Taqman probe qPCR primer set for detecting Ichthyophthirius multifeldianus and its application

Abstract

The invention discloses a Taqman probe qPCR detection primer set and application for detecting Ichthyophthirius multifelius, and belongs to the field of parasite detection. According to the gene sequence of the target molecule cathepsin L of Ichthyophthirius multifelius, the invention designs a pair of specific primers and a probe. The primers and probes have no amplification on Tetrahymena and Paramecium, which are closely related to Ichthyophthirius multifelius, as well as water samples and bottom mud of aquaculture water bodies, and have strong specificity; the detection sensitivity is high, the detection lower limit is 5.4 copies / μL, which is about 1 Ichthyophthirius multifelius, and the repeatability is good (the coefficient of variation of the technology is less than 5%); the method is convenient for detection, omitting the electrophoresis step in the conventional PCR method and eliminating the aerosol pollution of the traditional PCR, greatly saving time and improving the detection accuracy; it can be applied to the detection of Ichthyophthirius multifelius in water environments and on fish bodies, and provides technical support for the prevention of Ichthyophthirius multifelius disease.

Description

Technical Field

[0001] The invention belongs to the field of parasite detection, and particularly relates to a Taqman probe qPCR detection primer set for detecting Ichthyophthirius multifida and an application thereof. Background Art

[0002] Ichthyophthirius multifiliis is an important parasitic ciliate of freshwater and brackish water fish in the world. It mainly parasitizes on the gills and skin of fish, forming white spots, also known as white spot disease. The disease is very harmful, with a high mortality rate of diseased fish, causing serious economic losses. Therefore, the prevention and control of Ichthyophthirius multifiliis disease is an important task in aquaculture, and timely detection of the presence of Ichthyophthirius multifiliis in water bodies is the key to the prevention and control of Ichthyophthirius multifiliis disease, which requires a detection technology for detecting Ichthyophthirius multifiliis in water bodies. At present, the methods for detecting Ichthyophthirius multifiliis at home and abroad are mainly microscopic examination, conventional PCR, and fluorescent quantitative PCR. However, these existing detection methods are either limited in scope of application or have low detection sensitivity and cannot meet the needs. Taqman probe fluorescent quantitative PCR technology has strong specificity and high sensitivity, and is more suitable for quantitative detection. Therefore, it is very necessary to find a new target molecule to establish a Taqman probe fluorescent quantitative PCR method with good reproducibility, strong specificity, and high sensitivity to detect Ichthyophthirius multifiliis in water environments and on fish bodies. Summary of the invention

[0003] In order to overcome the shortcomings and deficiencies of the prior art, the object of the present invention is to provide an application of a cathepsin L gene in the preparation of a reagent for detecting, identifying or diagnosing Ichthyophthirius multifeldianus.

[0004] Another object of the present invention is to provide a Taqman probe qPCR detection primer set for detecting Ichthyophthirius multifida.

[0005] Another object of the present invention is to provide a Taqman probe qPCR detection kit for detecting Ichthyophthirius multifida.

[0006] Another object of the present invention is to provide an application of the above primer set or kit.

[0007] Another object of the present invention is to provide a method for quantitatively detecting Ichthyophthirius multiflesh in a sample.

[0008] The invention provides a kit and a detection method for detecting Ichthyophthirius multifellows by using a Taqman probe fluorescent quantitative PCR, and particularly provides a pair of specific primers Cathepsin F and Cathepsin R for Ichthyophthirius multifellows and a specific probe Cathepsin-Probe. The kit and the detection method can be used to quickly detect Ichthyophthirius multifellows in water environments and fish bodies.

[0009] The invention establishes a specific and sensitive Taqman probe real-time fluorescence quantitative PCR technology with a cathepsin L (CTSL) gene as a new target molecule, and assembles a kit for use with the technology. The cathepsin L gene has low similarity between different species, and the thermophilic Tetrahymena, which has the highest similarity with Ichthyophthirius polyspermum, has only 65.07% similarity at a coverage rate of 51%, but the cathepsin L gene similarity between Ichthyophthirius polyspermum is as high as 99.64%, which shows that the gene has the characteristics of large inter-species variation and intra-species conservation, and is suitable for use as a detection target.

[0010] The purpose of the present invention is achieved through the following technical solutions:

[0011] Application of cathepsin L gene as a molecular target in the preparation of reagents for detecting, identifying or diagnosing Ichthyophthirius multifeldianus.

[0012] Preferably, the GenBank of the cathepsin L gene is: XM_004039979.1.

[0013] The GenBank of the cathepsin L is XP_004040027.1.

[0014] A reagent for detecting Ichthyophthirius multifida, comprising primers capable of detecting a cathepsin L gene of Ichthyophthirius multifida;

[0015] Specifically, based on the Taqman probe fluorescence quantitative PCR method, a pair of specific primers and Taqman probes were designed for the cathepsin L gene of Ichthyophthirius multifida, and the sequences are as follows:

[0016] Cathepsin F: 5'-CAGGAGTTGCTTTCTTTG-3';

[0017] Cathepsin R: 5'-CTCCTTTAATTCTTTATAAGTTTTC-3';

[0018] Cathepsin-Probe: 5'-FAM-TTTCTCACCATAAAAGCTCCTACACATG-BHQ-3';

[0019] The 5' end of Cathepsin-Probe was labeled with a fluorescent group FAM, and the 3' end was labeled with a quenching group BHQ. The length of the amplified Ichthyophthirius multifungus cathepsin L gene was 192 bp.

[0020] The use of the reagent for detecting Ichthyophthirius multifellows in a kit for detecting, identifying or diagnosing Ichthyophthirius multifellows.

[0021] A kit for detecting Ichthyophthirius multifida contains the above reagent for detecting Ichthyophthirius multifida.

[0022] The kit also includes qPCR reaction reagents.

[0023] The primer concentration was 10 μmol / L, and the probe concentration was 10 μmol / L.

[0024] Reaction reagents: 10× PCR buffer (without MgCl2), magnesium chloride (MgCl2), a mixture of four deoxyribonucleotides (dNTPs), and Taq enzyme (5 units / μL).

[0025] A method for quantitatively detecting Ichthyophthirius multifida comprises the following steps: the method is a non-disease diagnosis method;

[0026] (1) extracting DNA from the sample to be tested;

[0027] (2) PCR amplified the cathepsin L gene, constructed a recombinant plasmid, used it as a template to establish a qPCR reaction system, and used the logarithmic values ​​of recombinant plasmids with different copy numbers (Sq) and their Ct values ​​to draw a standard curve; the relationship between the copy number Sq and the Ct value is Ct = -3.164 × lg (Sq) + 36.848, and the correlation coefficient R 2 =0.993;

[0028] (3) using the established detection method, adding the above reagent for detecting Ichthyophthirius multifida and the DNA of the sample to be tested to form a qPCR reaction system, performing a qPCR reaction, and obtaining the Ct value of the sample to be tested;

[0029] (4) Quantitative detection of Ichthyophthirius multifida

[0030] After qPCR is completed, the Ct value is used to judge: if there is no amplification, it is judged that there is no Ichthyophthirius multifleshed in the sample; if there is amplification, the corresponding cathepsin L gene copy number is calculated according to the established standard curve, and the Ct value of the negative control should be no amplification.

[0031] Among them, the copy number of cathepsin L gene in each Ichthyophthirius multifleus is about 7 copies.

[0032] Preferably, in step (1), the sample to be tested is water, sediment or fish tissue;

[0033] Preferably, in steps (2) and (3), the qPCR reaction system is: 2.5 μL of 10×PCR buffer, 25 mmol·mL magnesium chloride -1) 3.2μL, a mixture of four deoxynucleotides (2.5mM) 2μL, upstream and downstream primers (10μM) 1μL each, specific probe (10μM) 0.5μL, Taq enzyme (5U / μL) 0.25μL; template DNA 1μL, sterile double distilled water 12.55μL; the total volume of the reaction system is 24μL.

[0034] The qPCR reaction conditions are: 95℃ pre-denaturation for 2min, then 95℃ denaturation for 15s, 58℃ annealing for 30s, 72℃ extension for 20s, and 40 cycles. The fluorescence signal is collected at the end of 58℃ annealing each time; the Ct value of the negative control should not be displayed.

[0035] Preferably, in step (3), the recombinant plasmid with different copy numbers (Sq) is 5.4×10 0 ~5.4×10 9 Copies / μL of recombinant plasmid; specifically 5.4×10 0 , 5.4×10 1 , 5.4×10 2 , 5.4×10 3 , 5.4×10 4 , 5.4×10 5 , 5.4×10 6 , 5.4×10 7 , 5.4×10 8 , 5.4×10 9 Copies / μL of recombinant plasmid.

[0036] The main principle of the present invention is: according to the gene sequence of the target molecule cathepsin L of Ichthyophthirius multifida, a pair of specific primers and a probe are designed, the 5' end of the probe has a fluorescent group (such as: FAM), and the 3' end has a quenching group (BHQ). When the fluorescent probe is in a complete state, it is restricted by the quenching group and cannot excite fluorescence. However, when the probe is degraded by Taq enzyme, the fluorescent group at the 5' end will be freed and emit fluorescence, thereby detecting the copy number of the target gene in the sample.

[0037] Compared with the prior art, the present invention has the following advantages and effects:

[0038] (1) The method of the present invention is convenient for detection, omitting the electrophoresis step in the conventional PCR method and eliminating the aerosol contamination of the traditional PCR, thus greatly saving time and improving the detection accuracy;

[0039] (2) The detection sensitivity of the present invention is high, with a detection limit of 5.4 copies / μL, which is approximately one Ichthyophthirius multifleus, and good repeatability (the coefficient of variation of the technology is less than 5%);

[0040] (3) The primers and probes provided by the present invention have no amplification on Tetrahymena and Paramecium, which are closely related to Ichthyophthirius multifleuroides, as well as water samples and bottom mud of aquaculture water bodies, and have strong specificity;

[0041] (4) The present invention can be applied to the detection of Ichthyophthirius multiflorus in water environments and on fish bodies, providing technical support for the prevention of Ichthyophthirius multiflorus disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 It is the electrophoresis diagram of the extracted total DNA; among them, lane 1 is the DNA of Ichthyophthirius multifida larvae, lane 2 is the DNA of Tetrahymena thermophila, lane 3 is the DNA of Paramecium cerdata, lane 4 is the total DNA of mixed plankton in aquaculture water, lane 5 is the total DNA of mixed plankton in non-aquaculture water, lane 6 is the total DNA of mixed organisms in the sediment of aquaculture ponds, and M is the standard molecular weight DL10000.

[0043] Figure 2 These are the electrophoresis results of PCR products at different annealing temperatures; lane 1 is 54°C, lane 2 is 58°C, lane 3 is 60°C, lane 4 is the negative control, and M is the standard molecular weight DL500.

[0044] Figure 3 It is the electrophoresis result of PCR products of different DNA templates; among them, lanes 1 and 2 are DNA amplification products of Ichthyophthirius multifida larvae, lanes 3 and 4 are DNA amplification products of Tetrahymena thermophila, lanes 5 and 6 are DNA amplification products of Paramecium cerci, lanes 7 and 8 are DNA amplification products of mixed plankton in non-aquaculture water bodies, lanes 9 and 10 are DNA amplification products of mixed plankton in one aquaculture water body, lanes 11 and 12 are DNA amplification products of mixed plankton in another aquaculture water body, lanes 13 and 14 are DNA amplification products of mixed organisms in sediment of aquaculture water bodies, lane 15 is a negative control, and M is the standard molecular weight DL500.

[0045] Figure 4 It is the standard curve of the recombinant plasmid of TaqMan probe real-time fluorescence quantitative PCR; where Cq is the reaction cycle number.

[0046] Figure 5 This is the kinetic curve of real-time fluorescence quantitative PCR detection of recombinant plasmids using TaqMan probes; the template concentrations from left to right in the figure are: 5.4×10 9 Copies / μL, 5.4×10 8 Copies / μL, 5.4×10 7 Copies / μL, 5.4×10 6 Copies / μL, 5.4×10 5 Copies / μL, 5.4×10 4 Copies / μL, 5.4×10 3Copies / μL, 5.4×10 2 Copies / μL, 5.4×10 1 Copies / μL, 5.4×10 0 copies / μL, NTC was the negative control.

[0047] Figure 6 is the amplification curve of the TaqMan probe fluorescence quantitative PCR sensitivity test; where 1-10 respectively represent: 5.4×10 9 Copies / μL, 5.4×10 8 Copies / μL, 5.4×10 7 Copies / μL, 5.4×10 6 Copies / μL, 5.4×10 5 Copies / μL, 5.4×10 4 Copies / μL, 5.4×10 3 Copies / μL, 5.4×10 2 Copies / μL, 5.4×10 1 Copies / μL, 5.4×10 0 copies / μL, 11 was the negative control.

[0048] Figure 7 The electrophoresis results of PCR amplification products with different template concentrations are shown in Figure 1. Lanes 1 to 10 are 5.4×10 9 ~5.4×10 0 copies / μL template amplification product, lane 11 is the negative control, and M is the standard molecular weight DL2000.

[0049] Figure 8 It is a fluorescence quantitative PCR amplification curve of the DNA template amplification of Ichthyophthirius multifida without interference from other biological DNA, wherein 1-2 in the figure represents the amplification curve of the diluted pure recombinant plasmid, 3-4 represents the amplification curve after the recombinant plasmid is mixed with each sample template, 5-6 represents the amplification curve of Ichthyophthirius multifida DNA, 7-8 represents the amplification curve after the Ichthyophthirius multifida DNA template is mixed with other sample templates, and 9-11 represents the negative control and the mixed sample without the addition of Ichthyophthirius multifida DNA template and plasmid.

[0050] Fig. 9 Figure 1 is the specific amplification curve of Taqman probe fluorescence quantitative PCR, where 1 and 2 are the amplification curves of DNA of Ichthyophthirius multifida larvae and DNA of Ichthyophthirius multifida cysts, and 3-8 are the amplification curves of DNA of thermophilic Tetrahymena, DNA of Paramecium cerdata, DNA of mixed plankton in aquaculture water, DNA of mixed plankton in non-aquaculture water, DNA of mixed organisms in bottom mud of aquaculture ponds and negative control.

[0051] Fig.10There is a linear relationship between the number of Ichthyophthirius multifida and the gene copy number; among them, the tissue gene copy number corresponding to one insect is about 7 copies.

[0052] Fig.11 This is the amplification curve of DNA from aquaculture water samples in different regions; A: 1 is the positive control, 2-3 are positive samples from Huadu water, 4-14 are other water samples from Huadu and negative controls; B: 1, 2 are positive controls, 3-31 are aquaculture water samples from Panyu and Zhongshan and negative controls. DETAILED DESCRIPTION

[0053] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0054] The experimental methods in the following examples without specific experimental conditions are usually carried out under conventional experimental conditions or the experimental conditions recommended by the manufacturer. The materials and reagents used are commercially available unless otherwise specified.

[0055] The Ichthyophthirius multifida, Tetrahymena thermophila and Paramecium caudatum used in the examples are all conventional pathogens.

[0056] Example 1: A Taqman probe fluorescent quantitative PCR kit for Ichthyophthirius multifida was prepared according to the following formula:

[0057] (a) Primer and probe reagents: including upstream primer (Cathepsin F), downstream primer (Cathepsin R) and probe (Cathepsin-Probe) reaction solution.

[0058] Based on the published cathepsin L gene (CTSL gene) of Ichthyophthirius multifida (accession number: XM_004039979.1), a pair of specific primers and Taqman probe were designed. The specific sequences are as follows:

[0059] Upstream primer (Cathepsin F): 5'-CAGGAGTTGCTTTCTTTG-3'; (SEQ ID NO: 1)

[0060] Downstream primer (Cathepsin R): 5'-CTCCTTTAATTCTTTATAAGTTTTC-3'; (SEQ ID NO: 2)

[0061] The Taqman probe sequence is (Cathepsin-Probe):

[0062] 5'-FAM-TTTCTCACCATAAAAGCTCCTACACATG-BHQ-3'; (SEQ ID NO: 3)

[0063] The 5' end of the probe sequence is labeled with a fluorescent group FAM, and the 3' end is labeled with a quenching group BHQ. The length of the amplified Ichthyophthirius multifungus cathepsin gene is 192 bp.

[0064] (b) Polymerase chain reaction reagents: containing 10× PCR buffer (without MgCl2), MgCl2 (25 mmol·mL -1 ), a mixture of four deoxynucleotides (2.5 mM), Taq enzyme (5 units / μL), and sterile double-distilled water.

[0065] Follow the steps below to develop a method for detecting Ichthyophthirius multifida:

[0066] 1. DNA Extraction

[0067] Total DNA was extracted from larvae of Ichthyophthirius multifida, Tetrahymena thermophila, Paramecium caudatum, mixed plankton in water bodies, and bottom mud of aquaculture ponds and stored at -20°C for future use. The negative control template was sterile water.

[0068] The electrophoresis diagram of the extracted total DNA is as follows: Figure 1 As shown, it can be seen that the extracted total DNA has good integrity and can meet the needs of subsequent experiments.

[0069] 2. Determination of the appropriate annealing temperature of Taqman probe primers

[0070] Conventional PCR amplification was performed using DNA from Ichthyophthirius multifida larvae as a template, with annealing temperatures set at 54, 58, and 60°C. After the experiment, PCR products were analyzed by 3% agarose gel electrophoresis, and the appropriate annealing temperature was determined to be 58°C ( Figure 2 shown).

[0071] 3. Conventional PCR detection primer specificity

[0072] The extracted DNA of Ichthyophthirius multifida larvae, the thermophilic DNA of Tetrahymena thermophila, the DNA of Paramecium cerci, the DNA of mixed plankton in the water body, and the DNA of mixed organisms in the bottom mud were used as templates, the annealing temperature was 58℃, and conventional PCR amplification was performed to analyze the specificity of the primers. The electrophoresis of the amplified products showed that only the template of Ichthyophthirius multifida larvae was amplified, while the other templates were not amplified ( Figure 3 ), indicating that the primers have good specificity and can meet the needs of subsequent detection.

[0073] 4. Construction of recombinant target plasmid and preparation of standard

[0074] Using CTSL gene primers (Cathepsin F / Cathepsin R), with DNA of Ichthyophthirius multifida larvae as template, and using high-fidelity enzyme to amplify the target fragment of cathepsin L (CTSL) gene by PCR, a target band with a fragment length of 192bp was obtained (PCR products were purified, recovered, connected, transformed and other steps to construct a recombinant target gene fragment plasmid), the recombinant target plasmid strain was screened for positive clones, and then sent to BGI for sequencing. The results were consistent with the original sequence, and the homology with the corresponding protease sequence of Ichthyophthirius multifida was as high as 99%, and the recombinant plasmid was obtained. Then the recombinant plasmid was extracted using the Biotech plasmid extraction kit, and the concentration was measured by the BioTek Epoch micro nucleic acid quantifier to be 170.5ng / μL. According to the formula: copies / μL=6.02×10 23 ×(ng / μL×10 -9 ) / (DNAlength×660), the copy number is calculated as: 5.4×10 10 copies / μL, followed by 10-fold serial dilutions and stored at -20°C for later use.

[0075] 5. Optimization of Fluorescence Quantitative PCR Reaction System

[0076] Use diluted plasmid standard 5.4×10 5 With 100 copies / μL as template, gradient experiment was used to optimize the concentrations of primers, probes and Taq enzyme, as well as the mixture of four deoxynucleotides, magnesium ion concentration and buffer content in the reaction system under the optimal annealing temperature. The optimal reaction conditions of fluorescent quantitative PCR were determined by comprehensively considering the CT value, fluorescence intensity and repeatability.

[0077] The optimized reaction system was as follows: total volume 24 μL, including 10× PCR buffer 2.5 μL, magnesium chloride (25 mmol·mL -1 ) 3.2μL, a mixture of four deoxynucleotides (2.5mM) 2μL, upstream and downstream primers (10μM) 1μL each, specific probe (10μM) 0.5μL, Taq enzyme (5U / μL) 0.25μL, plasmid standard 1μL, deionized water 12.55μL.

[0078] Fluorescence quantitative PCR reaction conditions: 95℃ pre-denaturation for 2min, 95℃ denaturation for 15s; 58℃ annealing for 30s; 72℃ extension for 20s; 40 cycles. Collect the fluorescence signal at the end of 58℃ annealing each time; the Ct value of the negative control should not be displayed.

[0079] 6. Establishment of Fluorescence Quantitative PCR Standard Curve

[0080] The recombinant plasmid was used as the positive template for fluorescence quantitative PCR reaction, and 5.4×10 9 ~5.4×10 0 The plasmid with a copy / μL was detected on a Bio-Rad CFX96 fluorescence quantitative PCR instrument to obtain the amplification kinetic curve of the recombinant plasmid ( Figure 5 ), calculate the standard curve of the logarithmic value of the threshold cycle number (Ct) of the amplification product and the initial copy number (Sq) of the template ( Figure 4 ),Depend on Figure 4 The concentration of the known fluorescent quantitative PCR standard is 5.4×10 9 ~5.4×10 0 There is a good linear relationship in the range of copies / μL, Ct=-3.164×lg(Sq)+36.848, R 2 =0.993, amplification efficiency E=107%.

[0081] 7. Fluorescence quantitative PCR sensitivity test

[0082] The positive plasmid was expressed at 5.4 × 10 10 Copies / μL is the original concentration diluted 10 times to obtain 5.4×10 9 ~5.4×10 0 The plasmid with a copy / μL was used as a template to perform fluorescence quantitative PCR reaction and obtain the sensitivity kinetic amplification curve ( Figure 6 ), when the copy number of the standard is 5.4×10 0 The negative control showed no amplification, indicating that the minimum detection limit of this detection method can reach 5.4 copies / μL. In addition, conventional PCR can only detect 5.4×10 4 copies / μL( Figure 7 ), therefore, the detection sensitivity of Taqman probe fluorescent quantitative PCR is 10,000 times higher than that of conventional PCR.

[0083] 8. Specificity of fluorescent quantitative PCR and its resistance to interference of other DNA on specific amplification

[0084] In order to verify the specificity of the fluorescent quantitative PCR primers established in this experiment and their resistance to interference with specific amplification by other DNA, the thermophilic Tetrahymena DNA, Paramecium caudatum DNA, mixed plankton DNA in the water, mixed DNA of sediment organisms, and mixed DNA (2 μL of each DNA was mixed) were used as templates. The positive control was the DNA of Ichthyophthirius multifida, and the negative control was sterile water. The Taqman probe fluorescent quantitative PCR reaction system established in this study was used for detection to analyze the specificity of the primers and their resistance to interference with specific amplification by other DNA. The results showed that the primers could only amplify the Ichthyophthirius multifida DNA template ( Fig. 9 ), other DNA does not inhibit the specific primers from amplifying the DNA of Ichthyophthirius larvae ( Figure 8 ), indicating that this technology is suitable for the detection of Ichthyophthirius multifle in water bodies, sediments and fish.

[0085] 9. Repeatability and stability of fluorescence quantitative PCR

[0086] Repeatability analysis includes intra-group repeatability and inter-group repeatability. 8 Copies / μL~5.4×10 1 The plasmid with 8 different concentrations of copies / μL was used as the positive template. The test was repeated 3 times under the optimal reaction system conditions, once every 1 day, and repeated three times. The obtained CT values ​​were analyzed and the coefficient of variation was calculated. The results (such as Table 1 and Table 2) showed that the coefficient of variation within the group was less than 5%, and the maximum coefficient of variation within the group was 4.6%, and the coefficient of variation between groups was 0.9%.

[0087] Table 1 Intra-group repeatability test data of fluorescence quantitative PCR Ct values ​​on different days

[0088]

[0089] Table 2 Data of intergroup repeatability test of three-step fluorescence quantitative PCR Ct values

[0090]

[0091] 10. Linear relationship between the number of Ichthyophthirius multispermum and the number of gene copies

[0092] 100 and 200 larvae of Ichthyophthirius multifle were counted and collected under an optical microscope, and then total DNA was extracted. The total DNA of 100 larvae was diluted to 10, 8, 6, 4, 2, and 1 larvae, and the total DNA of 200 larvae was diluted to 200, 20, and 2 larvae. Fluorescence quantitative PCR detection was performed, and the results are shown in Tables 3 and 4. The linear regression line equation was obtained ( Fig.10 According to Tables 3 and 4, the gene copy number of one insect is about 7 copies.

[0093] Table 3 Gene copy number corresponding to the number of insects

[0094] Number of insects 10 8 6 4 2 1 0 Gene copy number 87.09 67.61 46.77 33.88 13.49 7.24 0

[0095] Table 4 Gene copy number corresponding to the number of insects

[0096] Number of insects 0 2 20 200 Gene copy number 0 14.13 123.03 1000

[0097] Example 2: Detection of aquaculture water samples

[0098] 1L water samples were collected from grass carp breeding ponds in Huadu, Guangzhou, Panyu and Zhongshan, Guangdong, respectively. The water samples were filtered and concentrated, and DNA was extracted from the water samples. Then, 40 samples of the breeding water were tested by TaqMan probe fluorescence quantitative PCR method using the detection method established by the present invention. Positive sample groups and negative sample groups were set at the same time. The test results are shown in Table 5 and Table 6. Fig.11 shown.

[0099] Table 5 Detection results of TaqMan probe fluorescence quantitative PCR on aquaculture water

[0100] Sampling location Number of ponds Number of positive samples Positive detection rate (%) Huadu 12 2 16.7 Panyu 12 0 0 Zhongshan City 16 0 0

[0101] From Table 5 and Fig.11 It can be seen that the detection rate of water samples in Huadu area is 16.7%, which is exactly the pond where grass carp fry are raised and infected with Ichthyophthirius multifiliis. Ichthyophthirius multifiliis was not detected in the water bodies of ponds in Panyu and Zhongshan, which may be related to the large individuals of the crispy-fleshed grass carp raised there, which are generally 2-3 years old and are difficult to be infected with Ichthyophthirius multifiliis.

[0102] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention. Sequence Listing <110> Jinan University <120> Taqman probe qPCR primer set for detecting Ichthyophthirius multifeldianus and its application <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Cathepsin F <400> 1 caggagttgc tttctttg 18 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Cathepsin R <400> 2 ctcctttaat tctttataag ttttc 25 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Cathepsin-Probe <220> <221> modified_base <222> (1) <223> FAM modification <220> <221> modified_base <222> (27)..(27) <223> BHQ Modification <400> 3 tttctcacca taaagctcct acacatg 27

Claims

1. Use of a reagent for detecting cathepsin L gene in the preparation of a reagent for detecting, identifying or diagnosing Ichthyophthirius multifida, characterized in that: The reagent for detecting the cathepsin L gene comprises primers and probes, and the sequences of the primers and probes are as follows: Cathepsin F: 5'-CAGGAGTTGCTTTCTTTG-3'; Cathepsin R: 5'-CTCCTTTAATTCTTTATAAGTTTTC-3'; Cathepsin-Probe: 5'-FAM-TTTCTCACCATAAAAGCTCCTACACATG-BHQ3'.

2. A reagent for detecting Ichthyophthirius multifida, characterized in that: Containing primers and probes for the cathepsin L gene of claim 1 capable of detecting Ichthyophthirius multifida; The primers and probes capable of detecting the cathepsin L gene of Ichthyophthirius multifida are as follows: Cathepsin F: 5'-CAGGAGTTGCTTTCTTTG-3'; Cathepsin R: 5'-CTCCTTTAATTCTTTATAAGTTTTC-3'; Cathepsin-Probe: 5'-FAM-TTTCTCACCATAAAAGCTCCTACACATG-BHQ3'.

3. Use of the reagent for detecting Ichthyophthirius multifellows according to claim 2 in a kit for detecting, identifying or diagnosing Ichthyophthirius multifellows.

4. A kit for detecting Ichthyophthirius multifida, characterized in that: A reagent for detecting Ichthyophthirius multifleus as claimed in claim 2.

5. A method for quantitatively detecting Ichthyophthirius multifida, characterized in that: The method comprises the following steps: the method is a non-disease diagnosis method; (1) Extract DNA from the sample to be tested; (2) PCR amplified the cathepsin L gene, constructed a recombinant plasmid, used it as a template to establish a qPCR reaction system, and used the logarithmic value of the recombinant plasmid with different copy numbers Sq and its Ct value to draw a standard curve; the relationship between the copy number Sq and the Ct value is Ct=-3.164×lg(Sq)+36.848, and the correlation coefficient R 2 =0.993; (3) using the established detection method, adding the reagent for detecting Ichthyophthirius multifungus as described in claim 2 and the DNA of the sample to be tested to prepare a qPCR reaction system, performing a qPCR reaction, and obtaining the Ct value of the sample to be tested; (4) Quantitative detection of Ichthyophthirius multifida After qPCR is completed, the Ct value is used to determine: if there is no amplification, it is determined that there is no Ichthyophthirius multifeldianus in the sample; If amplification occurs, the corresponding cathepsin L gene copy number is calculated based on the established standard curve, and the Ct value of the negative control should show no amplification.

6. The method according to claim 5, characterized in that: The number of copies of the cathepsin L gene in each Ichthyophthirius multifeldae was 7; In step (1), the sample to be tested is water, bottom mud or fish tissue.

7. The method according to claim 5, characterized in that: In steps (2) and (3), the qPCR reaction system is: 2.5 μL of 10× PCR buffer, 25 mmol mL -1 3.2 μL of magnesium chloride, 2 μL of a mixture of 2.5 mM four deoxynucleotides, 1 μL of 10 μM Cathepsin F / R each, 0.5 μL of 10 μM Cathepsin-Probe, 0.25 μL of 5 U / μL Taq enzyme; 1 μL of template DNA, 12.55 μL of sterile double distilled water; the total volume of the reaction system is 24 μL.

8. The method according to claim 5, characterized in that: The qPCR reaction conditions are as follows: pre-denaturation at 95°C for 2 min, followed by denaturation at 95°C for 15 s, annealing at 58°C for 30 s, extension at 72°C for 20 s, and 40 cycles; the fluorescence signal is collected at the end of annealing at 58°C each time; the Ct value of the negative control should not be displayed.

9. The method according to claim 5, characterized in that: In step (3), the recombinant plasmid with different copy number Sq is 5.4×10 0 ~5.4×10 9 Copies / μL of recombinant plasmid; specifically 5.4×10 0 , 5.4×10 1 , 5.4×10 2 , 5.4×10 3 , 5.4×10 4 , 5.4×10 5 , 5.4×10 6 , 5.4×10 7 , 5.4×10 8 , 5.4×10 9 Copies / μL of recombinant plasmid.

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