ABSTRACT
As of now, the global COVID-19 pandemic caused by SARS-CoV-2, which began in 2019, has been effectively controlled. However, the symptoms of influenza A virus infection were similar to those of SARS-CoV-2 infection, but they required different treatment approaches. To make the detection more accurate and the treatment more targeted. We developed a system that integrates RPA and CRISPR assays, allowing for the rapid, highly specific, and sensitive detection and differentiation of SARS-CoV-2, H1N1, and H3N2. Under isothermal amplification conditions, the RPA-CRISPR Cas12a detection system achieved a detection limit as low as 5 copies per µL, demonstrating excellent specificity. The measurement time was approximately 30 minutes. The RPA-CRISPR Cas12a detection system combined with the microfluidic chip we designed to simultaneously detect three viruses, providing a potential solution for efficient and reliable diagnosis.
Subject(s)
COVID-19 , Influenza, Human , SARS-CoV-2 , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Humans , Influenza, Human/diagnosis , COVID-19/diagnosis , Influenza A Virus, H1N1 Subtype/isolation & purification , Influenza A Virus, H1N1 Subtype/genetics , Nucleic Acid Amplification Techniques/methods , Influenza A Virus, H3N2 Subtype/isolation & purification , Influenza A Virus, H3N2 Subtype/genetics , CRISPR-Cas Systems/genetics , Lab-On-A-Chip Devices , Microfluidic Analytical Techniques/methods , Microfluidic Analytical Techniques/instrumentation , Limit of Detection , Molecular Diagnostic Techniques/methods , Influenza A virus/isolation & purification , Influenza A virus/genetics , Sensitivity and SpecificityABSTRACT
Aquaculture plays an increasingly important if not critical role in the current and future world food supply. Aeromonas hydrophila, a heterotrophic, Gram-negative, bacterium found in fresh or brackish water in warm climates poses a serious threat to the aquaculture industry in many areas, causing significant economic losses. Rapid, portable detection methods of A. hydrophila are needed for its effective control and mitigation. We have developed a surface plasmon resonance (SPR) technique to detect PCR (polymerase chain reaction) products that can replace agarose gel electrophoresis, or otherwise provide an alternative to costlier and more complicated real-time, fluorescence-based detection. The SPR method provides sensitivity comparable to gel electrophoresis, while reducing labor, cross-contamination, and test time, and employs simpler instrumentation with lower cost than real-time PCR.
Subject(s)
Aeromonas hydrophila , Surface Plasmon Resonance , Aeromonas hydrophila/genetics , Real-Time Polymerase Chain Reaction , Biological AssayABSTRACT
Seneca Valley virus (SVV) is related to vesicular disease in pigs, and its clinical symptoms are indistinguishable from other notifiable clinical symptoms of vesicular disease such as foot-and-mouth disease. The rapid and accurate detection of SVV is essential to confirm the pathogenic factors and initiate the implementation of control measures. The development of a rapid, simple, convenient, and low-cost molecular (nucleic acid amplification) test that can be used at the sample collection point has been identified as a key component for controlling SVV. This study describes the development and demonstration of recombinase polymerase amplification (RPA) test targeting the conserved regions of SVV for detection of SVV. The Primers and probes designed by us have shown good sensitivity and specificity in RPA test, which is helpful for RPA to be an effective tool for rapid diagnosis of SVV.