Article sharing | Establishment of Micropterus salmoides Rhabdovirus Detection Method Based on CRISPR/Cas13a System

《Establishment of a Micropterus salmoides rhabdovirus detection method based on CRISPR Cas13a system》

张敏琳, 黄枫淇, 左小玲, 梁建韬, 梁凯珊, 单金红, 李宗烊, 喻婕, 罗丽媛, 禤梓杰, 赵会宏, 王庆. 基于CRISPR/Cas13a系统建立大口黑鲈弹状病毒检测方法[J]. 水生生物学报, 2024, 48(2): 283-291. DOI: 10.7541/2023.2023.0199

Micropterus salmoides, also known as California bass or American black bass, is a freshwater fish native to North America. In recent years, it has become an important farmed fish worldwide because of its high market demand: micropterus salmoides has firm meat, few bones, delicious taste, rich nutrition, especially high protein and low fat, which meets consumers' demand for healthy food. Therefore, the market price is relatively high and it is often used in the high-end catering market. Fast growth: micropterus salmoides grows quickly and can generally reach commodity specifications in 6-8 months. It is suitable for short-cycle breeding and can quickly realize investment returns. Strong disease resistance: Compared with other farmed fish, micropterus salmoides has strong adaptability to the environment, less large-scale disease outbreaks, and reduces breeding risks. Ecological breeding: largemouth bass can be mixed with other freshwater fish such as silver carp and grass carp, which helps to achieve pond ecological balance and maximize resource utilization, further improve breeding efficiency and other factors. It is welcomed by diners and aquaculture farmers, and its breeding scale has grown rapidly in recent years.

Although micropterus salmoides are relatively disease-resistant, they may still suffer from some diseases under poor breeding conditions. Among them, Micropterus salmoides rhabdovirus (MSRV) is extremely harmful: it was first discovered in Florida, USA in 1991. The virus was initially detected in wild largemouth bass populations, and then gradually spread to many freshwater lakes and reservoirs in the United States, becoming one of the main pathogens of largemouth bass.

Clinical symptoms of diseased fish

A: Healthy Largemouth Bass, the arrow indicates the yolk sac; B: Diseased Largemouth Bass, the arrow indicates the yolk sac.

The source of transmission is often infected fry or adult fish, polluted water bodies, and wild fish carrying the virus; this virus can be transmitted through direct contact, fecal excretion, and water bodies, and is more likely to occur under poor water quality or high-density breeding conditions. diffusion. The virus can cause lethargy, wandering, abdominal swelling and body distortion in juvenile fish, as well as induce necrotic ulcers and multi-organ bleeding. Once infected, the mortality rate exceeds 90%.

Clinical symptoms of artificially infected micropterus salmoides

A: Healthy largemouth bass; B, C, D: Artificially infected largemouth bass.

Every year, more than 30% of Micropterus salmoides fry are lost due to MSRV. Currently, the treatment and detection methods for the virus are still immature, and there is no specific medicine to treat it. Therefore, it is very necessary to detect the virus in time before infection and take effective preventive measures, which can reduce the losses in the breeding process to a certain extent;

In addition, it is difficult to carry out on-site detection without complex detection equipment with existing detection technology. In the face of this challenge, the research team of South China Agricultural University has developed a new detection method combining MIRA with CRISPR-system. This method has strong specificity, high sensitivity and simple operation, which greatly improves the detection efficiency of MSRV and also helps to detect MSRV as early as possible and take prevention and control measures.

Products used in this article: RNA Isothermal Rapid Amplification Kit (Basic Type)-II

MIRA primer combination screening

Two pairs of different MIRA primers were used to amplify the gene fragment containing the target sequence, and the amplified products were subjected to agarose gel electrophoresis. The electrophoresis graph was located at the position of 100 bp, and bright bands appeared in all three lanes, indicating that there were non-specific amplification products. The amplified target bands at 250 bp in lanes 2 and 3 were respectively quantitatively analyzed by Image J software, and the results of the quantitative analysis were expressed by "gray value". From the analysis results, it can be concluded that the lower the gray value, the higher the primer utilization rate, that is, the higher the primer specific binding amplification efficiency.

The results showed that the specific binding amplification efficiency was high when the MIRA-F2/R2 primer pair was used.

MIRA primer pair screening electrophoresis gel image and gray value analysis

a. Electrophoresis lane M: 2000 bp DNA marker; Electrophoresis lane 1. Negative DNA sample amplification; Electrophoresis lane 2. MIRA-F1/R1 primer pair amplifies the target fragment (224 bp as shown in the box);Electrophoresis lane 3. MIRA-F2/R2 primer pair amplifies the target fragment (255 bp as shown in the box); b. Band gray value analysis (as shown in the box)

CRISPR/Cas13a detection system

After the reaction of CRISPR/Cas13a detection system is completed, it is irradiated with ultraviolet light to observe the results.

CRISPR/Cas13a detection system-fluorescence brightness diagram

1-3. The whole system with RNA standard added; 4. No crRNA added; 5. No Cas13a protein added; 6. No ssRNA reporter probe added; 7. Negative control

CRISPR/Cas13a-MSRV optimal reaction system

We conducted experiments using RNA standards and amplified sample RNA. The positioning speed of the target RNA was better with crRNA2 than with crRNA1. crRNA1 had a stronger final fluorescence signal than crRNA2. The mixed use of crRNA1+crRNA2 in equal proportions could combine the advantages of both and achieve better reaction efficiency.

Optimizing the CRISPR/Cas13a-MSRV detection reaction conditions and detection results

a. Comparison of detection results of standard RNA using crRNA with different spacer sequences;

b. Comparison of fluorescence signal data of standard RNA detected by the detection system at different temperatures;

c. Comparison of fluorescence signal data of standard RNA detected using RNA reporter probes with different concentrations;

d. Comparison of detection results of standard RNA detected using Cas13a/crRNA probe complexes with different concentration ratios;

To explore the effect of reaction temperature on the detection system, different reaction temperatures of 31℃, 34℃, 37℃ and 41℃ were set. The optimal reaction temperature of CRISPR/Cas13a-MSRV detection system was 37℃.

To explore the effect of ssRNA reporter probe concentration on the detection system, within the test range, the fluorescence intensity was positively correlated with the ssRNA reporter probe concentration. The detection effect of ssRNA reporter probe concentration was not much different at 500-700 nmol/L. Considering the actual economic problems, the optimal ssRNA reporter probe concentration was 500 nmol/L.

To explore the effect of different Cas13a and crRNA reaction concentration ratios on the detection system, when the ratio of Cas13a:crRNA was 4:1 and 3:1 based on the ratio of 100 nmol/L per portion, Cas13a reached saturation; when the amount of Cas13a was constant, the intensity of the fluorescence signal was not positively correlated with the increase in the amount of crRNA. Therefore, when the ratio of Cas13a to crRNA is 2:1, the CRISPR/Cas13a-MSRV detection system can achieve maximum detection activity.

Sensitivity evaluation

In order to explore the minimum detection concentration of MSRV by the CRISPR/Cas13a-MSRV detection system, the RNA standard was diluted 10 times in a series and detected by the CRISPR/Cas13a-MSRV detection system. The CRISPR/Cas13a-MSRV detection system can detect at least 100fM of MSRV target RNA. When the target RNA concentration is lower than 100fM, the fluorescence signal detected by the machine is not significantly different from that of the blank control. Therefore, the CRISPR/Cas13a-MSRV detection system can detect at least 100fM of MSRV ssRNA.

Evaluation of the specificity of the CRISPR/Cas13a-MSRV detection system for MSRV

Sample testing

For sick fish samples with obvious signs of illness collected from sea bass farms, we verified whether the pathogen infected by the sick fish was MSRV. We used primers specific to the MSRV capsid protein sequence for PCR amplification, and sent the corresponding bands in the electrophoresis diagram for testing. After comparison, we confirmed that it was MSRV.

Confirmation of MSRV sequence information

a. PCR amplification of 228 bp product band of MSRV primers; b. MSRV capsid protein sequence compared with NCBI, bold sequences are upstream and downstream primer binding sites, and shaded areas are target locations for CRISPR/Cas13a-MSRV crRNA1 binding

The diseased fish samples infected with MSRV were pretreated and preamplified for viral RNA, and then tested with the negative control group. At the same time, the cDNA of the samples was extracted for comparison with conventional qPCR. The positive samples (No. 3, 6, 7, 8, 9, 12, 13, 14 and 15) detected by the CRISPR/Cas13a-MSRV detection system were the same as the results of conventional qPCR, where the CQ value of qPCR was between 18 and 25; while the threshold cycle number of negative samples (No. 1, 2, 4, 5, 9, 10 and 11) was greater than 38, indicating that the samples did not carry MSVR virus. In summary, the detection data of the CRISPR/Cas13a-MSRV detection system and the conventional RT-qPCR were consistent, indicating that the CRISPR/Cas13a-MSRV detection system is feasible and can replace the original conventional and complicated detection methods to a certain extent.

Vertical comparison chart of sample qPCR detection data with qPCR and CRISPR

a. qPCR detection of MSRV clinical sample threshold cycle number diagram; the detected sample cycle number was compared with the negative reference cycle number using T-tests analysis (**P<0.01);

b. Cas13a combined with MIRA detection of MSRV clinical sample fluorescence diagram; the detected sample fluorescence value was compared with the negative reference fluorescence value using T-tests analysis (**P<0.01)

In summary, the CRISPR/Cas13a-MSRV detection method established in this study does not require expensive experimental instruments, making on-site detection fast, accurate and convenient. Therefore, if this detection method is used to timely discover MSRV in the actual breeding process and take targeted and effective measures, it can greatly reduce the losses caused by diseases in the breeding process. In addition, this detection method is fast, highly sensitive and specific, and has great application and promotion prospects.