Article Sharing | Rapid Detection of Hepatopancreatic microsporidium in Grass Shrimp Using Isothermal Recombinase Polymerase Amplification (MIRA)

Grass shrimp is an important economic species in China’s freshwater aquaculture industry. However, in recent years, the Hepatopancreatic microsporidium (EHP), a parasite infecting the shrimp’s hepatopancreas, has severely impacted the industry. Since there is currently no effective drug or vaccine to treat this disease, rapid and accurate detection is crucial to prevent its spread.

A. Magnified 9.0×103 times; B. Magnified 5.0×104 times.
Qiao Yi, Shen Hui, Wan Xihe, Fan Xianping, Jiang Ge, Li Hui, Wang Libao, Shi Wenjun, Cheng Jie. Isolation and Morphological Observation of Hepatopancreatic microsporidium in Litopenaeus vannamei. Journal of Fishery Sciences of China, 2018, 25(5): 1051-1058. DOI: 10.3724/SP.J.1118.2018.17430.

Detection of EHP primarily relies on molecular biology methods, with PCR technology being the most common. However, while PCR offers high sensitivity, it requires expensive PCR equipment and a specialized laboratory environment, making it unsuitable for field use. To address this issue, the research team developed two rapid detection methods based on MIRA technology. These methods offer high sensitivity, quick response, and easy operation, enabling efficient and rapid detection of EHP in shrimp on-site without the need for complex laboratory equipment, thus filling the gap in current detection methods.

Amplification Reagents Used in the Study:

• DNA Isothermal Rapid Amplification Kit (Basic)
• DNA Isothermal Rapid Amplification Kit (Colloidal Gold Strip Type)

MIRA technology is a multi-enzyme isothermal rapid nucleic acid amplification method. Its core principle involves the coordinated action of multiple functional proteins at ambient temperatures to achieve rapid nucleic acid amplification, enabling truly portable, on-site nucleic acid detection.

Research Methods

Sample Extraction

The research team collected both infected and healthy grass shrimp samples from three shrimp farms in Liaoning Province, extracting DNA from the shrimp’s hepatopancreas.

Primer Design

Three sets of MIRA primers were designed targeting the S8 serine protein gene of EHP (GenBank Accession Number: ON 182063). This gene has unique active sites, ensuring the specificity of detection.

MIRA-AGE and MIRA-LFD Detection

(A) Optimized RPA-AGE reaction temperatures. M: DL 500 marker; N: Negative control; Strains 1-5: 25°C, 30°C, 37°C, 39°C, and 42°C.
(B) Optimized RPA-AGE reaction times. M: DL 500 marker; N: Negative control; 1: 10 min; 2: 20 min; 3: 30 min; 4: 40 min.
(C) Optimized RPA-LFD reaction temperatures. N: Negative control; 1: 25°C; 2: 30°C; 3: 37°C; 4: 39°C; 5: 42°C.
(D) Optimized RPA-LFD reaction times. N: Negative control; 1: 5 min; 2: 10 min; 3: 20 min.

The two MIRA detection methods, MIRA-AGE (agarose gel electrophoresis) and MIRA-LFD (colloidal gold lateral flow immunoassay), were used to detect EHP. The research team optimized the reaction temperatures and times through experimentation. MIRA-AGE amplified the target DNA in 30 minutes at 37°C, while MIRA-LFD completed amplification in just 10 minutes at 37°C, with results readable within 5 minutes.

Experimental Results

Specificity and Sensitivity

(A) Specificity of RPA-AGE detection. M: DL 1000 marker; P: Nosema bombycis; 1: EHP; 2: Encephalitozoon; 3: Microsporidia; 4: White spot syndrome virus; 6: Abnormal; N: Negative control.
(B) Specificity of RPA-LFD detection. P: Nosema bombycis; N: Negative control; 1: EHP; 2: Encephalitozoon; 3: Microsporidia; 4: White spot syndrome virus; 6: Abnormal.
(C) Sensitivity of RPA-AGE with gradient dilutions of recombinant plasmids. M: DL 500 marker; N: Negative control; 1: 4.7×10^5 copies/µL; 2: 4.7×10^4 copies/µL; 3: 4.7×10^3 copies/µL; 4: 4.7×10^2 copies/µL; 5: 4.7 copies/µL; 6: 4.7 copies/µL.
(D) Sensitivity of RPA-LFD with gradient dilutions of recombinant plasmids. N: Negative control; 1: 4.7×10^5 copies/µL; 2: 4.7×10^4 copies/µL; 3: 4.7×10^3 copies/µL; 4: 4.7×10^2 copies/µL; 5: 4.7 copies/µL; and 6: 4.7 copies/µL.

The experiments demonstrated that MIRA technology has high specificity, accurately detecting EHP without reacting to other pathogens. In terms of sensitivity, both methods had a detection limit of 4.7 copies/µL, significantly higher than the sensitivity of traditional PCR.

Clinical Sample Testing

Results from testing 30 clinical samples showed that MIRA-AGE and MIRA-LFD had a positivity rate of 70%, slightly lower than qPCR's 80% but significantly higher than traditional PCR's 56.7%.

Application Prospects

MIRA-based detection methods allow for rapid amplification of target genes at constant temperatures. In particular, the MIRA-LFD method can deliver results in just 10 minutes without the need for complex laboratory equipment, making it ideal for on-site use in shrimp farms. The high sensitivity and specificity of this technology make it an efficient tool for early detection and control of EHP, thereby protecting the health and development of the grass shrimp farming industry.

Among these methods, the colloidal gold lateral flow immunoassay (MIRA-LFD) is particularly well-suited for on-site applications due to its simplicity and intuitive results. Grass shrimp farmers can detect and respond to EHP infections quickly, improving disease control capabilities, significantly boosting farming efficiency, and reducing economic losses.