Primer probe group, kit and detection method for detecting microsporidia of eriocheir sinensis

Abstract

The invention provides a primer probe group, a kit and a detection method for detecting microsporidia of eriocheir sinensis, and belongs to the technical field of pathogen detection. The invention provides a primer probe group for detecting microsporidia of eriocheir sinensis. The primer probe group comprises a primer pair and a TaqMan probe, the nucleotide sequences of the primer pair and the TaqMan probe are as shown in SEQ ID NO: 1 to SEQ ID NO: 3. In the invention, the primer probe group can specifically detect the eriocheir sinensis microsporidia, and does not have cross reaction with aeromonas veronii, vibrio parahaemolyticus, eriocheir sinensis spiroplasma, white spot syndrome virus and siniperca chuatsi iridovirus. The lowest detection limit of the primer probe group is 4.6 * 10 < 1 > copies / mu L, the sensitivity is improved by 1000 times compared with that of common PCR, and great detection advantages are shown.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of pathogen detection, and particularly relates to a primer probe set, a kit and a detection method for detecting Haplosporidium nihonense. BACKGROUND

[0002] Haplosporidium nihonense has high economic value and nutritional value, and a hepatopancreas necrosis disease caused by Haplosporidium nihonense has caused serious losses in the cultivation of Eriocheir sinensis. The main symptom of infection with Haplosporidium nihonense is that the appendages are long and empty, and although the crust can continue to be shed, the growth is slow, and the color of the hepatopancreas is gradually lightened from golden yellow to gray white, and some also have the symptom of "yellow discharge" (excretion of hepatopancreas).

[0003] So far, the detection of Haplosporidium nihonense still mainly adopts the method of dissection observation, and the method of dissection observation has higher requirements on the technical level of the operator and is not suitable for comprehensive promotion, and has obvious limitations. The fluorescent PCR technology is to detect the PCR reaction in real time by monitoring the change of the fluorescent signal in the PCR amplification process, and the whole process is completed in a closed tube, so it has the advantages of simple and rapid, high degree of automation, and not easy to cause cross contamination. Although the fluorescent PCR technology has been widely used in the detection and identification of pathogenic microorganisms, no primer for applying the fluorescent PCR technology to the detection of Haplosporidium nihonense has been reported. SUMMARY

[0004] In view of this, the application provides a primer probe set for detecting Haplosporidium nihonense, which can accurately detect Haplosporidium nihonense by the fluorescent PCR technology.

[0005] In order to achieve the above purpose, the application provides the following technical scheme:

[0006] The application provides a primer probe set for detecting Haplosporidium nihonense, which comprises a primer pair and a TaqMan probe.

[0007] The primer pair comprises a forward primer with a nucleotide sequence as shown in SEQ ID NO: 1 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO: 2.

[0008] The nucleotide sequence of the TaqMan probe is as shown in SEQ ID NO: 3.

[0009] Preferably, the 5 ′ end of the probe is labeled with a fluorescent group, and the 3 ′ end of the probe is labeled with a quenching group.

[0010] Preferably, the fluorescent group comprises FAM or TAMRA, and the quenching group comprises BHQ1.

[0011] The application provides application of the primer probe set in preparation of a kit for detecting H. eriocheiris.

[0012] The application provides a kit for detecting H. eriocheiris, which comprises the primer probe set and a real-time fluorescent quantitative PCR detection buffer.

[0013] Preferably, the real-time fluorescent quantitative PCR detection buffer is 2x5G qPCR Premix with UNG.

[0014] The application provides application of the kit in detection of H. eriocheiris for non-diagnostic purposes.

[0015] The application provides a detection method of H. eriocheiris for non-diagnostic purposes, which comprises the following steps.

[0016] Real-time fluorescent quantitative PCR detection is performed on DNA of the sample to be detected by using the primer probe set; if a positive amplification curve appears, it indicates that the sample to be detected is infected with H. eriocheiris; otherwise, it indicates that the sample to be detected is not infected with H. eriocheiris.

[0017] The Ct value of the positive amplification curve is less than or equal to 35.

[0018] Preferably, the reaction system of the real-time fluorescent quantitative PCR detection is 2x5G qPCR Premix with UNG 10 muL, 10 muM TaqMan probe 0.6 muL, 10 muM forward primer 1.2 muL, 10 muM reverse primer 1.2 muL, DNA template 1 muL, and ddH2O is supplemented to 20 muL.

[0019] Preferably, the reaction procedure of the real-time fluorescent quantitative PCR detection is as follows: 95 DEG C for 150 s; 94 DEG C for 15 s, 60 DEG C for 30 s, 40-45 cycles.

[0020] Compared with the prior art, the application has the following advantages:

[0021] The application provides a primer probe set for detecting H. chinense microsporidia, comprising a primer pair and a TaqMan probe; the primer pair comprises a forward primer with a nucleotide sequence as shown in SEQ ID NO: 1 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO: 2; and the nucleotide sequence of the TaqMan probe is as shown in SEQ ID NO: 3. In the application, the primer probe set can specifically detect H. chinense microsporidia, and the detection results of Aeromonas veronii, Vibrio parahaemolyticus, H. chinense spiroplasma, white spot syndrome virus and mandarin fish iridovirus are all negative. The primer probe set has high detection sensitivity, and the minimum detection limit is 4.6x10 1 copies / μL, which is 1000 times higher than the sensitivity of ordinary PCR, and has great detection advantages. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Fig. 1 is a specific detection result diagram of the primer probe set, wherein A-C are TaqMan fluorescence quantitative PCR detection results of different probes, A: CHI4, B: CHI4-1, C: CHI4-2, D is the amplification result of ordinary PCR detection of the primer pair CHI4-F and CHI4-R, and M in D: Trans2K DNA Marker, 1: H. chinense microsporidia; 2-7: negative control, Aeromonas veronii, Vibrio parahaemolyticus, H. chinense spiroplasma, white spot syndrome virus and mandarin fish iridovirus in sequence;

[0023] Figure 2 Fig. 2 is a TaqMan fluorescence quantitative PCR detection result diagram of the primer pair with a final concentration of 100 nmol / L;

[0024] Figure 3 Fig. 3 is a TaqMan fluorescence quantitative PCR detection result diagram of the primer pair with a final concentration of 200 nmol / L;

[0025] Figure 4 Fig. 4 is a TaqMan fluorescence quantitative PCR detection result diagram of the primer pair with a final concentration of 300 nmol / L;

[0026] Figure 5 Fig. 5 is a TaqMan fluorescence quantitative PCR detection result diagram of the primer pair with a final concentration of 400 nmol / L;

[0027] Figure 6 Fig. 6 is a TaqMan fluorescence quantitative PCR detection result diagram of the primer pair with a final concentration of 500 nmol / L;

[0028] Figure 7 Fig. 7 is a TaqMan fluorescence quantitative PCR detection result diagram of the primer pair with a final concentration of 600 nmol / L;

[0029] Figure 8 Fig. 9 is a standard curve and amplification curve of TaqMan fluorescent quantitative PCR for different concentrations of standard plasmid, A: 1-8 are 4.6x10 9 copies / μL, 4.6x10 8 copies / μL, 4.6x10 7 copies / μL, 4.6x10 6 copies / μL, 4.6x10 5 copies / μL, 4.6x10 4 copies / μL, 4.6x10 3 copies / μL and 4.6x10 2 copies / μL of standard plasmid;

[0030] Figure 9 Fig. 10 is a result of sensitivity detection of the Microsporidium eriocheiris, A: TaqMan fluorescent quantitative PCR detection result, 1-10 in A are 4.6x10 10 -4.6x10 1 copies / μL of standard plasmid, B: ordinary PCR detection result, 1-11 in B are 4.6x10 10 -4.6x10 0 copies / μL of standard plasmid, M: Trans2K DNA Marker;

[0031] Figure 10 Fig. 11 is a result of TaqMan fluorescent quantitative PCR detection of clinical samples of the Microsporidium eriocheiris. DETAILED DESCRIPTION

[0032] The application provides a primer probe set for detecting the Microsporidium eriocheiris, comprising a primer pair and a TaqMan probe.

[0033] The primer pair comprises a forward primer with a nucleotide sequence as shown in SEQ ID NO: 1 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO: 2.

[0034] The nucleotide sequence of the TaqMan probe is shown in SEQ ID NO: 3.

[0035] In the application, the primer probe set can specifically detect the Microsporidium eriocheiris. The primer probe set has high specificity and does not cross-react with other pathogenic bacteria (Aeromonas veronii, Vibrio parahaemolyticus, Spiroplasma eriocheiris, White Spot Syndrome Virus, Siniperca Huangshan Rainbow Virus). The primer probe set has high detection sensitivity, and the minimum detection limit is 4.6x10 1copies / μL. The source of the primer probe set is not particularly limited in the present application, and artificial synthesis of primers known in the art can be used. In the embodiments of the present application, the primer probe set is synthesized by Anhui General Biotech Co., Ltd., China.

[0036] In the present application, the 5 ′ end of the probe is preferably labeled with a fluorescent group, and the 3 ′ end of the probe is preferably labeled with a quenching group. The fluorescent group preferably includes FAM or TAMRA, and the quenching group preferably includes BHQ1.

[0037] The present application provides an application of the primer probe set in the preparation of a kit for detecting H. eriocheiris microspora.

[0038] The present application provides a kit for detecting H. eriocheiris microspora, comprising the primer probe set and a real-time fluorescent quantitative PCR detection buffer.

[0039] In the present application, the real-time fluorescent quantitative PCR detection buffer is preferably 2x5G qPCR Premix with UNG. In the embodiments of the present application, the 2x5G qPCR Premix with UNG is purchased from Toroivd, Shanghai, China.

[0040] The present application provides an application of the kit in the detection of H. eriocheiris microspora for non-diagnostic purposes.

[0041] The present application provides a detection method for H. eriocheiris microspora for non-diagnostic purposes, comprising the following steps:

[0042] Using the DNA of the sample to be detected as a template, real-time fluorescent quantitative PCR detection is performed using the primer probe set. If a positive amplification curve appears, it indicates that the sample to be detected is infected with H. eriocheiris microspora, otherwise, it indicates that the sample to be detected is not infected with H. eriocheiris microspora.

[0043] The Ct value of the positive amplification curve is ≤35.

[0044] The present application uses the DNA of the sample to be detected as a template. The extraction method of the DNA of the sample to be detected is not particularly limited in the present application, and the method for extracting DNA known in the art can be used. In the embodiments of the present application, DNA extraction is completed using an EasyPure Genomic DNA Kit (TransGen, Beijing, China).

[0045] After obtaining the DNA of the sample, real-time fluorescent quantitative PCR detection is carried out by using the primer probe set, if a positive amplification curve appears, it indicates that the sample to be detected is infected with Chinese mitten crab microsporidian, otherwise, it indicates that the sample to be detected is not infected with Chinese mitten crab microsporidian.

[0046] The reaction system of the real-time fluorescent quantitative PCR detection is preferably 2x5G qPCR Premix with UNG 10 muL, 10 muM TaqMan probe 0.6 muL, 10 muM forward primer 1.2 muL, 10 muM reverse primer 1.2 muL, DNA template 1 muL, and ddH2O is supplemented to 20 muL by adding ddH2O.

[0047] The reaction procedure of the real-time fluorescent quantitative PCR detection is preferably 95 DEG C for 150s; 94 DEG C for 15s, 60 DEG C for 30s, 40-45 cycles.

[0048] In order to further illustrate the present application, the scheme provided by the present application is described in detail below in combination with the drawings and examples, but they cannot be understood as limiting the protection scope of the present application.

[0049] 1. Experimental sample

[0050] The present application takes the liver and pancreas of Chinese mitten crab infected with microsporidian as experimental material.

[0051] 2. Main experimental instruments and equipment

[0052] (1) Ultra-low temperature refrigerator (Thermo, USA)

[0053] (2) Constant temperature water bath (Jinghong, Shanghai, China)

[0054] (3) Constant temperature shaking metal bath (Thermo, Germany)

[0055] (4) Constant temperature metal bath (MS Elite, Taiwan, China)

[0056] (5) Electrophoresis system (BIO-RAD, USA)

[0057] (6) Handheld homogenizer (TIANGEN, China)

[0058] (7) Pure water instrument (Saifei, Hong Kong, China)

[0059] (8) Electronic balance (OHAUS, USA)

[0060] (9) Gel imaging system (Jetta, Nanjing, China)

[0061] (10) Tabletop high speed centrifuge (Thermo Fisher, USA)

[0062] (11) PCR machine (BIO-RAD, USA)

[0063] (12) Rotator mixer (Xilinbail, Haimen, China)

[0064] (13) 4°C refrigerator (SEMENS, Germany)

[0065] (14) High pressure steam sterilization pot (Tomy, Japan)

[0066] (15) Nano Drop 2000c fluorescence spectrophotometer (Thermo, USA)

[0067] (16) Real-time fluorescent quantitative PCR analyzer (BIOER, Hangzhou, China)

[0068] (17) Roche fluorescent quantitative PCR machine (LightCycle 96, Switzerland)

[0069] (18) Shaking incubator (Shanghai, China)

[0070] 3. Experimental reagents and preparation

[0071] (1) EasyPure Genomic DNA Kit (TransGen, Beijing, China)

[0072] (2) DNA marker (TransGen, Beijing, China)

[0073] (3) YeaRed Nucleic Acid Gel Stain (Yixing, Shanghai, China)

[0074] (4) Flash Master Mix (Dye Plus) (Novozyme, Nanjing, China)

[0075] (5) 5G qPCR PreMix with UNG (Toroivd, Shanghai, China)

[0076] (6) -T1 Cloning Kit (TransGen, Beijing, China)

[0077] (7) Trans1-T1 competent cells (TransGen, Beijing, China)

[0078] (8) FastPure Plasmid Mini Kit-BOX 2 (Vazyme, Nanjing, China)

[0079] (9) Agarose (Hydragene)

[0080] (10) Nucleic acid electrophoresis solution (1 x TAE): Measure 10 mL of 50 x TAE solution in a container, and add 490 mL of ultrapure water and mix well.

[0081] (11) Preparation of 2% agarose gel: Weigh 0.6 g of agarose into a 250 mL conical flask, add 30 mL of 1 x TAE, heat in a microwave oven for about 1 min until the agarose is completely melted, add 3 μL of nucleic acid dye, pour into the gel preparation tank with a comb, and use after cooling and solidification.

[0082] Example 1

[0083] Screening of TaqMan fluorescent quantitative PCR primers for H. eriocheir

[0084] 1. Design of TaqMan fluorescent quantitative PCR primers

[0085] According to the conserved sequence of CHI4 gene and the design principles of TaqMan primers: design 18-25 bp primers without secondary structure, and the size of the amplified fragment is 70-200 bp. The primer pairs were designed by Oligo 6 software, and the sequences of the primer pairs are shown in Table 1.

[0086] Table 1 Nucleotide sequences of primer pairs

[0087]

[0088] 2. Verification of the specificity of the primers by conventional PCR

[0089] The specificity of the primer pairs in Table 1 was verified by conventional PCR detection using H. eriocheir microsporidian total DNA as the template.

[0090] Method for extracting H. eriocheir microsporidian total DNA: Weigh about 4-6 mg of animal tissue infected with microsporidia into a sterile 1.5 mL centrifuge tube, and prepare two replicate samples for each. The specific operation for DNA extraction is described in the EasyPure Genomic DNA Kit manual.

[0091] PCR reaction system: 10 μM forward primer 1 μL, 10 μM reverse primer 1 μL, Flash MasterMix (Dye Plus) 12.5 μL, template DNA 1 μL, sterile deionized water (PCR grade) to 25 μL.

[0092] PCR amplification program: pre-denaturation: 98°C for 30 s, 1 cycle; denaturation: 98°C for 10 s, annealing: 57°C for 5 s, extension: 72°C for 30 s, 30 cycles; complete extension: 72°C for 1 min, 1 cycle; cooling: 4°C for 30 min, 1 cycle.

[0093] After amplification, PCR products were obtained. 3 μL of PCR products were taken and subjected to electrophoresis using 2% agarose gel at 120 V for 22 min. The results were viewed on an imaging system.

[0094] Amplification results of primer pair CHI4-F and CHI4-R ( Figure 1 Middle (D) shows that only when microsporidium DNA is used as a template will the specifically amplified target band appear on the agarose gel electrophoresis diagram, while the other groups do not show any bands, indicating that the primers have good specificity and will not cross-react with other pathogens.

[0095] Example 2

[0096] 1. Design and synthesis of TaqMan fluorescent quantitative probe for Eriocheir sinensis

[0097] Based on the optimal primer pair CHI4-F and CHI4-R screened in Example 1, combined with the TaqMan probe design principles: between the upstream and downstream primers, the length is generally between 25 and 32 bp, the 5' end is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group. The probe was designed using Oligo 6 software and sent to a biological company for synthesis for subsequent experiments.

[0098] Table 2 TaqMan fluorescent quantitative PCR probe nucleotide sequences

[0099]

[0100] 2. Establishment of TaqMan Fluorescence Quantitative PCR Reaction System for Microsporidia in Eriocheir sinensis

[0101] According to the Tianluo diagnostic group 5G qPCRPreMix with UNG kit instructions, set up a 20μL TaqMan fluorescent quantitative PCR reaction system:

[0102]

[0103] The TaqMan fluorescence quantitative PCR reaction procedure is shown in Table 3.

[0104] Table 3 TaqMan fluorescence quantitative PCR reaction procedures

[0105]

[0106] 3. Verification of TaqMan fluorescent quantitative PCR primer specificity

[0107] In order to ensure the specificity of the TaqMan detection method in detecting microsporidia, the DNA of Chinese mitten crab microsporidia was used as a positive template, and the DNA of five pathogenic bacteria, i.e., Veillonella dispar, Vibrio parahaemolyticus, Chinese mitten crab spiroplasma, white spot syndrome virus, and mandarin fish iridovirus, was used as a control template to perform specific detection according to the above reaction system and reaction procedure.

[0108] The fluorescent quantitative results of primer pair CHI4-F and CHI4-R and probe CHI4-P Figure 1 Fig. 2A shows that only when the microsporidia DNA was used as a template, a specific amplification curve appeared, and the other groups did not show an amplification curve, indicating that the primer specificity was good and did not cross-react with other pathogens.

[0109] The fluorescent quantitative PCR amplification results of primer pair HE-CHI4-sF1 and HE-CHI4-sR1 and probe CHI4-P1 Figure 1 Fig. 2B shows that in addition to microsporidia DNA, Vibrio parahaemolyticus also amplified a curve, indicating that the primer specificity was poor and cross-reacted with Vibrio parahaemolyticus.

[0110] The fluorescent quantitative PCR amplification results of primer pair HE-CHI4-sF2 and HE-CHI4-sR2 and probe CHI4-P2 Figure 1 Fig. 2C shows that in addition to microsporidia DNA, the other groups of pathogens also amplified a curve, indicating that the primer specificity was poor and cross-reacted with other pathogens.

[0111] Therefore, primer pair CHI4-F and CHI4-R and probe CHI4-P were used as the final primer pair and probe for subsequent detection.

[0112] Example 3

[0113] Optimization method of primer pair and probe concentration

[0114] The optimal reaction condition was determined by considering the parameters of low Ct value, smooth amplification curve and strong fluorescence signal. The final concentration of probe CHI4-P in the TaqMan fluorescence quantitative PCR reaction system was 100 nmol / L, 200 nmol / L and 300 nmol / L, respectively, and the fluorescence quantitative PCR amplification was carried out under the condition that the final concentration of primer pair was 100 nmol / L, 200 nmol / L, 300 nmol / L, 400 nmol / L, 500 nmol / L and 600 nmol / L, respectively. The amount of other components in the reaction system was added according to the TaqMan fluorescence quantitative PCR reaction system in Example 2.

[0115] The reaction procedure was the same as that of TaqMan fluorescence quantitative PCR reaction in Example 2.

[0116] The optimization results showed that (Table 4 and Table 5): Figures 2-7 When the final concentration of primer pair was 600 nmol / L, the Ct value was smaller than that of other concentrations; and in the group of primer pair final concentration of 600 nmol / L, the Ct value was the smallest (19.63) and the fluorescence intensity was the largest (11819.12) when the final concentration of probe CHI4-P was 300 nmol / L, so the optimal final concentration of primer was 600 nmol / L and the optimal final concentration of probe was 300 nmol / L.

[0117] Table 4 Ct value of fluorescence quantitative PCR reaction with different concentrations of primers and probes

[0118]

[0119] Table 5 Fluorescence intensity of fluorescence quantitative PCR reaction with different concentrations of primers and probes

[0120]

[0121] After optimization of the concentrations of primer pair and probe, the optimal TaqMan fluorescence quantitative PCR reaction system was obtained:

[0122]

[0123] Example 4

[0124] Sensitivity detection of TaqMan fluorescence quantitative PCR for H. dromiacearum

[0125] 1. Preparation method of recombinant plasmid CHI4-T standard

[0126] 1) Purification of target gene: The target gene fragment amplified by primers CHI4-F and CHI4-R in Example 1 was gel-cut recovered and purified by a multifunctional DNA purification and recovery kit, and the specific steps were referred to Gel DNA Extraction Mini Kit instruction. The concentration was determined by Nano Drop 2000c fluorescence spectrophotometer after recovery, which was used for the next step of T cloning vector construction.

[0127] 2) Construction of T cloning vector of target gene

[0128] Cloning reaction system:

[0129] Target gene 1 μL

[0130] T1 Cloning Vector 1 μL

[0131] Enzyme-free sterile water to 5 μL

[0132] Gently mix, room temperature or PCR instrument reaction for 10 min, and then place on ice.

[0133] Note: The optimal insertion amount of DNA fragment (molar ratio of vector to target fragment = 1:7, which can be roughly calculated according to the ratio of 20 ng per 1 kb). The optimal amount of vector is 1 μL, and the optimal reaction system is 3-5 μL, and the insufficient amount is supplemented with sterile water.

[0134] Reaction time: 5 min;

[0135] Reaction temperature: 25°C.

[0136] The constructed recombinant plasmid is CHI4-T.

[0137] 3) Recombinant plasmid CHI4-T is transformed into cloning competent cells:

[0138] A. Add the ligation product to 50 μL Trans1-T1 competent cells (add the ligation product when the competent cells are just thawed), gently mix, and ice bath for 20-30 min;

[0139] B. 42°C water bath heat shock for 30 s, immediately placed on ice for 2 min;

[0140] C. Add 250 μL of LB liquid medium (without antibiotics) balanced to room temperature, and place in a shaking incubator at 200 rpm, 37°C for 1 h;

[0141] D. Mix 8 μL IPTG (500 mM) and 40 μL X-Gal (20 mg / mL), and evenly spread on the prepared LB solid medium plate, and place in a 37°C incubator for 30 min;

[0142] E. After the IPTG and X-Gal are absorbed, take 200 μL of bacterial solution and evenly spread on the plate, and place in a 37°C incubator for overnight culture.

[0143] 4) Positive clone detection and sequencing analysis

[0144] Each of the 10 white single colonies was picked into 10 μL sterile enzyme-free water and vortexed. 1 μL of the mixture was used as a template with 25 μL PCR system, M13 Forward Primer and M13 Reverse Primer to identify positive clones.

[0145] The PCR reaction program was as follows: pre-denaturation: 98°C for 30 s, 1 cycle; denaturation: 98°C for 10 s, annealing: 57°C for 5 s, extension: 72°C for 30 s, 30 cycles; complete extension: 72°C for 1 min, 1 cycle; cooling: 4°C for 30 min, 1 cycle.

[0146] Agarose gel electrophoresis was performed, and the bacterial liquid with a bright single band was selected and added into 5 ml LB liquid medium (with antibiotics) for culture at 37°C in a shaking bed for 12 h; 1 mL of fresh bacteria was subpackaged by General Biotech (Anhui) Co., Ltd. for sequencing; the sequencing results were analyzed by comparison using software DNAMAN, and the recombinant plasmid was extracted from the correct sequencing results using FastPure Plasmid Mini Kit-BOX 2 (see the instruction for specific operation) to obtain the recombinant plasmid CHI4-T.

[0147] 2. Construction of standard curve to determine the minimum detection concentration

[0148] The concentration was determined using a Nano Drop 2000c spectrophotometer, and the plasmid copy number was calculated. The plasmid copy number was calculated according to formula I.

[0149] Plasmid copy number (copies / μL) = (6.02 x 10 23 ng / μL) / (DNA length x 660 x 10 9 ) Formula I

[0150] The recombinant plasmid CHI4-T was serially diluted with sterile enzyme-free water, with 10 times as a gradient, and the recombinant plasmid with a final concentration of 4.6 x 10 9 ~ 4.6 x 10 2 copies / μL was used as a detection sample to prepare a reaction system according to the optimal TaqMan fluorescent quantitative PCR reaction system obtained in Example 3, and amplification was performed according to the reaction program in Example 2. The standard curve was constructed according to the initial copy number (SQ) of the recombinant plasmid and the corresponding cycle threshold (Ct), the correlation coefficient and amplification efficiency were analyzed, the minimum detection concentration was determined, and the sensitivity was analyzed.

[0151] As Figure 8The standard curve equation of the recombinant plasmid CHI4-T is y=-3.1654x+43.925, the correlation coefficient R=0.9977, and the amplification efficiency E=107%. The PCR efficiency is between 90%-110%, and R is greater than 0.99, indicating that the primers and probes have good performance. 2 2

[0152] The recombinant plasmid CHI4-T (4.6x10 10 ~4.6x10 0 copies / μL) was diluted by 10 times, and the optimal TaqMan fluorescent quantitative PCR reaction system obtained in Example 3 was used to prepare the reaction system. The amplification was performed according to the reaction procedure in Example 2 to determine the minimum detection concentration.

[0153] Figure 9 The results show that the minimum detection limit of the recombinant plasmid by TaqMan fluorescent quantitative PCR is 4.6x10 1 copies / μL. The minimum detection limit is 4.6x10 4 copies / μL by ordinary PCR using the same primers. The TaqMan detection method is 1000 times more sensitive than ordinary PCR, and the TaqMan fluorescent quantitative PCR method has high sensitivity.

[0154] Example 5

[0155] Clinical sample detection of Chinese mitten crab (Eriocheir sinensis) by TaqMan fluorescent quantitative PCR

[0156] In order to verify the feasibility of the method for clinical application, 19 nucleic acid DNAs of river crabs verified as positive, 1 recombinant plasmid CHI4-T in Example 4, and 1 negative control were used as templates, the optimal TaqMan fluorescent quantitative PCR reaction system obtained in Example 3 was used to prepare the reaction system, and the clinical samples were detected according to the reaction procedure in Example 2.

[0157] The clinical sample detection results Figure 10 show that the TaqMan fluorescent quantitative PCR detection method detects 20 amplification curves (including 1 positive control), indicating that the method has high accuracy for clinical detection.

[0158] Although the above examples have described the present application in detail, it is only a part of the embodiments of the present application, but not all the embodiments, and other embodiments can be obtained according to the present embodiments without creativity, which belong to the protection scope of the present application.​​

Claims

1. A primer probe set for detecting microsporidia in Chinese mitten crab, characterized in that: Includes primer pairs and TaqMan probes; The primer pair includes a forward primer having a nucleotide sequence as shown in SEQ ID NO: 1 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO: 2; The nucleotide sequence of the TaqMan probe is shown in SEQ ID NO:

3.

2. The primer probe set according to claim 1, characterized in that: The 5' end of the probe is labeled with a fluorescent group, and the 3' end of the probe is labeled with a quencher group.

3. The primer probe set according to claim 1, characterized in that: The fluorescent group includes FAM or TAMRA, and the quenching group includes BHQ1.

4. Use of the primer-probe set according to any one of claims 1 to 3 in preparing a kit for detecting microsporidia in Chinese mitten crab.

5. A kit for detecting microsporidia in Chinese mitten crab, characterized in that: The method comprises the primer probe set according to any one of claims 1 to 3 and a real-time fluorescence quantitative PCR detection buffer.

6. The kit according to claim 5, characterized in that The real-time fluorescence quantitative PCR detection buffer is 2×5G qPCR Premix with UNG.

7. Use of the kit according to claim 5 or 6 in the detection of Microsporidia in Eriocheir sinensis for non-diagnostic purposes.

8. A method for detecting microsporidia in Chinese mitten crab for non-diagnostic purposes, characterized in that: The following steps are involved: Using the DNA of the sample to be tested as a template, the primer probe set according to any one of claims 1 to 3 is used to perform real-time fluorescence quantitative PCR detection. If a positive amplification curve appears, it indicates that the sample to be tested is infected with Chinese mitten crab microsporidia; otherwise, it indicates that the sample to be tested is not infected with Chinese mitten crab microsporidia; The Ct value of the positive amplification curve is ≤35.

9. The detection method according to claim 8, characterized in that: The reaction system for the real-time fluorescence quantitative PCR detection is 2×5G qPCR Premix with UNG 10 μL, 10 μM TaqMan probe 0.6 μL, 10 μM forward primer 1.2 μL, 10 μM reverse primer 1.2 μL, DNA template 1 μL, and ddH2O added to 20 μL.

10. The detection method according to claim 8 or 9, characterized in that: The reaction procedure of the real-time fluorescence quantitative PCR detection is: 95° C. for 150 s; 94° C. for 15 s, 60° C. for 30 s, for 40 to 45 cycles.

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