capable of high throughput testing with a low turnaround time for rabies diagnosis.

Figure 1. Amplification of rabies virus RNA by reverse transcription loop-mediated isothermal amplification (RT-LAMP).

For each reaction 1 µg RNA, prepared using TriZol (Invitrogen) from normal mouse brain (1A, circles; 1B −) or infected mouse brain (1A, triangles; 1B, +) was added to a reaction mixture containing all six primers at concentrations indicated in Table 1, Thermopol buffer (New England Biolabs, USA), 0.2 mM dNTPs (Promega, UK), 2 mM MgSO4, 1 M betaine (Sigma, UK), 16 units Bst 1 polymerase (New England Biolabs), 0.12 units Thermoscript reverse transcriptase, 50 nM ROX dye (Invitrogen) and 1/1000 dilution of the intercolating dye picogreen (Molecular Probes) in a final volume of 25 µl. The reaction was incubated at 65°C for 1 hour in an MX3000P thermal cycler with data collection at 80 second intervals. Samples were analysed in real time (Figure 1A) or by separation in a 1% agarose gel (Figure 1B), the arrow indicates a marker band with a size of 1.35 kilobase pairs.

Figure 2. Microarray identification of rabies virus RNA prepared from a brain sample from a confirmed case of human rabies.

Total RNA was extracted using TriZol (Invitrogen) and treated with DNase I prior to conversion to double stranded DNA . Non-specific amplification was achieved using a DNA polymerase (Klen Taq, Sigma) and the products were labelled through binding of Alexa Fluor 555 reactive dye (Invitrogen) to amplicons. Labelled target DNA was denatured at 95°C and chilled on ice before dilution in hybridization buffer and addition to the microarray slide. Hybridization occurred at 50°C overnight. Slides were washed and the target-probe binding was captured using GenePix Pro 6.1 software (Molecular Devices). Statistical analysis of the data was conducted using DetectiV software .