Method for synchronously and rapidly detecting macrobrachium rosenbergii iridovirus and multiple pathogens

Abstract

The invention discloses a synchronous rapid detection method for macrobrachium rosenbergii iridovirus and multiple pathogens, and belongs to the technical field of biology. According to the method, macrobrachium rosenbergii tissue gene DNA is extracted by adopting a Chelex method, then PCR amplification is performed, and an amplification product is detected for judgment. According to the method, the genome DNA is extracted by adopting an optimized CHELEX method, enough DNA samples for detection can be extracted only by using a trace amount of tissue samples, meanwhile, the DNA extraction time is greatly shortened, and only 20 minutes are needed from sample treatment to extraction of enough macrobrachium rosenbergii tissue DNA. PCR (Polymerase Chain Reaction) specific amplification and gel electrophoresis are carried out by taking the macrobrachium rosenbergii as a template, so that the aim of quickly detecting common diseases (especially iridovirus) of macrobrachium rosenbergii can be fulfilled.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for the simultaneous and rapid detection of iridovirus and multiple pathogens in giant freshwater prawns. Background Technology

[0002] The giant freshwater prawn (Macrobrachium rosenbergii), also known as the Malaysian prawn, freshwater long-armed prawn, and Thai prawn, is one of the largest freshwater prawns in the world. It is characterized by rapid growth, omnivorous diet, short farming cycle, and high nutritional value, making it a widely farmed and high-quality species globally. However, in recent years, due to high-density and intensive farming methods, outbreaks of iridovirus disease in giant freshwater prawn farming have posed a serious threat to the industry.

[0003] Iridovirus disease is a general term for a group of infectious diseases in aquatic animals caused by viruses of the Iridoviridae family. These viruses are named for their iridescent appearance; under an electron microscope, the virus particles often exhibit a regular icosahedral structure and may display an iridescent sheen. In recent years, it has become one of the most threatening diseases to the global shrimp and fish farming industries, characterized by high mortality rates and rapid outbreaks, often causing enormous economic losses.

[0004] Currently, PCR is used to detect iridovirus in giant freshwater prawns, but the conventional gene extraction process is time-consuming and costly. Summary of the Invention

[0005] The purpose of this invention is to provide a method for the simultaneous and rapid detection of multiple pathogens in giant freshwater prawns, comprising the following steps:

[0006] Step 1: Extract gene DNA from giant freshwater prawn tissue using the Chelex method: Take giant freshwater prawn tissue, add ultrapure water, homogenize, centrifuge and remove the supernatant, add Chelex-100 and proteinase K to the precipitate, incubate and centrifuge, and collect the supernatant.

[0007] Step 2: Dilute the supernatant obtained in Step 1, use it as a DNA template, perform PCR amplification, and detect the amplification product for judgment.

[0008] Furthermore, the tissue of the giant freshwater prawn is either gills or hepatopancreas.

[0009] Further, in step 1, the ratio of the amounts of giant freshwater prawn tissue, ultrapure water, Chelex-100, and proteinase K is as follows: for every 0.1g of giant freshwater prawn tissue, add 200μL of ultrapure water, 200μL of 5% Chelex-100, and 2.5μL of 20mg / mL proteinase K.

[0010] Furthermore, in step 1, the incubation is first carried out in a metal bath at 56°C for 10 minutes, and then incubated in a metal bath at 100°C for 5 minutes.

[0011] Furthermore, the primers for PCR amplification include primers for amplifying iridovirus I, iridovirus II, iridovirus III, Vibrio parahaemolyticus, Aeromonas hydrophila, white spot virus I, white spot virus II, and white spot virus III.

[0012] This invention employs an optimized CHELEX method to extract genomic DNA. This method requires only a small amount of tissue sample to extract sufficient DNA for detection, while significantly reducing the DNA extraction time. From sample processing to extracting a sufficient amount of Macrobrachium rosenbergii tissue DNA, only 20 minutes are needed. Using this as a template for PCR-specific amplification and gel electrophoresis, the aim is to rapidly detect common diseases in Macrobrachium rosenbergii (especially iridoviruses).

[0013] This invention utilizes the Chelex method for rapid tissue DNA extraction, which offers several advantages:

[0014] I. Chelex Rapid Tissue DNA Extraction

[0015] 1. The operation process is extremely simple and fast.

[0016] Fewer steps: Usually, you only need to add the sample to a centrifuge tube containing Chelex-100, heat to boiling, centrifuge, and take the supernatant to obtain the DNA template.

[0017] Short processing time: The entire DNA extraction process can be completed within 20 minutes, which is much faster than the traditional phenol-chloroform method (which requires multiple centrifugation and transfer steps and takes about 40-60 minutes) and many commercial kits (which take about 30 minutes).

[0018] 2. Low cost

[0019] Chelex-100 resin itself is inexpensive and requires only a small amount each time; just 5g is needed to prepare 100mL of extraction suspension, which can be used for DNA extraction from 500-1000 samples.

[0020] It eliminates the need for expensive proteinase K (which can be omitted in simplified protocols), toxic reagents such as phenol-chloroform, and costly centrifuge column kits, making it convenient, fast, and low-cost.

[0021] 3. High security

[0022] It completely avoids the use of highly toxic and corrosive organic solvents such as phenol and chloroform in the traditional phenol-chloroform method, making it more friendly to laboratory personnel and the environment.

[0023] 4. Particularly suitable for PCR reactions

[0024] Effective removal of inhibitors: Chelex-100 is a chelating resin that can efficiently chelate (bind) metal ions, such as magnesium ions, under high temperature and alkaline conditions. These ions are cofactors of DNase; removing them effectively inactivates DNase, preventing DNA degradation during extraction.

[0025] Product purity: The obtained DNA solution does not contain impurities that interfere with subsequent PCR reactions. In the traditional phenol-chloroform method, if the phenol is not completely removed, residual phenol can inhibit Taq enzyme activity, while the Chelex method does not have this problem.

[0026] II. Applicable to trace and difficult samples

[0027] Because the entire process is carried out in a single tube, sample transfer losses are reduced, resulting in high extraction efficiency for trace samples.

[0028] It is widely used for DNA extraction from forensic samples (such as bloodstains, saliva stains, hair follicles), cell clusters, oral swabs, and other samples.

[0029] In the testing of broodstock, seed shrimp, and larvae in aquaculture such as shrimp and crab, the amount of shrimp body sample required is small. DNA can be successfully extracted with only a tissue sample the size of a mung bean or a grain of sand. This causes minimal damage to the broodstock or larvae, effectively reducing broodstock damage and costs, and has broad application prospects in rapid disease detection. Attached Figure Description

[0030] Figure 1. Pathogen detection results of giant freshwater prawns. 1 was infected with Vibrio parahaemolyticus, 2 with Aeromonas hydrophila, 3 with iridovirus I, 4 with iridovirus II, 5 with iridovirus III, 6 with white spot syndrome virus I, 7 with white spot syndrome virus II, 8 with white spot syndrome virus III, 9 with Vibrio parahaemolyticus, and 10 with Aeromonas hydrophila. Detailed Implementation

[0031] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0033] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0034] Example 1

[0035] I. Chelex Method for Extracting Tissue Gene DNA

[0036] 1. Sample preparation: Take about 0.1g each of gills and hepatopancreas, quickly put them into a 1.5mL centrifuge tube, add 200μL of ultrapure water, homogenize quickly with an electric homogenizer for 1min, centrifuge and discard the supernatant.

[0037] 2. Add 200 μL of 5% Chelex-100 suspension to the obtained precipitate, and supplement with 2.5 μL of proteinase K (20 mg / mL) to enhance the lysis effect.

[0038] 3. Incubate in a 56°C metal bath (Dalung, HC110-Pro) for 10 minutes to allow proteinase K to fully digest the protein.

[0039] 4. A 100℃ metal bath for 5 minutes, combined with the high temperature and the chelating effect of Chelex, rapidly lyses cells, destroys protein structures, and separates DNA.

[0040] 5. Centrifuge at 13,000 rpm for 3 minutes at 4°C, and collect the supernatant (containing single-stranded DNA) into a new centrifuge tube. For short-term storage, store at 4°C; for long-term storage, freeze at -20°C.

[0041] II. Pathogen Detection

[0042] The extracted DNA concentration was determined using a micro-spectrophotometer and diluted to 40-100 ng / μL. Using this as a template, amplification was performed on a PCR instrument using the primers listed in the table. The primer sequences are as follows:

[0043]

[0044] The PCR reaction system is as follows:

[0045]

[0046] 2 × Taq Plus Master Mix Ⅱ (Dye Plus): Nanjing Novizan Biotechnology Co., Ltd.

[0047] Ultrapure water: sterile deionized water.

[0048] The PCR reaction procedure is as follows:

[0049] Experiments have shown that the common annealing temperature (Tm) for iridovirus, vibrio, Aeromonas hydrophila, and white spot virus is 56℃, which allows for the simultaneous detection of multiple pathogens.

[0050]

[0051] III. Gel Detection

[0052] After PCR, the amplification products were electrophoresed on a 1% agarose gel for 30 minutes and then detected and photographed on a gel imaging analyzer.

[0053] IV. Experimental Results

[0054] 1. Chelex gene extraction results

[0055]

[0056] The results above show that the genomic DNA extracted using the optimized CHELEX method of this invention has high purity.

[0057] 2. Comparison of Chelex gene extraction time and sensitivity with other methods:

[0058]

[0059] 3. Results of multiple pathogen detection

[0060] The results of the electrophoresis showed that, as shown in Figure 1, 10 tested individuals of *Macrobrachium rosenbergii* (Giant River Prawn) exhibited DNA bands of the expected pathogen-specific length. This indicates that sample 1 was infected with *Vibrio parahaemolyticus*, sample 2 with *Aeromonas hydrophila*, sample 3 with iridovirus I, sample 4 with iridovirus II, sample 5 with iridovirus III, sample 6 with white spot syndrome virus I, sample 7 with white spot syndrome virus II, sample 8 with white spot syndrome virus III, sample 9 with *Vibrio parahaemolyticus*, and sample 10 with *Aeromonas hydrophila*.

Claims

1. A method for simultaneous and rapid detection of multiple pathogens in giant freshwater prawns, characterized in that, Includes the following steps: Step 1: Extract gene DNA from giant freshwater prawn tissue using the Chelex method: Take giant freshwater prawn tissue, add ultrapure water, homogenize, centrifuge and remove the supernatant, add Chelex-100 and proteinase K to the precipitate, incubate and centrifuge, and collect the supernatant. Step 2: Dilute the supernatant obtained in Step 1, use it as a DNA template, perform PCR amplification, and detect the amplification product for judgment.

2. The method according to claim 1, characterized in that, The tissues of the giant freshwater prawn are gills or hepatopancreas.

3. The method according to claim 1, characterized in that, In step 1, the ratio of the following amounts of giant freshwater prawn tissue, ultrapure water, Chelex-100, and proteinase K is as follows: for every 0.1g of giant freshwater prawn tissue, add 200μL of ultrapure water, 200μL of 5% Chelex-100, and 2.5μL of 20mg / mL proteinase K.

4. The method according to claim 1, characterized in that, In step 1, the incubation is first carried out in a metal bath at 56°C for 10 minutes, and then in a metal bath at 100°C for 5 minutes.

5. The method according to claim 1, characterized in that, The primers used for PCR amplification include primers for amplifying iridovirus I, iridovirus II, iridovirus III, Vibrio parahaemolyticus, Aeromonas hydrophila, white spot virus I, white spot virus II, and white spot virus III.

6. The method according to claim 5, characterized in that, The primers for the PCR amplification are as follows: 。 7. The method according to claim 6, characterized in that, The PCR reaction system consists of: PCR reaction reagents, upstream primer, downstream primer, template DNA, and water.

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