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Multiplex Pan_lyssavirus/β-actin Real-time RT-PCR Assays for Rabies Diagnostics

Description:

Fast-Track proposals will not be accepted.
Number of anticipated awards: 1
Budget (total costs):
Phase I: up to $150,000 for up to 12 months
PROPOSALS THAT EXCEED THE BUDGET OR PROJECT DURATION LISTED ABOVE MAY NOT BE FUNDED.
Background
Rabies, caused by Rabies virus (RABV) and related lyssaviruses, is one of the most deadly zoonotic diseases responsible for up to 70,000 estimated human deaths worldwide each year. Rapid and accurate laboratory diagnosis of rabies is essential for timely administration of post-exposure prophylaxis in humans and control of the disease in animals. Currently, only the direct fluorescent antibody (DFA) test is recommended for routine rabies diagnostics. DFA is a rapid and sensitive method, but its accuracy depends on the quality of brain tissue, availability of high-quality anti-rabies diagnostic conjugates, accessibility to a fluorescence microscope and, most importantly, an experienced diagnostician. Reverse Transcription-Polymerase Chain Reaction (RT-PCR)-based diagnostic methods have been widely adapted for the diagnosis of other viral pathogens, but there is currently no widely accepted rapid real-time RT-PCR assay for the detection of all lyssaviruses. The CDC rabies molecular diagnostic team has developed and validated a new multiplex real-time RT-PCR assay named LN34, which uses a combination of degenerate primers and probes along with probe modifications to achieve superior phylogenetic breadth, while maintaining sensitivity and specificity. The primers and probes of the LN34 assay target the highly conserved non-coding leader region and part of the nucleoprotein (N) coding sequence of the lyssavirus genome to maintain assay robustness. The probes were further modified by locked nucleotides to increase their melting temperature to meet the requirements for an optimal real-time RT-PCR assay. The LN34 assay was able to detect all rabies-causing variants in a validation panel that included representative RABV isolates from most regions of the world and 13 additional lyssavirus species. The LN34 assay was successfully used for both ante-mortem and post-mortem diagnosis using over 200 clinical samples as well as field derived surveillance samples. An algorithm of using the LN34 assay for rabies diagnostics has been developed based on the international validation data. The algorithm also comprised of a beta-actin real-time RT-PCR assay to measure the quality of the sample tested. This combined assay represents a major improvement over previously published rabies-specific PCR or RT-PCR assays because of its ability to universally detect RABV and other lyssaviruses, its high throughput capability and its simplicity of use, which can be quickly adapted in a laboratory to enhance the capacity of rabies molecular diagnostics. The LN34 assay provides an alternative approach for rabies diagnostics, especially in rural areas and rabies endemic regions that lack the conditions and broad experience to run the standard DFA assay. Nevertheless, the cost of the assay will be an important factor for its adaptation both in US (rabies surveillances test more 100,000 samples each year) and developing countries. We are looking for a products to further combined the LN34/beta-actin real-time RT-PCR assays and reduce the reaction volume to simplify the assay cost and set-up.
Project Goals
1.
Develop a reaction kit combining the LN34/beta-actin real-time RT-PCR assays into a single reaction.
2.
Select enzymes and reaction volumes to further reduce the cost for the assay.
3.
Develop a dry-bead format and optimize the reaction conditions for diagnostic laboratories.
Phase I Activities and Expected Deliverables
1.
Utilize artificial positive control RNA and rabies-negative brain samples to optimize the multiple assay combining LN34/beta-actin real-time RT-PCR assays (end of the 4th month).
2.
Test low cost enzymes to further reduce the cost of the reaction kit, develop a dry-bead format for the reaction kits to improve the stabilities of the reaction kits (end of the 6th month).
3.
Optimize the reaction in a low volume format to further reduce the cost of the reaction (end of the 6th month)
For Successful Phase I Awardees ONLY (Expected Phase II Deliverables)

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1.
Large-scale production of the optimized real-time RT-PCR reaction kits and the start of large scale validation of these kits in multiple laboratories where both CLIA samples and field collected samples will be tested. The validation data must lead to the development of a standardized algorithm of using this assay for rabies diagnostics.
2.
Based on the validation data through multiple laboratories, a standardized commercial kit will be optimized and finalized at the end of 24 months. A widely available and utilized new PCR-based rabies diagnostic assay will enhance rabies diagnostics and surveillances, and make a key contribution to the goal of canine rabies eradication by 2030.
Impact
A PCR-based rabies diagnostic is expected to be recommended later this year as multiple research and validation data for using PCR-based rabies diagnostics are highly supportive. Laboratories in the US and in most developing countries have real-time PCR capability and have the expertise required for conducting real-time PCR assays for the diagnosis of viral infections and, therefore, for rabies molecular diagnostics.
Successful commercialization of a real-time PCR-based rabies diagnostic will further improve the rabies diagnostic capacities in many laboratories domestically and internationally. An assay that can detect highly variable rabies viruses and other lyssaviruses can be used in areas endemic to both rabies viruses and other lyssaviruses and will enhance clinical rabies diagnostics and surveillance.
Commercialization Potential
This new multiplex assay should be able to detect all the available rabies virus variants and other lyssaviruses. It can be used for rabies diagnostics domestically and around the world, especially in regions with both canine rabies and lyssaviruses. Development of this assay could represent a significant advancement compared to previously published PCR-based rabies diagnostics assays, which only detected a limited number of rabies variants or limited other lyssaviruses.

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