Taqman probe qPCR primer set for detecting shrimp hepatocellular carcinoma and its application

Abstract

The present invention discloses a Taqman probe qPCR detection primer set for detecting shrimp hepatoenteric cysts and its application. The present invention uses the cysteine ​​desulfurase gene sequence of EHP as a new target sequence, and designs a pair of specific primers and Taqman probes. The total DNA of the hepatopancreas of the shrimp to be tested or the shrimp pond water sample or the shrimp pond mud sample is extracted respectively, and the total DNA is used as a template after quantification. The specific primers and Taqman probe designed are used to perform qPCR to detect the copy number of the EHP cysteine ​​desulfurase gene, and the corresponding relationship between the EHP spores and the cysteine ​​desulfurase gene is established, and the EHP spores can be quantified by the gene copy number. The present invention is based on new molecular target detection, and there is no amplification signal for the detection samples that do not contain shrimp hepatoenteric cysts, and the specificity is good; the method is fast, accurate, and highly sensitive, and the detection limit is as low as 10 1 It can not only avoid nonspecific amplification, but also be simple and quick to operate.

Description

Technical Field

[0001] The invention relates to the field of biological detection, and in particular to a Taqman probe qPCR detection primer set for detecting shrimp hepatoenterocystis and an application thereof. Background Art

[0002] Enterocytozoon hepatopenaei (EHP) belongs to the family Microsporidia and the genus Enterocytozoa. EHP is an intracellular parasite that infects shrimp exclusively. Its infection can cause severe growth retardation in shrimp, causing huge losses to the aquaculture industry. EHP mostly parasitizes in the hepatopancreatic tubule cells of shrimp. The size of mature spores is about 1 μm, which is difficult to observe using conventional methods. Therefore, it is very important to establish a rapid and sensitive EHP quantitative detection method that can be applied to aquaculture environments.

[0003] At present, a variety of detection methods for EHP have been developed, including tissue sections, PCR detection, in situ hybridization, LAMP, etc. Among them, PCR detection methods include conventional PCR detection, nested PCR, SYBR GREEN fluorescent quantitative PCR, TaqMan probe fluorescent quantitative PCR detection technology, etc. Among these methods, the histopathological method is time-consuming and has low detection efficiency; the ordinary PCR method is not sensitive enough and cannot be quantified; the accuracy of the SYBR GREEN fluorescent dye qPCR method is easily affected by nonspecific amplification; LAMP detection is prone to false positive results; and the existing PCR methods are mostly based on the 18s rDNA gene, and the homology of this gene in closely related species can reach more than 90%. Studies have shown that once a similar species exists, the method based on the SSUrDNA target will produce false positive results. Therefore, it is urgent to establish a specific, sensitive and stable TaqMan probe fluorescent quantitative PCR detection method that can be used for EHP detection in shrimp and aquaculture environments. Summary of the invention

[0004] In order to overcome the shortcomings and deficiencies of the prior art, the present invention aims to provide an application of a cysteine ​​desulfurase gene of EHP in the preparation of a reagent for detecting, identifying or diagnosing shrimp hepatoenteric cysts.

[0005] Another object of the present invention is to provide a Taqman probe qPCR detection primer set for detecting shrimp hepatoenteric cysts.

[0006] Another object of the present invention is to provide a Taqman probe qPCR detection kit for detecting shrimp hepatoenteric cysts.

[0007] Another object of the present invention is to provide an application of the above primer set or kit.

[0008] Another object of the present invention is to provide a method for quantitatively detecting shrimp hepatocellular carcinoma.

[0009] The present invention establishes a Taqman probe fluorescent quantitative PCR method for detecting shrimp hepatocystis, and provides a kit of all reagents required for detection. The present invention extracts the total DNA of the hepatopancreas of shrimp to be tested or the water sample of shrimp pond or the mud sample of shrimp pond respectively, and uses it as a template after quantification, and uses suitable primers and Taqman probes designed according to the cysteine ​​desulfurase gene sequence to perform real-time fluorescent quantitative PCR (qPCR) to detect the copy number of EHP cysteine ​​desulfurase gene, and establishes the corresponding relationship between EHP spores and cysteine ​​desulfurase gene, and can quantify EHP spores by gene copy number. Based on this, it is judged whether shrimp hepatocystis exists in the sample, and accurate information is provided for preventing the spread of shrimp hepatocystis disease.

[0010] Among them, the cysteine ​​desulfurase gene sequence of EHP is very conservative. The cysteine ​​desulfurase gene sequence amplified from the EHP-containing shrimp samples collected from different regions is exactly the same as the shrimp hepatocystis gene sequence in the shrimp samples uploaded by NCBI in Thailand and other places. At the same time, the nucleotide sequence homology of cysteine ​​desulfurase between microsporidia is less than 70%. Selecting a specific sequence region can completely avoid nonspecific amplification and obtain specific detection results. Therefore, the present invention uses the cysteine ​​desulfurase gene sequence of EHP as a new target sequence to establish a TaqMan probe fluorescent quantitative PCR detection method.

[0011] The purpose of the present invention is achieved through the following technical solutions:

[0012] The invention discloses an application of the cysteine ​​desulfurase gene (NFS1 gene) of EHP as a target in the preparation of a reagent for detecting, identifying or diagnosing shrimp hepatocystis.

[0013] Preferably, the GenBank of the cysteine ​​desulfurase gene of EHP is: MNPJ01000001.1, located at sites 38693-39937, Nfs1.

[0014] A reagent for detecting shrimp hepatocellular carcinoma, comprising primers capable of detecting a cysteine ​​desulfurase gene of EHP;

[0015] Specifically, based on the Taqman probe fluorescent quantitative PCR method, a pair of specific primers and Taqman probes were designed for the cysteine ​​desulfurase gene of EHP, and the sequences were as follows:

[0016] NFS177F: 5'-GCAAGGAACACAACATAG-3';

[0017] NFS177R: 5'-GCTTGGTTATCCTTACAC-3';

[0018] NFS177P: 5'-FAM-TTCATACAGACGCCGCACAGG-BHQ1-3';

[0019] Among them, NFS177P was modified with the fluorescent group FAM at the 5′ end and the fluorescent group BHQ1 at the 3′ end.

[0020] The use of the above reagent for detecting shrimp hepatoenteric cytozoa in a kit for detecting, identifying or diagnosing shrimp hepatoenteric cytozoa.

[0021] A kit for detecting shrimp hepatoenteric cysts contains the above reagent for detecting shrimp hepatoenteric cysts.

[0022] The kit also includes reagents required for qPCR reaction.

[0023] Reagents required for qPCR reaction: including reaction premix reagent A, reagent B and reagent C;

[0024] Reagent A: Contains 2.5 μL 10× PCR buffer, 2 μL dNTP mixture (10 mM), 1.5 μL MgCl 2 (25mmol·mL -1 ), 1.0 μL NFS177F (10 μM), 1.0 μL NFS177R (10 μM) and 15 μL sterile double distilled water (ddHO) 2 O).

[0025] Reagent B: 0.5 μL Taqman probe NFS177P (10 μM).

[0026] Reagent C: Taq enzyme, 0.5 μL (5 units / μL).

[0027] The preferred composition of the qPCR reaction system is: a total volume of 25 μL, including 2.5 μL 10× PCR buffer, 2.0 μL dNTP mixture (10 mM), 1.5 μL MgCl 2 (25mmol·mL -1 ), 1.0μL NFS177F (10μmol / L), 1.0μL NFS177R (10μmol / L), 0.5μL NFS177P (10μmol / L), 15μL ddH 2 O, 0.5 μL Taq enzyme, and 1 μL DNA template to be tested.

[0028] A method for quantitatively detecting shrimp hepatoenteric cysts comprises the following steps: the method is a non-disease diagnosis method;

[0029] (1) extracting DNA from the sample to be tested;

[0030] (2) adding the above-mentioned reagent for detecting shrimp hepatoenterozoa 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;

[0031] (3) The cysteine ​​desulfurase (NFS1) gene was amplified by PCR and connected to pMD18-T to construct the recombinant plasmid pMD18T-NFS1. The standard curve was drawn based on the relationship between the logarithmic values ​​of the recombinant plasmid pMD18T-NFS1 with different copy numbers (Sq) and their Ct values; the relationship between the copy number Sq and the Ct value was Ct = -3.231lg (Sq) + 40.638, and the correlation coefficient R 2 =0.998;

[0032] (4) Quantitative detection of shrimp hepatocellular carcinoma

[0033] After qPCR is completed, the Ct value is used to judge: if there is no amplification, it is judged that there is no shrimp hepatocellular carcinoma EHP in the sample; if there is amplification, the corresponding cysteine ​​desulfurase gene copy number is calculated according to the established standard curve, and the Ct value of the negative control and the blank control should have no amplification.

[0034] Among them, the copy number of cysteine ​​desulfurase gene (NFS1 gene) of each shrimp hepatocystis is about 2 copies.

[0035] Preferably, in step (1), the sample to be tested is shrimp tissue, shrimp pond water sample or shrimp pond mud sample;

[0036] Preferably, the method for processing shrimp pond water samples comprises the following steps:

[0037] Take a shrimp pond water sample, filter it using a 0.22μm filter membrane, rinse the filter membrane with an appropriate amount of sterile water, centrifuge the washing solution, discard the supernatant, and weigh the precipitate; use a DNA extraction kit or phenol / chloroform extraction method to extract the sample DNA and measure the DNA concentration.

[0038] Preferably, the method for processing shrimp tissue comprises the following steps:

[0039] Take shrimp tissue (such as shrimp hepatopancreas) and put it into a centrifuge tube. Use scissors to cut it into pieces or homogenize it as much as possible; use a DNA extraction kit or phenol / chloroform extraction method to extract the sample DNA and measure the DNA concentration.

[0040] Preferably, in step (2), the qPCR reaction system includes 1 μL of the sample DNA to be tested as a template, 23 μL of reagent A, 0.5 μL of reagent B, and 0.5 μL of reagent C mixed to a total volume of 25 μL.

[0041] Reagent A: Contains 2.5 μL 10× PCR buffer, 2 μL dNTP mixture (10 mM), 1.5 μL MgCl 2 (25mmol·mL -1 ), 1.0 μL NFS177F (10 μM), 1.0 μL NFS177R (10 μM) and 15 μL sterile double distilled water (ddHO) 2 O).

[0042] Reagent B: 0.5 μL Taqman probe NFS177P (10 μM).

[0043] Reagent C: Taq enzyme, 0.5 μL (5 units / μL).

[0044] qPCR reaction conditions: pre-denaturation at 95°C for 5 min; denaturation at 94°C for 20 s, annealing at 58°C for 20 s, extension at 72°C for 15 s, and amplification for 40 cycles; the fluorescence signal was collected at the end of annealing at 58°C each time; the Ct values ​​of the negative control and blank control should not be displayed.

[0045] Preferably, in step (3), the recombinant plasmid pMD18T-NFS1 with different copy numbers (Sq) is 1.67×10 1 ~1.67×10 9 The copy number / μL of the recombinant plasmid pMD18T-NFS1 is 1.67×10 1 , 1.67×10 2 , 1.67×10 3 , 1.67×10 4 , 1.67×10 5 , 1.67×10 6 , 1.67×10 7 , 1.67×10 8 , 1.67×10 9 The copy number / μL of the recombinant plasmid pMD18T-NFS1.

[0046] The main principle of the present invention is as follows: reagents A, B and C are polymerase chain reaction reagents. The 5' end of the Taqman probe in reagent B is labeled with a fluorescent group, and the 3' end is labeled with a quenching group. Under normal circumstances, the spatial distance between the two groups is very close, and the fluorescent group cannot emit fluorescence due to quenching. During PCR amplification, the primer and the probe can specifically bind to the EHP cysteine ​​desulfurase gene sequence. As the amplification proceeds, the DNA thermal polymerase (Taq enzyme) cuts off the fluorescent group on the 5' end of the probe through the 5' exonuclease activity, thereby emitting a fluorescent signal. The sample DNA is specifically amplified by the EHP cysteine ​​desulfurase gene specific primer contained in reagent A and the Taq enzyme in reagents B and C. Since the primer designed by the present invention is a specific primer, if the sample contains EHP, the Taqman probe will bind to the amplified specific fragment. When the amplification reaches the probe binding position, the 5' exonuclease activity of the Taq enzyme is used to cut off the fluorescent group, thereby separating it from the quenching group and releasing a fluorescent signal. After each cycle, the fluorescence signal will increase exponentially like the target fragment. According to the relationship between the starting concentration of different templates and the fluorescence signal intensity of PCR amplification, the starting copy number of the cysteine ​​desulfurase gene of the unknown sample can be calculated.

[0047] Compared with the prior art, the present invention has the following advantages and effects:

[0048] (1) The present invention can be applied to the detection of environmental samples from various aquaculture ponds, and can also be used to detect EHP infection in cultured shrimps;

[0049] (2) The method of the present invention is fast, accurate, and highly sensitive, with a detection limit as low as 10 1 level copies;

[0050] (3) The present invention is based on new molecular target detection, and no amplification signal is found for test samples that do not contain shrimp hepatocystis, and the specificity is good;

[0051] (4) The present invention uses the TaqMan probe fluorescent quantitative PCR method, which can not only avoid nonspecific amplification but also is simple and quick to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 It is the electrophoresis diagram of the PCR amplification product specific to EHP primers; wherein, M: DL500 DNA Marker (TaKaRa); 1: positive control; 2: IHHNV DNA; 3: HPV DNA; 4: WSSV DNA; 5: BP DNA; 6: Vibrio parahaemolyticus JGB080708-1 DNA; 7, 8: negative control (healthy shrimp tissue DNA); 9, 10: blank control.

[0053] Figure 2 It is a graph of the qPCR amplification curve of the EHP primer-specific Taqman probe; wherein, 1: positive control; 2: IHHNV DNA; 3: HPV DNA; 4: WSSV DNA; 5: BP DNA; 6: Vibrio parahaemolyticus JGB080708-1 DNA; 7: negative control (healthy shrimp tissue DNA); 8: blank control.

[0054] Figure 3 It is a graph of qPCR amplification of EHP primers and aquaculture water-specific Taqman probes; wherein, 1, 2: positive controls; 3, 4: positive controls mixed with aquaculture water; 5-8: aquaculture water; 9: blank control.

[0055] Figure 4 It is the electrophoresis diagram of the recombinant plasmid pMD18T-NFS1; wherein, M: DL10000 DNA Marker (TaKaRa); 1, 2: recombinant plasmid pMD18T-NFS1.

[0056] Figure 5 The standard plasmids with different concentrations (1.67×10 1 ~1.67×10 9 qPCR amplification curve of Taqman probe (0.1447 copies / μL).

[0057] Figure 6 It is the quantitative standard curve of Taqman probe qPCR detection of standard plasmid pMD18T-NFS1; where Cq is the reaction cycle number.

[0058] Figure 7 The standard plasmids with different concentrations (1.67×10 1 ~1.67×10 8 copies / μL) electrophoresis of conventional PCR amplification products, where M: DL2000; 1: 1.67×10 8 Copies / μL; 2: 1.67×10 7 Copies / μL; 3: 1.67×10 6 Copies / μL; 4: 1.67×10 5 Copies / μL; 5: 1.67×10 4 Copies / μL; 6: 1.67×10 3 Copies / μL; 7: 1.67×10 2 Copies / μL; 8: 1.67×10 1 Copies / μL; 9: negative control.

[0059] Figure 8This is the morphological observation of EHP spores; where, (a): EHP spores; (b): EHP spores stained with Giemsa stain; scale bar: 10 μm. DETAILED DESCRIPTION

[0060] 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.

[0061] 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.

[0062] The shrimp hepatocytozoon, infectious hypodermal and hematopoietic necrosis virus (IHHNV), hepatopancreatic parvovirus (HPV), and white spot syndrome virus (WSSV) used in the examples are all disclosed in the document "Liu Zhen, Zhang Qingli, Wan Xiaoyuan, Ma Fang, Huang Jie. Establishment of real-time fluorescence quantitative PCR detection method for shrimp hepatocytozoon (Enterocytozoon hepatopenaei) and detection of shrimp samples [J]. Advances in Fishery Science, 2016, 37(02): 119-126."

[0063] Baculovirus (BP) of shrimp was disclosed in the literature "Lei Yan, Xiao Yang, Zhang Huijun, Zhang Xiang, Wang Juan, Zhang Wenwen, Wang Xuepeng. Establishment and preliminary application of PCR detection method for baculovirus of Penaeus vannamei [J]. Journal of Guangdong Ocean University, 2015, 35(04): 63-66."

[0064] Vibrio parahaemolyticus JGB080708-1 is disclosed in the document "Zhang Xiaojun, Liang Liguo, Yan Binlun, Qin Lei, Qi Yaozhi. Research on double PCR detection method of pathogenic Vibrio parahaemolyticus in aquatic animals [J]. Marine Science, 2010, 34(10): 7-12."

[0065] Example 1 A TaqMan probe qPCR kit for detecting shrimp hepatocellular carcinoma was prepared according to the following formula:

[0066] (a) Taqman probe and specific primers:

[0067] According to the published cysteine ​​desulfurase gene (NFS1 gene) of shrimp hepatocystis (GenBank: MNPJ01000001.1, located at position 38693-39937, Nfs1), a pair of specific primers and Taqman probe were designed, and the specific sequences are as follows:

[0068] NFS177F: 5'-GCAAGGAACACAACATAG-3'; (SEQ ID NO: 1)

[0069] NFS177R: 5'-GCTTGGTTATCCTTACAC-3'; (SEQ ID NO: 2)

[0070] NFS177P: 5'-FAM-TTCATACAGACGCCGCACAGG-BHQ1-3'; (SEQ ID NO: 3)

[0071] Among them, NFS177P was modified with the fluorescent group FAM at the 5′ end and the fluorescent group BHQ1 at the 3′ end.

[0072] (b) Reagents required for qPCR reaction: including reaction premix reagent A, reagent B and reagent C.

[0073] Reaction premix A contains 2.5 μL 10× PCR buffer, 2 μL dNTP mixture (10 mM), 1.5 μL MgCl 2 (25mmol·mL -1 ), 1.0 μL NFS177F (10 μM), 1.0 μL NFS177R (10 μM) and 15 μL sterile double distilled water (ddHO) 2 O).

[0074] Reagent B: 0.5 μL TaqMan probe NFS177P (10 μM).

[0075] Reagent C: Taq enzyme, 0.5 μL (5 units / μL).

[0076] The preferred composition of the qPCR reaction reagent is: a total volume of 25 μL, including 2.5 μL 10× PCR buffer, 2.0 μL dNTP mixed solution (10 mM), 1.5 μL MgCl 2 (25mmol·mL -1 ), 1.0μL NFS177F (10μmol / L), 1.0μL NFS177R (10μmol / L), 0.5μL NFS177P (10μmol / L), 15μL ddH 2 O; 0.5 μL Taq enzyme, 1 μL DNA template to be tested.

[0077] (c) Specificity test

[0078] Conventional PCR and Taqman probe fluorescence quantitative PCR were used to test the specificity of the primers and probes. The nucleic acids of common shrimp pathogens were selected as templates for specificity testing, including infectious hypodermal and hematopoietic necrosis virus (IHHNV), hepatopancreatic parvovirus (HPV), white spot syndrome virus (WSSV), shrimp baculovirus (BP), and total DNA of Vibrio parahaemolyticus JGB080708-1. The negative control used the total DNA of healthy Penaeus vannamei without virus infection, the positive control used the total DNA of the hepatopancreas of Penaeus vannamei infected with EHP, and the blank control was sterile water.

[0079] The common PCR reaction system is: 2.5 μL of 10×PCR buffer, 2 μL of dNTP mixture (10 mM), 0.5 μL of upstream and downstream primers (10 μmol / L), 18.375 μL of sterile water, 1 μL of DNA template, 0.125 μL of TaKaRaTaq enzyme (5 U / μL), a total of 25 μL; the PCR reaction conditions are: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 20 s, a total of 35 cycles. Finally, 72℃ extension for 10 min. Electrophoresis detection, the results are as follows Figure 1 As shown, only the positive control (hepatopancreas DNA from shrimp infected with EHP) had an amplified band.

[0080] The Taqman probe qPCR-EHP method established in the present invention was tested for specificity, and the results were as follows: Figure 2 As shown, only the positive control (hepatopancreas DNA from shrimp infected with EHP) had an amplification curve.

[0081] The specificity of the Taqman probe qPCR method established in the present invention was tested using the DNA of the pond mixed water sample. The total DNA of the hepatopancreas of Penaeus vannamei infected with EHP was used as a positive control. Figure 3 As shown in the figure, when the positive control and the DNA of different pond water samples were mixed in a certain ratio as the template, there was an amplification curve. When the template was the aquaculture water DNA or sterile water (blank control), there was no amplification curve.

[0082] (d) Standard quantitative equations and conversion formulas;

[0083] The target fragment of NFS1 gene (177 bp in length) was amplified using the DNA sample of shrimp hepatocystis using NFS1 gene primers (NFS177F / NFS177R), connected to the pMD18-T vector and transformed into DH5α. After sequencing verification, the recombinant plasmid pMD18T-NFS1 was obtained. The electrophoresis diagram of the recombinant plasmid pMD18T-NFS1 is as follows: Figure 4 As shown, the recombinant plasmid pMD18T-NFS1 was successfully constructed.

[0084] Extract the recombinant plasmid pMD18T-NFS1, measure its A260 / A28, and calculate the copy number according to the following formula:

[0085]

[0086] Where M represents the average molecular mass per base pair, and X represents the size of the recombinant plasmid.

[0087] The DNA concentration of the extracted plasmid standard was 52.53 ng / μL. According to the above formula, the copy number of the plasmid was 1.67×10 10 copies / μL.

[0088] The plasmid standard with known concentration was diluted 10-fold in series, starting from 10 9 Up to 10 1 The number of copies / μL was 9 gradients, and each gradient was replicated 3 times. The optimized system was used for qPCR to establish a standard curve corresponding to the number of copies of the plasmid standard and the Ct value. The correlation coefficient (R 2 ) and amplification efficiency to judge the quality of the standard curve.

[0089] According to the experimental results, a standard curve of the threshold cycle number (Ct) of the amplification product and the logarithmic value of the initial copy number of the template was established ( Figure 6 ) equation is Ct = -3.231lg (Sq) + 40.638. Correlation coefficient R 2 =0.998, the amplification efficiency was 103.9%. Different concentrations of standard plasmid (1.67×10 1 ~1.67×10 9 The Taqman probe qPCR amplification curve of Figure 5 As shown in the figure, this standard curve is applicable to the standard template concentration of 1.67×10 9 ~1.67×10 1 When the template concentration is between 1.67 × 10 1 copies / μL.

[0090] Different concentrations of standard plasmid (1.67×10 1 ~1.67×10 8 Copies / μL) conventional PCR amplification product electrophoresis diagram, such as Figure 7 As shown in the figure, the minimum template concentration that can be detected by conventional PCR with this pair of primers is 1.67×10 3copies / μL, and the bands are darker. Taqman-qPCR can detect 1.67×10 1 This indicates that the sensitivity of Taqman-qPCR is 2 orders of magnitude higher than that of conventional PCR qualitative detection.

[0091] (e) Repeatability test of Taqman probe qPCR method

[0092] Repeatability analysis includes intra-group repeatability and inter-group repeatability. 1 to 1.67×10 8 The recombinant plasmid pMD18T-NFS1 standard template with 8 concentrations of copies / μL was used to calculate the mean, standard deviation and coefficient of variation (CV) of its Ct value. The results are shown in Table 1. The intra-group repetition was analyzed by using the mean and variance of the Ct of three parallel gradients in the same experiment, and the intra-group coefficient of variation was calculated; the inter-group repetition was performed after qPCR detection at different times, and the Ct value and variance of each experiment were analyzed. The results showed that the maximum coefficient of variation of intra-group repetition and inter-group repetition was 1.31%, both less than 5%, indicating that the detection method has good repeatability.

[0093] Table 1 Intra-group and inter-group repeatability experiments

[0094]

[0095]

[0096] (f) EHP spore staining and counting methods and their relationship with gene copy number

[0097] EHP spores were counted and the relationship between spores and NFS1 gene copy number was established. The purification method of frozen hepatopancreas tissue spores was based on the reported method (Zhao Ruoheng, Gao Wen, Qiu Liang, Li Chen, Xie Guosi, Huang Jie. Purification and counting of shrimp hepatopancreatocystis (EHP) spores [J]. Hebei Fisheries, 2019(01):16-20.). 20%, 30%, 40%, 50%, and 60% sucrose solutions were prepared by weight, spread in centrifuge tubes and left to stand at 4°C overnight. About 1g of frozen EHP-containing shrimp hepatopancreas was placed in ice-cold sterile seawater (salinity 25‰, pH 6.0) and homogenized and broken. Centrifuged at 4°C, 2000r / min for 10min. The supernatant was collected, and the precipitate was homogenized and broken in ice-cold sterile seawater again. After centrifugation at 4°C, 3000r / min for 10min, the two supernatants were combined and filtered with a 10μm filter membrane. The obtained spore crude extract was added to the top of the sucrose concentration gradient solution, centrifuged at 4°C, 13000r / min for 30min, the white band in the sucrose concentration gradient solution was aspirated, and the precipitate was collected by centrifugation at 4°C, 8000r / min for 10min, and the spores were suspended with an appropriate amount of 0.1M PBS buffer (pH 7.4), that is, the purified spore suspension. The purified EHP spore suspension was stained with Giemsa staining solution. The sample was observed under a microscope (Nikon, E200).

[0098] Take 5 different purified spore liquid samples (sample 1, sample 2, sample 3, sample 4, sample 5), and use a hemocytometer (Shanghai, Qiujing) to count the initial spore concentration of the spore liquid, and repeat 3 times. Dilute sample 1 4 times, sample 2 4 times, sample 3 10 times, sample 4 2 times, and sample 5 2 times. Use a hemocytometer to count the spores of each dilution to obtain the actual spore concentration of each dilution, and repeat three times. The initial spore concentration of the sample is divided by the dilution multiple to obtain the theoretical spore concentration of the dilution. The actual spore concentration of the same sample dilution is compared with the theoretical spore concentration. The NFS1 copy number of the dilutions of these 5 samples was quantified by qPCR, repeated 3 times, and the copy number corresponding to 1 spore in each sample was calculated. The corresponding copy numbers of single spores obtained from different samples were compared. All data are in the form of mean ± standard deviation.

[0099] Spore concentration (number of spores / μL) = number of spores in 80 cells / 80×400×10×dilution factor

[0100] Sample EHP loading (copies / μL) = 1.67×10 (Ct-40.638) / -3.2321 , where the Ct value is the number of cycles required for the reaction to reach a set threshold.

[0101] SPSS 17.0 was used to analyze the data. Paired T test was performed on the actual spore count and theoretical spore count of each sample dilution, and one-way analysis of variance (Turkey test) was performed on the corresponding copy numbers of single spores obtained from each sample dilution. There were three levels of significance analysis: significant difference (*P < 0.05), extremely significant difference (**P < 0.01), and no significant difference (P > 0.05).

[0102] Purify spores, stain spores Figure 8 As shown, EHP spores are oval in shape and about 1 × 0.7 μm in size.

[0103] Take 5 different spore liquid samples and count them with a hemocytometer, repeat 3 times. Dilute sample 1 4 times, sample 2 4 times, sample 3 10 times, sample 4 2 times, sample 5 2 times, and then use a hemocytometer to count the dilutions of each sample, repeat 3 times. Paired T tests were performed on the actual spore concentration and theoretical spore concentration of each sample dilution, and the results showed that there was no significant difference between the actual spore liquid concentration and the theoretical spore liquid concentration of each sample (P>0.05). The dilution method is feasible.

[0104] The NFS1 copy number of the five sample dilutions obtained in the previous step was quantified by qPCR and repeated three times. The copy number of each dilution and the spore concentration were used to calculate the copy number corresponding to one spore. The copy number corresponding to a single spore calculated in each group was subjected to a one-way analysis of variance (Turkey test). The results showed that there was no significant difference between the groups (P>0.05). The NFS1 gene copy number corresponding to a single spore in different samples ranged from 1.67 to 1.86, and one spore corresponded to approximately 2 NFS1 gene copies.

[0105] Example 2 Rapid detection of shrimp hepatocellular carcinoma according to the following procedure

[0106] The detection of shrimp hepatocystis in the sample using the kit includes the following steps:

[0107] (a) Extract DNA from samples:

[0108] ① Take 1L of pond water sample, filter it with a 0.22μm filter membrane, rinse the filter membrane with an appropriate amount of sterile water, centrifuge the washing solution and discard the supernatant, weigh about 25mg of precipitate; or take about 25mg of shrimp tissue (shrimp hepatopancreas) and add it to a centrifuge tube, chop it as much as possible or homogenize it with scissors.

[0109] ②Extract sample DNA using a DNA extraction kit or phenol / chloroform extraction method and measure the DNA concentration.

[0110] (b) Taqman probe qPCR:

[0111] Add 23 μL of reagent A to the PCR tube, add 0.5 μL of reagent B under light-proof conditions, and then add 0.5 μL of reagent C. 1 μL of the extracted sample DNA is used as a PCR amplification template.

[0112] The fluorescence quantitative PCR amplification procedure is as follows: 95°C pre-denaturation for 5 min; 94°C denaturation for 20 s, 58°C annealing for 20 s, 72°C extension for 15 s, amplification for 40 cycles; each time the fluorescence signal is collected at the end of 58°C annealing; after the reaction is completed, the Ct value reading is substituted into the standard curve equation Ct = -3.231lg (Sq) + 40.638 to calculate the NFS gene copy number of EHP contained in 1 μL sample DNA. If there is no reading, it is judged that the sample does not contain EHP.

[0113] Example 3 EHP monitoring of farmed shrimp and water environment in various regions

[0114] Sampling was carried out in coastal areas where shrimp were cultured, and the EHP detection method established by the present invention was used for detection. In 2019, EHP-positive shrimp samples were collected in Fuyan Village, Changsha Town, Rudong County, Nantong City, Jiangsu Province, and Hengshan Village, Lianzhou Town, Doumen District, Zhuhai City, Guangdong Province, which were stored in liquid nitrogen and sent back to the laboratory. In 2020, shrimp samples and shrimp pond water samples were collected from a farm in Chuandao Town, Jiangmen City, Guangdong Province, and a breeding area in Shenjing Town. 1L of water sample was collected, filtered through a 0.22μm filter membrane, the filter membrane was rinsed with an appropriate amount of sterile water, the supernatant was discarded after centrifugation of the washing solution, and part of the precipitate was taken to extract DNA; the shrimp sample was placed in the preservation solution and taken back to the laboratory, and DNA was extracted according to the method described above. The test results are shown in Table 2. Detailed data are shown in Tables 3 to 6. As can be seen from Tables 2 to 6, the EHP load in the hepatopancreas of shrimp ranges from 8.40×10 0 to 8.73×10 5 The EHP load in shrimp pond water ranged from 2.14×10 1 to 2.59×10 5 The detection method established by the invention has strong applicability to shrimp tissue samples and environmental samples.

[0115] Table 2 Field test results and positive detection rate

[0116] Sample type Number of samples Positive detection number Positive rate (%) Nantong shrimp samples 19 19 100％ Zhuhai shrimp samples 19 19 100％ Jiangmen City Prawn Samples 30 23 76.67％ Water samples from shrimp ponds in Jiangmen City 10 8 80％

[0117] Table 3 Detection of shrimp hepatopancreas samples in Nantong, Jiangsu

[0118] serial number Ct value ± SD EHP load ± SD (copy / μL) NT190726001 24.37±0.11 <![CDATA[1.81×10 5 ±1.80×10 4 ]]> NT190726002 27.60±0.05 <![CDATA[1.81×10 4 ±8.11×10 2 ]]> NT190726003 22.31±0.07 <![CDATA[7.84×10 5 ±4.47×10 4 ]]> NT190726004 23.87±0.06 <![CDATA[2.57×10 5 ±1.30×10 4 ]]> NT190726005 22.16±0.03 <![CDATA[8.73×10 5 ±2.10×10 4 ]]> NT190726006 24.16±0.09 <![CDATA[2.09×10 5 ±1.69×10 4 ]]> NT190726007 27.46±0.05 <![CDATA[1.99×10 4 ±9.28×10 2 ]]> NT190726008 24.54±0.01 <![CDATA[1.60×10 5 ±1.98×10 3 ]]> NT190726009 30.92±0.01 <![CDATA[1.70×10 3 ±1.40×10 1 ]]> NT190726010 29.71±0.05 <![CDATA[4.20×10 3 ±1.72×10 2 ]]> NT190726011 27.39±0.04 <![CDATA[2.10×10 4 ±7.51×10 2 <!-- 8 -->]]> NT190726012 26.67±0.07 <![CDATA[3.50×10 4 ±2.21×10 3 ]]> NT190726013 30.49±0.13 <![CDATA[2.30×10 3 ±2.71×10 2 ]]> NT190726014 30.11±0.04 <![CDATA[3.30×10 3 ±1.19×10 2 ]]> NT190726015 31.76±0.09 <![CDATA[9.32×10 2 ±7.71×10 1 ]]> NT190726016 30.53±0.05 <![CDATA[2.24×10 3 ±9.20×10 1 ]]> NT190726017 31.23±0.04 <![CDATA[1.36×10 3 ±4.93×10 1 ]]> NT190726018 27.91±0.07 <![CDATA[1.45×10 4 ±9.37×10 2 ]]> NT190726019 23.61±0.11 <![CDATA[3.11×10 5 ±3.20×10 4 ]]>

[0119] Table 4 Detection of shrimp hepatopancreas samples in Zhuhai, Guangdong

[0120] serial number Ct value ± SD EHP load ± SD (copy / μL) ZH190816001 25.38±0.10 <![CDATA[8.82×10 4 ±7.78×10 3 ]]> ZH19081602 26.70±0.08 <![CDATA[3.43×10 4 ±2.48×10 3 ]]> ZH19081603 23.27±0.20 <![CDATA[3.96×10 5 ±5.85×10 3 ]]> ZH19081604 24.51±0.40 <![CDATA[1.63×10 5 ±6.38×10 3 ]]> ZH19081605 27.53±0.08 <![CDATA[1.91×10 4 ±1.40×10 3 ]]> ZH19081606 28.62±0.20 <![CDATA[8.73×10 3 ±1.64×10 2 ]]> ZH190816007 27.72±0.07 <![CDATA[1.66×10 4 ±1.20×10 3 ]]> ZH190816008 29.58±0.40 <![CDATA[4.41×10 3 ±1.45×10 2 ]]> ZH190816009 27.43±0.10 <![CDATA[2.40×10 4 ±1.75×10 3 ]]> ZH190816010 27.64±0.40 <![CDATA[1.75×10 4 ±6.30×10 2 ]]> ZH190816011 25.50±0.60 <![CDATA[8.09×10 4 ±4.30×10 3 ]]> ZH190816012 25.73±0.40 <![CDATA[6.86×10 4 ±2.29×10 3 ]]> ZH190816013 29.49±0.40 <![CDATA[4.69×10 3 ±1.63×10 2 ]]> ZH190816014 29.12±0.00 <![CDATA[6.10×10 3 ±2.51×10 1 ]]> ZH190816015 29.76±0.40 <![CDATA[3.88×10 3 ±1.21×10 2 ]]> ZH190816016 26.65±0.07 <![CDATA[3.57×10 4 ±2.19×10 3 ]]> ZH190816017 26.42±0.07 <![CDATA[4.18×10 4 ±2.45×10 3 ]]> ZH190816018 24.17±0.60 <![CDATA[2.09×10 5 ±1.60×10 4 ]]> ZH190816019 23.73±0.60 <![CDATA[2.85×10 5 ±1.45×10 4 ]]>

[0121] Table 5 Hepatopancreas detection of shrimp in Jiangmen, Guangdong

[0122]

[0123]

[0124] Note: The mark “ / ” indicates that there is no amplification signal and the test result is negative.

[0125] Table 6 Water sample testing of shrimp ponds in Jiangmen, Guangdong

[0126] Shrimp pond water sample Ct±SD EHP load ± SD (copy / μL) SJ2011120106 30.76±0.09 <![CDATA[1.90×10 3 ±1.49×10 2 <!-- 10 -->]]> SJ2011120206 29.96±0.02 <![CDATA[3.36×10 3 ±6.31×10 1 ]]> SJ2011120306 34.31±0.02 <![CDATA[1.52×10 2 ±2.25×10 0 ]]> SJ2011120406 37.06±0.08 <![CDATA[2.14×10 1 ±1.45×10 0 ]]> SJ2011120506 23.86±0.04 <![CDATA[2.59×10 5 ±8.12×10 3 ]]> SJ2011120606 34.41±0.07 <![CDATA[1.41×10 2 ±9.13×10 0 ]]> CD2011090101 31.00±0.06 <![CDATA[1.60×10 3 ±8.10×10 1 ]]> CD2011090201 34.18±0.06 <![CDATA[1.66×10 2 ±9.28×10 0 ]]> CD2011090301 / / CD2011090401 / /

[0127] Note: The mark “ / ” indicates that there is no amplification signal and the test result is negative.

[0128] 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 shrimp hepatocellular carcinoma and its application <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> NFS177F <400> 1 gcaaggaaca caacatag 18 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> NFS177R <400> 2 gcttggttat ccttacac 18 <210> 3 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> NFS177P <220> <221> modified_base <222> (1) <223> FAM modification <220> <221> modified_base <222> (21)..(21) <223> BHQ1 modification <400> 3 ttcatacaga cgccgcacag g 21

Claims

1. Use of a reagent for detecting the cysteine ​​desulfurase gene of shrimp hepatoenteric cysts in the preparation of a reagent for detecting, identifying or diagnosing shrimp hepatoenteric cysts, characterized in that: The cysteine ​​desulfurase gene of the shrimp hepatocystis is located at position 38693-39937 of GenBank: MNPJ01000001.1; The reagents for detecting the cysteine ​​desulfurase gene of shrimp hepatoenteric cysts are primers and Taqman probes capable of detecting the cysteine ​​desulfurase gene of shrimp hepatoenteric cysts, and the sequences are as follows: NFS177F: 5'-GCAAGGAACACAACATAG-3'; NFS177R: 5'-GCTTGGTTATCCTTACAC-3'; NFS177P: 5'-FAM-TTCATACAGACGCCGCACAGG-BHQ1-3'.

2. Use of a reagent for detecting shrimp hepatoenteric cytosis in the preparation of a kit for detecting, identifying or diagnosing shrimp hepatoenteric cytosis, characterized in that: The reagents for detecting shrimp hepatoenteric cysts are primers and Taqman probes capable of detecting the cysteine ​​desulfurase gene of the shrimp hepatoenteric cysts described in claim 1, and the sequences are as follows: NFS177F: 5'-GCAAGGAACACAACATAG-3'; NFS177R: 5'-GCTTGGTTATCCTTACAC-3'; NFS177P: 5'-FAM-TTCATACAGACGCCGCACAGG-BHQ1-3'.

3. A method for quantitatively detecting shrimp hepatocellular carcinoma, 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) adding the reagent for detecting shrimp hepatoenterozoa 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; (3) PCR amplified the cysteine ​​desulfurase NFS1 gene and connected it to pMD18-T to construct the recombinant plasmid pMD18T-NFS1. The standard curve was drawn based on the relationship between the logarithmic value of the recombinant plasmid pMD18T-NFS1 with different copy numbers Sq and its Ct value; the relationship between the copy number Sq and the Ct value was Ct=-3.231 lg(Sq)+40.638, and the correlation coefficient R 2 =0.998; (4) Quantitative detection of shrimp hepatocellular carcinoma After qPCR is completed, the Ct value is used to judge: if there is no amplification, it is judged that there is no shrimp hepatocellular carcinoma EHP in the sample; if there is amplification, the corresponding cysteine ​​desulfurase gene copy number is calculated according to the established standard curve, and the Ct value of the negative control and the blank control should have no amplification.

4. The method according to claim 3, characterized in that: The copy number of cysteine ​​desulfurase gene in each shrimp hepatocystis is 2 copies; In step (1), the sample to be tested is a shrimp pond water sample or a shrimp pond mud sample.

5. The method according to claim 3, characterized in that: In step (2), the qPCR reaction system includes 1 μL of the sample DNA as a template, 23 μL of reagent A, 0.5 μL of reagent B, and 0.5 μL of reagent C mixed to a total volume of 25 μL; Reagent A: Contains 2.5 μL 10× PCR buffer, 2 μL 10 mM dNTP mixture, 1.5 μL 25 mmol·mL -1 MgCl2, 1.0 μL 10 μM NFS177F, 1.0 μL 10 μM NFS177R, and 15 μL ddH2O; Reagent B: 0.5 μL 10 μM NFS177P; Reagent C: 5 units / μL Taq enzyme, 0.5 μL.

6. The method according to claim 3, characterized in that: qPCR reaction conditions: pre-denaturation at 95°C for 5 min; denaturation at 94°C for 20 s, annealing at 58°C for 20 s, extension at 72°C for 15 s, and amplification for 40 cycles; the fluorescence signal was collected at the end of annealing at 58°C each time; there was no amplification in the negative control and blank control, and their Ct values ​​should not be displayed.

7. The method according to claim 3, characterized in that: In step (3), the recombinant plasmid pMD18T-NFS1 with different copy numbers Sq is 1.67×10 1 ~1.67×10 9 The number of copies / µL of the recombinant plasmid pMD18T-NFS1 is 1.67×10 1 , 1.67×10 2 , 1.67×10 3 , 1.67×10 4 , 1.67×10 5 , 1.67×10 6 , 1.67×10 7 , 1.67×10 8 , 1.67×10 9 The number of copies / µL of the recombinant plasmid pMD18T-NFS1.

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