A system for PCR-based diagnostic detection of rabies virus in brain tissue samples

Abstract

A system for PCR-based diagnostic detection of rabies virus in brain tissue samples with high sensitivity, specificity and accuracy, consisting of: (a) a sample preparation unit configured to handle brain tissue samples obtained from dogs using the rapid occipital technique or the autopsy sampling technique; b) an RNA extraction unit configured to extract and quantify total RNA from the brain tissue samples; c) a cDNA synthesis unit configured to convert extracted RNA into complementary DNA; (d) a PCR amplification unit configured to amplify target sequences using region-specific primers targeting positions 92 to 348 of the N gene of rabies virus; (e) a thermal cycler configured to perform PCR amplification under optimized conditions; f) an electrophoresis unit configured to visualise PCR results by agarose gel electrophoresis; and g) a gel documentation system configured to record and analyze electrophoresis results.

Description

FIELD OF THE INVENTION

[0001] The present disclosure relates to a system for PCR-based diagnostic detection of rabies virus in brain tissue samples with high sensitivity, specificity and accuracy. BACKGROUND OF THE INVENTION

[0002] Rabies is a neglected tropical disease that causes acute neurological damage in warm-blooded animals with a 100% mortality rate. Worldwide, it kills approximately 59,000 people annually, and the economic damage amounts to $8.6 billion. Current diagnostic methods face significant limitations, particularly in Africa, where the high genetic diversity of rabies viruses compromises the effectiveness of existing universal diagnostic tools. The direct fluorescent antibody test, although considered the gold standard, has limited efficacy for early detection of the disease and may fail to identify viral antigens in the early stages of infection.

[0003] Existing PCR-based diagnostic methods, including hemisphere reverse transcription PCR and real-time PCR, exhibit reduced sensitivity due to the high genetic diversity of African lyssavirus strains, which is attributable to single nucleotide polymorphisms in the target regions of the primers. Universal primers developed over a decade ago cannot adequately detect current and emerging lyssavirus variants, leading to false-negative results and misdiagnoses. Many African laboratories lack access to sophisticated real-time PCR equipment but do have conventional PCR capacity. Therefore, there is a need for region-specific diagnostic solutions tailored to local viral genetic diversity.

[0004] The challenge is further exacerbated by the limited availability of representative virus isolates from different geographical regions for validating primer design. This makes it difficult to detect all circulating lyssavirus strains with single tests. Previous approaches to region-specific solutions have been inadequate because they have not adequately accounted for local genetic diversity and the dynamic nature of virus evolution in African lyssavirus populations.

[0005] In view of the previous discussion, it is clear that there is a need for a system for PCR-based diagnostic detection of rabies virus in brain tissue samples with high sensitivity, specificity and accuracy. Summary of the invention

[0006] The present disclosure relates to a system for PCR-based diagnostic detection of rabies virus in brain tissue samples with high sensitivity, specificity, and accuracy. The present invention relates to a novel PCR-based diagnostic system specifically developed for the detection of rabies virus in canine brain tissue samples. The system uses region-specific primers targeting the N gene of the rabies virus to overcome the limitations of existing universal primers, whose sensitivity is reduced due to the genetic diversity of lyssaviruses in Africa. The system comprises integrated units for sample preparation, RNA extraction, cDNA synthesis, PCR amplification, electrophoresis visualization, and result documentation, thus providing improved diagnostic accuracy for rabies detection in West and Central Africa.

[0007] The present disclosure aims to provide a system for PCR-based diagnostic detection of rabies virus in brain tissue samples with high sensitivity, specificity and accuracy.The system includes: a sample preparation unit for processing brain tissue samples from dogs obtained using the rapid occipital technique or autopsy sampling methods; an RNA extraction unit for extracting and quantifying total RNA from the brain tissue samples; a cDNA synthesis unit for converting extracted RNA into complementary DNA; a PCR amplification unit for amplifying target sequences with region-specific primers targeting positions 92 to 348 of the N gene of the rabies virus; a thermocycler for performing PCR amplification under optimized conditions; an electrophoresis unit for visualizing PCR results using agarose gel electrophoresis; and a gel documentation system for recording and analyzing electrophoresis results.

[0008] Another objective of the present disclosure is to provide a system for PCR-based diagnostic detection of rabies virus in brain tissue samples with high sensitivity, specificity and accuracy.

[0009] Another objective of the present disclosure is to develop a highly sensitive and specific PCR-based diagnostic system that can accurately detect rabies virus in brain tissue samples.

[0010] A further objective of the present disclosure is to provide a system for region-specific diagnostics tailored to the genetic diversity of West and Central African lyssaviruses, using primers designed based on a comprehensive genomic data analysis of 52 whole-genome sequences to ensure optimal performance in these geographical regions.

[0011] Another objective of this disclosure is to provide a system for medical professionals and veterinary diagnosticians that offers them a reliable tool for the rapid detection and confirmation of rabies.

[0012] To further clarify the advantages and features of the present disclosure, the invention will be explained in more detail with reference to specific embodiments illustrated in the accompanying drawings. These drawings illustrate only typical embodiments of the invention and are therefore not to be considered as limiting its scope. The invention will be described and explained in more detail with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE CHARACTERS

[0013] These and other features, aspects, and advantages of the present disclosure will become better understood when the following detailed description is read with reference to the accompanying drawings, in which like characters represent like parts throughout. Fig. 1 shows a block diagram of a system for PCR-based diagnostic detection of rabies virus in brain tissue samples with high sensitivity, specificity, and accuracy according to an embodiment of the present disclosure; and Fig. 2 shows a block diagram illustrating the workflow of the proposed system according to an embodiment of the present disclosure.

[0014] Those skilled in the art will also appreciate that the elements in the drawings are shown for convenience and are not necessarily to scale. For example, the flowcharts illustrate the method by key steps to enhance understanding of aspects of the present disclosure. Furthermore, with respect to device construction, one or more components of the device may be represented in the drawings by conventional symbols. The drawings may show only the specific details relevant to understanding embodiments of the present disclosure in order not to clutter the drawings with details that would be readily apparent to those skilled in the art from the present description. DETAILED DESCRIPTION:

[0015] To facilitate understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the drawings and will be clearly described. However, the scope of the invention is not limited thereby. Changes and further modifications to the illustrated system, as well as further applications of the principles of the invention, are possible, as would normally occur to one skilled in the art to which the invention pertains.

[0016] It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be limiting thereof.

[0017] References in this specification to "one aspect," "another aspect," or similar language mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Therefore, the language "in one embodiment," "in another embodiment," and similar language throughout this specification may or may not refer to the same embodiment.

[0018] The terms "comprises," "comprising," or other variations thereof are intended to cover non-exclusive inclusion, such that a process or method comprising a list of steps may include not only those steps, but also additional steps not expressly listed or inherent in that process or method. Likewise, the statement "comprises" for one or more devices, subsystems, elements, structures, or components does not exclude, without further limitation, the existence of other devices, subsystems, elements, structures, components, or additional devices, subsystems, elements, structures, or components.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. The systems, methods, and examples provided herein are for illustrative purposes only and should not be considered limiting.

[0020] Embodiments of the present disclosure will be described in detail below with reference to the accompanying drawings.

[0021] Fig. 1 shows a block diagram of a system (100) for PCR-based diagnostic detection of rabies virus in brain tissue samples with high sensitivity, specificity and accuracy according to an embodiment of the present disclosure.

[0022] Referring to Fig. 1, the system (100) comprises: a) a sample preparation unit (102) configured to process brain tissue samples taken from dogs using the Rapid Occipital Technique or the autopsy sampling technique; an RNA extraction unit (104) configured to extract and quantify total RNA from the brain tissue samples; a cDNA synthesis unit (106) configured to convert extracted RNA into complementary DNA; a PCR amplification unit (108) configured to amplify target sequences using region-specific primers targeting positions 92 to 348 of the N gene of the rabies virus; a thermocycler (110) configured to perform PCR amplification under optimized conditions; an electrophoresis unit (112) configured to visualize PCR results by agarose gel electrophoresis;and a gel documentation system (114) configured to record and analyze electrophoresis results;

[0023] In one embodiment, the RNA extraction unit (104) is configured to utilize phenol / chloroform extraction or commercially available RNA extraction kits for complete RNA isolation.

[0024] In one embodiment, the cDNA synthesis unit (106) is configured to use commercially available cDNA synthesis kits for reverse transcription of extracted RNA.

[0025] In one embodiment, the PCR amplification unit (108) is configured to use a forward primer designated ACENTDFB@ABU ZARIA1 and a reverse primer designated ACENTDFB@ABU ZARIA2 to amplify rabies virus genotype I sequences, wherein the region-specific primers are configured to have high specificity with a query coverage of 100%, a percent identity of 100% to 99.61%, and E-values ​​of less than 0.001 when analyzed with the Basic Local Alignment Search Tool.

[0026] In one embodiment, the PCR amplification unit (108) is further configured to generate amplicons having a size of 256 base pairs when the rabies virus is present in the sample.

[0027] In one embodiment, the system is configured to detect rabies viruses in both fresh brain tissue samples and archived brain tissue samples dating back ten years.

[0028] In one embodiment, the system is configured for the clinical diagnosis and surveillance of rabies in the West and Central African subregions.

[0029] In one embodiment, the system (100) further comprises a sequencing unit (116) configured to sequence gel-purified PCR amplicons for definitive confirmation of rabies virus detection.

[0030] In one embodiment, the system is configured to overcome diagnostic limitations caused by single-nucleotide polymorphisms in primer target regions of universal lyssavirus primers. This is accomplished by using region-specific primers designed based on genetic diversity data from 52 complete genome sequences of rabies virus isolates.

[0031] The present invention relates to a system for PCR-based diagnostic detection of rabies virus in brain tissue samples with high sensitivity, specificity, and accuracy. The system comprises several functional units configured to provide improved diagnostic performance with superior sensitivity, specificity, and accuracy compared to conventional diagnostic methods.

[0032] The system includes a sample preparation unit configured for processing brain tissue samples from suspected infected canine species. This sample preparation unit was collected using either the rapid occipital technique or conventional necropsy sampling procedures. This flexibility in sample handling ensures the system's applicability under diverse field conditions and in laboratory environments, such as those common in West and Central African diagnostic facilities. The sample preparation unit is specifically designed to maintain sample integrity while preparing the tissue for subsequent molecular analysis.

[0033] The RNA extraction unit utilizes advanced extraction techniques that can utilize either traditional phenol / chloroform extraction methods or modern, commercially available RNA extraction kits. This unit ensures efficient extraction and quantification of total RNA from brain tissue samples, thus laying the foundation for successful subsequent molecular analysis.

[0034] A dedicated cDNA synthesis unit converts the extracted RNA into complementary DNA using commercially available reverse transcription kits. This critical conversion step enables subsequent PCR amplification of the viral target sequences. The unit is configured to ensure optimal conditions for efficient reverse transcription while maintaining the integrity of the genetic material.

[0035] The core innovation lies in the PCR amplification unit, which uses novel region-specific primers designated ACENTDFB@ABU ZARIA1 (forward primer) and ACENTDFB@ABU ZARIA2 (reverse primer). These primers were carefully designed based on a comprehensive analysis of 52 whole genome sequences of rabies virus isolates from Nigeria and Cameroon, supplemented by reference sequences from neighboring West and Central African countries. The primers target positions 92 to 348 of the N gene and generate amplicons of 256 base pairs when rabies virus is present in the sample. The system incorporates a precision thermal cycler configured to perform PCR amplification under carefully optimized conditions that maximize sensitivity while maintaining specificity.The thermocycling parameters were specifically adapted to accommodate the unique properties of the region-specific primers and the genetic diversity of African lyssavirus strains. Visualization of PCR results is achieved by an integrated electrophoresis unit that performs agarose gel electrophoresis, allowing unambiguous identification of the expected 256 bp amplicon. The system includes a gel documentation system that captures, records, and analyzes electrophoresis results, enabling permanent documentation of diagnostic results for clinical and monitoring purposes.

[0036] The region-specific primers are characterized by exceptional performance characteristics: they achieve 100% query coverage, a percent identity between 99.61% and 100%, and E-values ​​below 0.001 when analyzed with the Basic Local Alignment Search Tool. These values ​​confirm the high specificity of the primers and their superior binding affinity to target sequences compared to existing universal primers, whose performance decreases due to single nucleotide polymorphisms in African lyssavirus strains.

[0037] The system's versatility extends to its ability to process both fresh brain tissue samples and archived samples from the past ten years. This makes it indispensable for retrospective studies and longitudinal surveillance programs. This capability enables comprehensive epidemiological investigations and supports the development of evidence-based rabies control strategies in endemic areas.

[0038] An optional sequencing unit can be integrated into the system to definitively confirm rabies virus detection by sequencing gel-purified PCR amplicons. This additional feature increases diagnostic confidence and supports phylogenetic analysis for understanding viral evolution and transmission patterns.

[0039] The invention specifically addresses the technical challenges posed by the high genetic diversity of lyssaviruses in Africa, which have historically compromised the accuracy of universal diagnostic primers. By integrating region-specific design principles and comprehensive genomic analyses, the system overcomes these limitations and provides reliable diagnostic performance tailored to the unique epidemiological context of West and Central African rabies strains.

[0040] Fig. 2 shows a block diagram illustrating the workflow of the proposed system according to an embodiment of the present disclosure.

[0041] Fig.Figure 2 shows the comprehensive functionality of the PCR-based rabies diagnostic system. This includes sampling from slaughtered dogs at dog markets and slaughterhouses, as well as archived rabies-positive samples from the National Veterinary Research Institute (NVRI), Nigeria, and the National Veterinary Laboratory (LANAVET), Cameroon. The system's sample preparation unit processed brain tissue samples obtained from dogs using rapid smear and necropsy techniques. The collected samples were processed in the system's RNA extraction unit for total RNA isolation and quantification, followed by the cDNA synthesis unit for reverse transcription. The PCR amplification unit, equipped with the region-specific primers ACENTDFB@ABU ZARIA1 and ACENTDFB@ABU ZARIA2, performed amplification at positions 92 to 348 of the N gene to generate 256 bp amplicons.The thermocycler component performed optimized PCR conditions, while the electrophoresis unit visualized the results using agarose gel electrophoresis. The gel documentation system recorded the amplicons, while the optional sequencing unit provided final confirmation by sequencing the gel-purified amplicons. This integrated systems approach enabled the molecular characterization and validation of the region-specific diagnostic tool, which was specifically developed for the genetic diversity of West and Central African lyssaviruses.

[0042] Single nucleotide polymorphisms have been detected in the primer target regions of the N gene, particularly affecting the universal lyssavirus primers (JW6 UNI and JW12 / N165-1N165-146) commonly used for rabies diagnosis. In silico and in vitro experiments showed that these polymorphisms significantly impair the sensitivity of existing primers. This necessitated the development of a novel PCR-based diagnostic system.

[0043] The system's sample preparation unit is designed to process two different sample sets to enable a comprehensive understanding of circulating rabies virus strains in West and Central Africa. The first set included 700 recent canine brain tissue samples from Cameroon and Nigeria, while the second set contained 100 archived brain tissue samples dating back 10 years from specialized rabies diagnostic facilities in Nigeria and Cameroon. The system's RNA extraction unit processed these samples using serological and molecular analysis methods, generating 52 complete genome sequences of rabies virus isolates from both countries. This extensive genome dataset, combined with reference genome sequences available in GenBank from neighboring West and Central African countries, formed the basis for the development of the region-specific primers integrated into the PCR amplification unit.

[0044] The PCR amplification unit was configured with synthesized primers designated ACENTDFB@ABU ZARIA1 and ACENTDFB@ABU ZARIA2, which account for the genetic diversity of African lyssavirus strains while targeting conserved genomic regions. The thermal cycler component was optimized to enable detection of rabies virus in brain tissue samples and generate the expected amplicon size of 256 base pairs. The specificity of the system was validated using analysis with the Basic Local Alignment Search Tool. This demonstrated that the region-specific primers exhibited higher binding affinity to target sequences compared to universal primers.

[0045] BLAST analysis confirmed the primer's exceptional performance characteristics. It demonstrated 100% query coverage, percent identity between 99.61% and 100%, and E-values ​​below 0.001, indicating highly specific target sequence matches and improved diagnostic accuracy. The sequencing unit provided final confirmation of rabies virus detection by sequencing gel-purified PCR amplicons processed by the electrophoresis unit. The system's detection capabilities were thoroughly characterized through serial dilution analyses, while comprehensive performance metrics such as sensitivity, specificity, accuracy, kappa value, negative and positive predictive value were determined through comparative analysis with the gold standard of the direct fluorescent antibody technique.

[0046] The diagnostic system demonstrated robust validation using both archived and current field samples from Nigeria and Cameroon. The sample preparation unit successfully processed samples obtained using both the rapid occipital sampling method and the conventional autopsy sampling method, thereby improving the system's utility for rabies detection under diverse field conditions.

[0047] The system starts its operation with the sample preparation unit, which processes brain tissue samples from suspected infected dogs or other canine species using the rapid occipital technique or autopsy technique. The RNA extraction unit extracts and quantifies total RNA using Nanodrop technology. It is configured to use either phenol-chloroform extraction or commercially available RNA extraction kits such as the Zymo RNA extraction kit according to the manufacturer's instructions. The cDNA synthesis unit converts the extracted RNA into complementary DNA using commercially available cDNA synthesis kits, including the Superscript III first-strand synthesis kit, according to the manufacturer's instructions. The PCR amplification unit determines whether the extracted RNA contains rabies virus genotype I of lyssavirus using the region-specific primers configured in the system.The unit uses two sets of primers: the forward primer ACENTDFB@ABU ZARIA1 and the reverse primer ACENTDFB@ABU ZARIA2. These primers alone are sufficient to confirm the presence of rabies virus genotype 1 in the sample. The primers were designed using comprehensive genetic data from 52 complete rabies virus genome sequences obtained during development, supplemented by reference sequences, predominantly from the West and Central African subregions. The PCR amplification unit targets positions 92 to 348 of the rabies virus N gene, which serves as the diagnostic gene, and generates amplicons with a size of 256 base pairs. The integrated thermal cycler performs PCR analysis under optimized conditions using the primers ACENTDFB@ABU ZARIA1 and ACENTDFB@ABU ZARIA2 to detect rabies virus infection in suspected canine species.The system's performance was validated through testing with canine brain tissue samples, confirming the accuracy predicted by in silico analysis. The electrophoresis unit visualizes PCR results using agarose gel electrophoresis, while the optional sequencing unit can sequence gel-purified amplicons for definitive confirmation of rabies virus detection.

[0048] The present invention comprises a comprehensive PCR-based diagnostic system with region-specific primers specifically tailored to the genetic diversity of rabies virus strains in West and Central Africa for the detection of canine rabies virus. The PCR amplification unit uses novel primers designated ACENTDFB@ABU ZARIA1 (forward primer) and ACENTDFB@ABU ZARIA2 (reverse primer), which can detect canine rabies virus strains in West and Central African subregions using conventional RT-PCR amplification. The system offers optimized operating conditions for the ACENTDFB@ABU ZARIA1 and ACENTDFB@ABU ZARIA2 primer sets, which are configured via the thermocycler component to ensure maximum diagnostic performance. The primers are specifically tailored to conserved regions throughout the viral genome and detect rabies virus in brain tissue samples with exceptional sensitivity and specificity.The diagnostic system demonstrates superior performance characteristics compared to existing RT-PCR-based diagnostic tools for rabies, with a diagnostic sensitivity of 96.2%, a specificity of 88.9%, an accuracy of 94.3%, and a detection limit of 4.5 × 10. -9ng / µl. The system's region-specific primers exhibit 100% query coverage, percent identity ranging from 99.61% to 100%, and E-values ​​of less than 0.001, confirming high specificity and superior binding affinity to target sequences. The system offers significant cost advantages over existing diagnostic approaches and represents a cost-effective solution with lower reagent and consumable costs compared to seminested RT-PCR systems, which require three primers and involve dual PCR runs, and real-time RT-PCR systems, which require expensive probe-based detection. This cost-effectiveness makes the system particularly suitable for resource-constrained environments where financial constraints often limit access to diagnostic tools.The PCR amplification unit generates amplicons of 256 base pairs, significantly smaller than the 508 bp amplicons generated by conventional diagnostic tools. This reduced amplicon size enables the system to detect rabies virus even in RNA-depleted samples and specimens with low viral loads, improving diagnostic performance under challenging sample conditions. Its versatility extends to the ability to process both archived brain tissue samples that are ten years old and fresh field samples, demonstrating superior performance with fresh samples while maintaining effectiveness across diverse sample types and storage conditions.This comprehensive validation with samples from Nigeria and Cameroon confirms the effectiveness of the system in detecting rabies virus infections in different operational contexts and makes it an optimal solution for improving rabies diagnosis and surveillance in West and Central African subregions.

[0049] The integrated system architecture, which includes units for sample preparation, RNA extraction, cDNA synthesis, PCR amplification, electrophoresis visualization, and documentation, provides a complete diagnostic workflow specifically tailored to the unique epidemiological and genetic characteristics of African lyssavirus strains. This overcomes the limitations of universal diagnostic approaches that fail to account for regional genetic diversity.

[0050] The drawings and the foregoing description illustrate examples of embodiments. Those skilled in the art will recognize that one or more of the described elements may well be combined into a single functional element. Alternatively, certain elements may be separated into multiple functional elements. Elements of one embodiment may be added to another embodiment. For example, the order of the processes described herein may be changed and is not limited to the manner described herein. Furthermore, the actions of a flowchart need not be performed in the order shown; nor do all actions need to be performed. Also, actions that are not dependent on other actions may be performed in parallel with the other actions. The scope of the embodiments is in no way limited by these specific examples.Numerous variations, whether explicitly stated in the specification or not, such as differences in structure, dimensions, and use of materials, are possible. The scope of the embodiments is at least as broad as indicated in the following claims.

[0051] Advantages, further benefits, and solutions to problems have been described above with reference to specific embodiments. However, the advantages, advantages, solutions to problems, and any components that may result in or enhance an advantage, advantage, or solution are not to be construed as critical, required, or essential features or components of any or all of the claims. REFERENCES 100 The system also includes a sequencing unit. 102 Sample preparation unit 104 RNA extraction unit 106 Cdna synthesis unit 108 PCR amplification unit 110 thermal cyclers 112 Electrophoresis unit 114 Gel documentation system 116 sequencing unit 202 Sample of brain tissue 204 Detection of Rabv antigen by (DFAT) 206 Virus replication in Balb / C mice (Mit) 208 Total RNA extraction 210 Cdna synthesis 212 Multiplex PCR 214 Whole genome sequencing 216 Sequence Analysis Using Bioinformatics Tools 218 Molecular Characterization and Phylogenetic Analysis 220 Geospatial mapping of rabies virus infection in the study areas 222 Optimization of a region-specific diagnostic tool

Claims

[1] A system for PCR-based diagnostic detection of rabies virus in brain tissue samples with high sensitivity, specificity and accuracy, consisting of: (a) a sample preparation unit configured to handle brain tissue samples obtained from dogs using the rapid occipital technique or the autopsy sampling technique; b) an RNA extraction unit configured to extract and quantify total RNA from the brain tissue samples; c) a cDNA synthesis unit configured to convert extracted RNA into complementary DNA; (d) a PCR amplification unit configured to amplify target sequences using region-specific primers targeting positions 92 to 348 of the N gene of rabies virus; (e) a thermal cycler configured to perform PCR amplification under optimized conditions; f) an electrophoresis unit configured to visualise PCR results by agarose gel electrophoresis; and g) a gel documentation system configured to record and analyze electrophoresis results. [2] The system of claim 1, wherein the RNA extraction unit is configured to use phenol / chloroform extraction or commercially available RNA extraction kits for complete RNA isolation. [3] The system of claim 1, wherein the cDNA synthesis unit is configured to use commercially available cDNA synthesis kits for reverse transcription of extracted RNA. [4] The system of claim 1, wherein the PCR amplification unit is configured to use a forward primer designated ACENTDFB@ABU ZARIA1 and a reverse primer designated ACENTDFB@ABU ZARIA2 to amplify rabies virus genotype I sequences, wherein the region-specific primers are configured to have high specificity with a query coverage of 100%, a percent identity of 100% to 99.61%, and E-values ​​of less than 0.001 when analyzed with the Basic Local Alignment Search Tool. [5] The system of claim 1, wherein the PCR amplification unit is further configured to generate amplicons having a size of 256 base pairs when rabies virus is present in the sample. [6] The system of claim 1, wherein the system is configured to detect rabies viruses in both fresh brain tissue samples and archived brain tissue samples dating back ten years. [7] The system of claim 1, wherein the system is configured for the clinical diagnosis and monitoring of rabies in the West and Central African sub-regions. [8] The system of claim 1, further comprising a sequencing unit configured to sequence gel-purified PCR amplicons to definitively confirm detection of rabies virus. [9] The system of claim 1, wherein the system is configured to overcome diagnostic limitations caused by single nucleotide polymorphisms in primer target regions of universal lyssavirus primers by using region-specific primers designed based on genetic diversity data from 52 complete genome sequences of rabies virus isolates.

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