Equine Rotavirus Group B Detection Primers and Probes
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Solution Overview
Problem
Current methods are inadequate for diagnosing Equine Rotavirus Group B, particularly in cases of foal diarrhea in central Kentucky, where existing techniques have not been successful in detection.
Innovation Solution
Development of specific primers and probes for detecting Equine Rotavirus Group B, including forward and reverse primers and probes targeting various segments of the virus, used in conjunction with PCR techniques such as real-time quantitative PCR for accurate detection and quantification of viral RNA in samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If existing diagnostic techniques are used for Equine Rotavirus Group B, then the diagnostic process can be maintained with current methods, but detection success rate is insufficient and diagnosis remains inadequate
Solution Approach 1:
The diagnostic approach segments the rotavirus detection into multiple targeted regions across different viral segments (VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4, NSP5, NSP6), allowing specific primers and probes to be designed for each segment. This segmentation enables comprehensive coverage of the viral genome, improving detection reliability by targeting multiple conserved regions that may vary between strains.
Solution Approach 2:
The invention changes the diagnostic parameters by developing a customized primer and probe set specifically optimized for Equine Rotavirus Group B, with sequences tailored to match the viral genome segments. The primers are designed with specific melting temperatures and binding affinities, while probes incorporate fluorescent reporters and quenchers for sensitive detection. This parameter optimization significantly improves detection accuracy compared to generic diagnostic methods.
2Reliability
If a comprehensive primer and probe set targeting multiple viral segments is developed, then detection reliability improves, but the complexity of the diagnostic system increases
Solution Approach 1:
The primer and probe set is designed with multi-functionality, where each primer pair and probe combination can target multiple viral segments simultaneously. The universal design allows a single diagnostic system to detect various rotavirus segments (VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4, NSP5, NSP6) using the same basic platform, reducing the need for separate diagnostic assays for each segment and thereby managing system complexity while maintaining comprehensive detection capability.
Solution Approach 2:
The invention uses copied and adapted primer sequences that are derived from known rotavirus genome regions but specifically optimized for equine rotavirus group B. By copying and modifying existing primer designs from other rotavirus species or strains, the system achieves high detection reliability without requiring entirely new diagnostic tools for each viral segment, thus reducing overall system complexity.
3Measurement precision
If real-time quantitative PCR is used for viral RNA detection, then quantification accuracy improves, but the time and resources required for analysis increase
Solution Approach 1:
The primer and probe sets are pre-designed and pre-optimized for real-time quantitative PCR, with sequences and concentrations determined in advance through extensive characterization. The probes are pre-labeled with fluorescent reporters and quenchers, and the reaction conditions are pre-established. This preliminary preparation allows for rapid, accurate quantification of viral RNA without requiring time-consuming optimization steps during actual diagnosis, thus reducing analysis time while maintaining high quantification accuracy.
Solution Approach 2:
The invention replaces traditional mechanical or manual detection methods with real-time quantitative PCR technology, which uses fluorescent signal detection instead of manual analysis. This substitution enables automated, high-throughput quantification of viral RNA, significantly improving measurement precision while reducing the time and labor resources required compared to conventional diagnostic techniques.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables effective detection and quantification of Equine Rotavirus Group B, improving diagnostic capabilities and aiding in the management of foal diarrhea cases by providing a reliable method for identifying the virus in clinical samples.
Implementation Method 1
used in conjunction with PCR techniques such as real-time quantitative PCR for accurate detection and quantification of viral RNA in samples
Data Source
AI summary
Provided herein are a set of primers and probes for detecting rotavirus. The set of primers and probes includes at least one forward primer selected from the group including SEQ ID NOS: 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, 119, 122, 125, 128, 131, 134, 137, 140, 143, 146, 149, 152, 155, 158, 161, 164, 167, 204, 207, 210, and combinations thereof; at least one reverse primer selected from the group including SEQ ID NOS: 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153, 156, 159, 162, 165, 168, 205, 208, 211, and combinations thereof; and at least one probe selected from the group including SEQ ID NOS: 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139, 142, 145, 148, 151, 154, 157, 160, 163, 166, 169, 206, 209, 212, and combinations thereof. Also provided herein is a method for detecting rotavirus using the set of primers and probes.