Article sharing | A real-time fluorogenic recombinase polymerase amplification microfluidic chip (on-chip RPA) for multiple detection of pathogenic microorganisms of penaeid shrimp

The article proposes a new microfluidic chip technology for multiplex detection of shrimp pathogenic microorganisms. The research team integrated multi-enzyme isothermal rapid nucleic acid amplification (MIRA) technology and real-time fluorescence detection methods. This microfluidic chip provides a fast, sensitive and efficient solution for on-site detection of major shrimp pathogens.

Research background and significance

The global shrimp farming industry is one of the most important economic components in aquaculture, but the industry has long faced threats from a variety of pathogenic microorganisms. These pathogens often lead to large-scale death of shrimps, seriously affecting production and economic benefits. Including acute hepatopancreatic necrosis disease (AHPND), white spot syndrome virus (WSSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), shrimp hemocyte iridovirus (SHIV), and enterohepaticus (EHP), etc. Viruses, bacteria and parasites pose major threats to the shrimp industry.

Traditional pathogen detection methods, such as polymerase chain reaction (PCR technology), have high sensitivity and specificity, but are complex to operate, take a long time, and rely on laboratory equipment. These limitations make PCR technology unsuitable for rapid on-site testing. MIRA technology has become a molecular detection tool suitable for on-site application due to its rapid amplification characteristics under constant temperature and normal temperature.

MIRA technology principle

Multi-enzyme constant-temperature rapid nucleic acid amplification technology (MIRA technology) is a constant-temperature amplification technology that relies on the synergy of multiple functional proteins at room temperature to achieve rapid amplification of nucleic acids. It is a truly portable on-site rapid nucleic acid detection technology. Compared with PCR, MIRA has more advantages.

Design and innovation of microfluidic chips

Schematic diagram of RPA detection platform on chip for several important shrimp pathogenic microorganisms

(A) Structure of a disc-type microfluidic chip. 1: Reaction chamber; 2: Ball valve; 3: Exhaust port; 4: Sampling port;

(B) Exploded view. 5: Heat sealing film; 6: Chip substrate.

(C) Detailed layout of the six sets of primers within the sector. Each primer set is pre-fixed in a designated reaction chamber.
(D) Complete operating procedure on-chip RPA detection platform.

The article proposes a microfluidic chip integrated with MIRA technology for multiplex detection of shrimp pathogenic microorganisms. Microfluidic technology can control liquids within micron-level channels for precise manipulation, thereby achieving efficient and low-cost multi-target detection.

The chip has the following key design features:

●Centrifugal microfluidic chip

The chip adopts a centrifugal drive design, using centrifugal force to evenly distribute the sample into each reaction chamber. This design simplifies the operation process, and the operator can complete the entire reaction by simply injecting the sample.

●Multi-target detection

The chip is pre-embedded with the bacterial types that need to be detected: acute hepatopancreatic necrosis (V-AHPND), white spot syndrome virus (WSSV), infectious subcutaneous necrosis virus (IHHNV), shrimp hemocyte iridovirus (SHIV) and Enterohepatitis hepatica (EHP). ) Primer probes for five pathogens. This design can simultaneously detect six gene targets in one reaction, enabling rapid screening of multiple pathogens.

●Real-time fluorescence detection

The MIRA technology amplification reaction in the chip is combined with a fluorescent probe, which can monitor the progress of the amplification reaction in real time. The probe design allows the fluorescent signal to be released only when the amplification product is generated, thus achieving rapid and sensitive quantitative detection.

●low concentration reaction system

Each reaction unit of the chip only requires a reaction volume of 5 μL, which greatly reduces reagent consumption. When the entire system is running, up to four samples can be processed simultaneously, and each sample can detect five pathogens, greatly improving detection efficiency.

Sensitivity, specificity and repeatability

The performance of the chip system includes detection of symmetry, resonance and repeatability.

●Sensitivity

Experimental results show that the chip can detect target gene copy numbers as low as 10 copies/μL, with sensitivity that is comparable or even higher than traditional PCR methods. This high sensitivity ensures that the technology can detect early-infected individuals in practical applications, thereby enabling early screening of pathogens.

●Specificity

The experiment conducted cross-reaction tests on a variety of non-target pathogens, and the results showed that the chip is highly specific to the target pathogens and can accurately distinguish various types of pathogens. All tests for Acute Hepatopancreatic Necrosis Disease (V-AHPND), White Spot Syndrome Virus (WSSV), Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV), Shrimp Hemacytic Iridescence Virus (SHIV) and Enterohepatitis Hepatica (EHP) The results all showed no cross-reaction, indicating that this method can effectively avoid non-specific amplification.

●Repeatability

In order to evaluate the stability of the method, the research team conducted multiple repeated experiments. The results showed that the coefficient of variation (CV value) between different experiments was less than 10%, showing good experimental repeatability and consistency.

Field applications and advantages

The design of the MIRA technology microfluidic chip gives it significant field application advantages, especially suitable for rapid detection and monitoring of pathogens in shrimp farming:

●Convenient operation process

The entire detection process only requires injecting the sample into the chip and starting the reaction device. It requires almost no manual operation and is suitable for non-professionals to use on site.

●Quick response capability

The system can obtain test results within 20 minutes, greatly shortening the test time. This is crucial for rapid disease response in farms and reducing the spread of pathogens.

●Multiplex detection capability

Five major pathogens can be identified in one test, which greatly improves detection efficiency and saves detection costs and time.

MIRA technology prospects

Although PCR technology has long been rated as the gold standard for nucleic acid amplification detection, MIRA technology has demonstrated great application potential due to its rapid reaction at constant temperature, simple operation, and low equipment requirements. By comparing with PCR combined with DNA sequencing, it was found that the detection results of the isothermal amplification chip are consistent with the PCR results in terms of sensitivity and specificity, and the reaction time of MIRA technology is significantly shortened. Therefore, MIRA technology microfluidic chips are considered to be a technology more suitable for field applications, especially in environments that require rapid and large-scale pathogen detection.

Microfluidic chips based on multi-enzyme isothermal rapid nucleic acid amplification (MIRA) technology provide an effective tool for the detection of pathogenic microorganisms in shrimp farming. This technology has the advantages of high sensitivity, good specificity, easy operation, and fast detection speed, and has broad application prospects in aquaculture disease prevention and control. With further development and optimization of the technology, the system is expected to be applied to the detection of more aquatic pathogens, enabling broader disease prevention and monitoring.

The research results provide a reliable early detection method for the aquaculture industry, which can effectively reduce the risk of disease outbreaks, improve the production efficiency and output of farms, and lay a technical foundation for the sustainable development of the aquaculture industry.