CRISPR-Cas13a ALV-J Detection Kit With RAA Signal Amplification
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Solution Overview
Problem
Current methods for detecting Avian Leukosis Virus Subgroup J (ALV-J) are cumbersome, costly, and require sophisticated equipment, making them unsuitable for remote areas, while existing CRISPR-Cas systems have shown promise in detecting other viruses but lack specificity and sensitivity for ALV-J.
Innovation Solution
A CRISPR/Cas13a system is developed with crRNA designed for the gp85 gene in ALV-J's whole genome env sequence, combined with recombinase aided amplification (RAA) and a lateral flow test strip for rapid, sensitive, and specific detection of ALV-J.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If virus isolation method is used for ALV-J detection, then detection accuracy is improved, but detection time increases to 7-9 days and cost increases
Solution Approach 1:
The patent replaces the mechanical virus isolation method (cell culture) with a molecular detection system combining CRISPR-Cas13a and RAA. The CRISPR-Cas13a system provides specific recognition and cleavage of ALV-J viral RNA, while RAA amplifies the target sequence, achieving rapid detection within hours rather than days without requiring cell culture facilities.
Solution Approach 2:
The patent introduces an intermediary detection system that bridges the gap between virus isolation and direct detection. The CRISPR-Cas13a system acts as an intermediary by first amplifying viral RNA through RAA, then using Cas13a-mediated cleavage of a fluorescent reporter RNA to generate a detectable signal, thus avoiding the need for direct virus isolation while maintaining high specificity.
2Speed
If CRISPR-Cas12a system is used for detection, then detection speed is improved, but specificity deteriorates due to inability to directly recognize RNA
Solution Approach 1:
The patent segments the detection process into two distinct modules: (1) RAA amplification module that converts viral RNA to DNA and amplifies the target sequence, and (2) CRISPR-Cas13a detection module that specifically recognizes and cleaves the amplified DNA. This segmentation allows each module to optimize for its specific function while maintaining overall system specificity and speed.
Solution Approach 2:
The patent introduces DNA as an intermediary molecule between the viral RNA target and the CRISPR-Cas13a detection system. The RAA process first converts viral RNA to cDNA and amplifies it, creating a DNA intermediate that the Cas13a system can then specifically recognize and process, thus enabling both speed and specificity.
3Reliability
If conventional detection methods are used, then detection capability is achieved, but equipment cost and operational complexity increase
Solution Approach 1:
The patent employs disposable, single-use reagent components including pre-mixed RAA reagents, crRNA molecules, and fluorescent reporter RNAs that can be used once and discarded. This eliminates the need for expensive, complex equipment while maintaining reliable detection capability, making the system suitable for resource-limited settings.
Solution Approach 2:
The patent replaces complex mechanical detection equipment (flow cytometers, plate readers, cell culture incubators) with a simple optical detection system that measures fluorescent signal intensity. The CRISPR-Cas13a-mediated cleavage of fluorescent reporter RNA generates a measurable signal that can be detected with basic fluorescence equipment or even visual inspection, dramatically reducing equipment requirements.
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
The system achieves rapid, sensitive, and cost-effective detection of ALV-J with a minimum limit of 5.4×100 copies/μL, requiring no expensive equipment, and can differentiate ALV-J from other avian viruses, suitable for on-site detection.
Implementation Method 1
a CRISPR/Cas13a system
Implementation Method 2
recombinase aided amplification (RAA)
Data Source
AI summary
Provided is a kit for rapid detection of avian leukosis virus subgroup J (ALV-J) based on a CRISPR/Cas13a system. The method is based on the combination of the CRISPR/Cas13a system and recombinase aided amplification (RAA) for ALV-J detection. An oligonucleotide probe is designed as a substrate for CRISPR/Cas13a trans-cleavage and produces a detectable signal. The method can substantially improve detection sensitivity by amplifying a detection signal twice by RAA and T7 transcription. The detection method further exhibits excellent specificity, allowing for clear differentiate from other avian viruses. It does not require expensive experimental equipment and special laboratory environment, and it is rapid and efficient. The method is of great significance for biological research and on-site detection of ALV-J.