CN118006802B - Primer pair for detecting Charybdis japonica crab drop disease and application thereof - Google Patents

Abstract

The invention discloses a primer pair for detecting Charybdisjaponica (Charybdisjaponica) Anomalae and application thereof, and belongs to the technical field of biological detection. The invention provides a primer pair and a PCR detection system for detecting the ND4 and/or ND5 genes of Charybdis japonica, and the results of examples show that the primer pair has specific fragments only in Charybdis japonica, has no cross reaction with other common Charybdis japonica and bacteria, and has good specificity. The sensitivity experiment of the PCR detection system shows that the minimum limit of the first primer pair ND4-F/R for detecting the Charybdis japonica is 16.251/mu L, the minimum limit of the second primer pair ND5-F/R for detecting the Charybdis japonica is 7.759/mu L, and the kit has good sensitivity. The invention provides an effective technical means for detecting or diagnosing the Charybdis japonica.

Description

A primer pair for detecting Charybdis japonica crab disease and its application

Technical Field

The invention belongs to the field of biological detection technology, and specifically relates to a primer pair for detecting Charybdis japonica crab disease and its application

Background Art

Japanese crab disease is a common and seriously harmful parasitic disease of the aquatic animal Japanese crab (Charybdis japonica), which causes serious damage to the growth, physiology and behavior of the host crab. For example, the diseased crabs grow slowly and cannot molt normally, and they suffer from hepatopancreatic lesions and atrophy of the reproductive system. A large number of diseased crabs lose their reproductive ability and edible value, endangering population breeding and industrial development.

The parasitic process of Japanese crabs is to first drill into the crab body and grow, and then generate a large number of branched endosomes in the host crab's tissues to absorb nutrients. After a few weeks, the endosomes in the lower abdomen of the host crab break out of the shell to generate spherical exosomes, which are the reproductive system of Japanese crabs. At present, whether Japanese crabs are infected with crab disease can only be judged by checking whether there are exosomes in the abdomen of Japanese crabs, which has a serious lag. In the early stage of Japanese crab infection with crab disease, Japanese crabs have endosomes in the host crab, but when the exosomes have not yet formed, it is difficult to confirm the diagnosis through clinical diagnosis.

In summary, there is currently a lack of accurate methods for early detection of Japanese crab disease in this field.

Summary of the invention

The purpose of the present invention is to provide primers and application methods for detecting Japanese crab disease. The primers provided by the present invention have high sensitivity and strong specificity in detecting Japanese crab endometrium, and can accurately detect the endometrium growing in the hepatopancreas of host crabs suffering from Japanese crab disease.

In order to achieve the above object, the present invention provides the following technical solutions:

The present invention provides a primer pair for detecting Japanese crab disease. The primer pair detects Japanese crab ND4 and/or ND5 genes. The nucleotide sequence of the ND4 gene is shown in SEQ ID NO.1, and the nucleotide sequence of the ND5 gene is shown in SEQ ID NO.2.

Preferably, the primer pair for detecting the ND4 gene of Crabella japonica is ND4-F and ND4-R, and their sequences are shown in SEQ ID NOs. 3 and 4.

Preferably, the primer pair for detecting the ND5 gene of Crabella japonica is ND5-F and ND5-R, and their sequences are shown in SEQ ID NOs. 5 and 6.

The present invention also provides a PCR system for detecting Japanese crab disease, and the PCR system comprises the above primer pair.

Preferably, the PCR system further comprises PCR Mix and dd water.

Preferably, the PCR system also includes a positive control sample and a negative control sample.

Preferably, the annealing temperature in the PCR reaction of the PCR system is 51°C-59°C.

Preferably, the annealing temperature in the PCR reaction of the PCR system is 55°C.

Preferably, the PCR reaction amplification program is: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s; annealing at 51°C-59°C for 30 s; extension at 72°C for 30 s; final extension at 72°C for 7 min; wherein the denaturation, annealing and extension are 35 cycles.

The present invention also provides the application of the primer pair and PCR system in detecting Japanese crab disease.

Beneficial effects:

The present invention provides a primer pair for the specific gene fragments ND4 and ND5 of Japanese crab disease, which can be used for the detection of Japanese crab disease. The primer pair ND4-F/R and ND5-F/R has the advantages of good specificity and sensitivity.

The present invention establishes a relevant detection method, which can quickly and accurately detect the infection of Japanese crab disease through PCR detection, greatly improves the detection rate of Japanese crab disease, makes the diagnosis more accurate, has broad application prospects, and is of great significance for the prevention and control of Japanese crab disease and healthy aquaculture.

The specificity experimental results of the present invention show that the primer pair of the present invention has a specific fragment only in Japanese crab disease, and has no cross reaction with other common crab diseases and bacteria, and has good specificity. The sensitivity experiment shows that the minimum limit of the first primer ND4-F/R for detecting Japanese crab disease is 16.251 cells/μL, and the minimum limit of the second primer ND5-F/R for detecting Japanese crab disease is 7.759 cells/μL, which has good sensitivity.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

Figure 1 shows the PCR amplification results of the primers for the ND4 gene of the Japanese crab Crabella nipponensis; wherein: Lane M is a DNA marker; Lane P is a positive control; Lane N is a negative control; Lanes 1-2 are the amplification results of the hepatopancreas tissue samples of the Japanese crab suffering from Crabella nipponensis.

Figure 2 shows the PCR amplification results of the primers for the ND5 gene of the Japanese crab Crabella nipponensis; Lane M is a DNA marker; Lane P is a positive control; Lane N is a negative control; Lanes 1-2 are the amplification results of the hepatopancreas tissue samples of the Japanese crab suffering from Crabella nipponensis.

Figure 3 shows the PCR amplification results of the ND4 gene at different amplification temperatures using the hepatopancreas cDNA of Japanese crab as a template, wherein: lane M is a DNA marker; lane N is a negative control; lanes 1-2, 3-4, 5-6, 7-8, and 9-10 are the amplification results when the annealing temperature is 51°C, 53°C, 55°C, 57°C, or 59°C, respectively.

Figure 4 shows the PCR amplification results of the ND5 gene at different amplification temperatures using the hepatopancreas cDNA of Japanese crab as a template, wherein: Lane M is a DNA marker; Lane N is a negative control; Lanes 1-2, 3-4, 5-6, 7-8, and 9-10 are the amplification results when the annealing temperature is 51°C, 53°C, 55°C, 57°C, or 59°C, respectively.

Figure 5 is the sensitive PCR amplification result of the ND4 gene of Crabella japonica; wherein: lane M is a DNA marker; lane N is a negative control; lanes 1-10 have copy numbers of 16.251×10 ⁹ , 16.251×10 ⁸ , 16.251×10 ⁷ , 16.251×10 ⁶ , 16.251×10 ⁵ , 16.251×10 ⁴ , 16.251×10 ³ , 16.251×10 ² , 16.251×10 ¹ , and 16.251×10 ⁰ /μL, respectively.

Figure 6 is the sensitive PCR amplification result of the ND5 gene of Crabella japonica; wherein: lane M is a DNA marker; lane N is a negative control; the ND5 plasmid copy numbers corresponding to lanes 1-10 are 7.759×10 ⁹ , 7.759×10 ⁸ , 7.759×10 ⁷ , 7.759×10 ⁶ , 7.759×10 ⁵ , 7.759×10 ⁴ , 7.759×10 ³ , 7.759×10 ² , 7.759×10 ¹ , 7.759×10 ⁰ , respectively.

Figure 7 shows the specific detection results of the specific primer pair of the ND4 gene of Japanese crab; wherein: lane M is a DNA marker: lane P is a positive control; lane N is a negative control; 1: Enterocytosis hepatocellularis (EHP) DNA; 2: Microsporidia DNA; 3: Rotifer Microsporidia DNA; 4: Metschnikowia bicuspids DNA; 5: White spot syndrome virus (WSSV) DNA; 6: Saccharomyces cerevisiae DNA; 7: Candida albicans DNA; 8: Artemia DNA; 9: hepatopancreas cDNA of Japanese crab with crab disease; 10: exosome cDNA of Japanese crab with crab disease; 11: hepatopancreas cDNA of healthy Japanese crab; 12: hepatopancreas cDNA of river crab with crab disease; 13: exosome cDNA of river crab with crab disease; 14: hepatopancreas cDNA of healthy river crab; 15: hepatopancreas cDNA of narrow-grass mitten crab with crab disease; 16: exosome cDNA of narrow-grass mitten crab with crab disease.

Figure 8 shows the specific detection results of the specific primer pair of the ND5 gene of Japanese crab; wherein: lane M is a DNA marker; lane P is a positive control; lane N is a negative control; 1: Enterocytosis hepatocellularis (EHP) DNA; 2: Microsporidia DNA; 3: Rotifer Microsporidia DNA; 4: Metschnikowia bicuspids DNA; 5: White spot syndrome virus (WSSV) DNA; 6: Saccharomyces cerevisiae DNA; 7: Candida albicans DNA; 8: Artemia DNA; 9: hepatopancreas cDNA of Japanese crab with crab disease; 10: exosome cDNA of Japanese crab with crab disease; 11: hepatopancreas cDNA of healthy Japanese crab; 12: hepatopancreas cDNA of river crab with crab disease; 13: exosome cDNA of river crab with crab disease; 14: hepatopancreas cDNA of healthy river crab; 15: hepatopancreas cDNA of narrow-grass mitten crab with crab disease; 16: exosome cDNA of narrow-grass mitten crab with crab disease.

FIG9 shows the results of the clinical samples of the first pair of specific primers ND4-F/ND4-R for Japanese crab disease; wherein: lane M is a DNA marker; lane P is a positive control; lane N is a negative control; lanes 1-9 are the amplification results of the hepatopancreas tissue of diseased Japanese crabs.

FIG. 10 shows the results of the clinical samples of the second pair of specific primers ND5-F/ND5-R for Japanese crab disease; wherein: lane M is a DNA marker; lane P is a positive control; lane N is a negative control; lanes 1-9 are the amplification results of the hepatopancreas tissue of diseased Japanese crabs.

DETAILED DESCRIPTION

The present invention provides a primer pair for detecting Japanese crab disease, which can specifically detect Japanese crab ND4 and/or ND5 genes. The primer pairs for detecting ND4 and ND5 genes are ND4-F/ND4-R and ND5-F/ND5-R, respectively. The present invention also provides a PCR system comprising the above primer pair and specific PCR reaction conditions.

The PCR amplification procedure in the present invention is: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds; annealing at 51°C-59°C for 30 seconds; extension at 72°C for 30 seconds; final extension at 72°C for 7 minutes; wherein, denaturation, annealing and extension are 35 cycles. Preferably, the annealing temperature in the PCR reaction is 55°C.

In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

The production processes, experimental methods or detection methods involved in the embodiments of the present invention, unless otherwise specified, are all conventional methods in the prior art, and their names and/or abbreviations are all conventional names in the field, and are very clear and unambiguous in the relevant application fields. Technical personnel in the field can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

The various instruments, equipment, raw materials or reagents used in the embodiments of the present invention are not particularly limited in terms of their sources, and are all conventional products that can be purchased through regular commercial channels, or can be prepared according to conventional methods well known to those skilled in the art.

Example 1

Design and validation of primer pairs for detecting Japanese crab disease

1.1 Plasmids, strains, and samples

The PMD19T vector was purchased from Baoriyi Biotechnology Co., Ltd.; the Escherichia coli DH5α competent cells were purchased from Quanshijin Biotechnology Co., Ltd.; the positive samples of Japanese crab were collected from Dalian, Liaoning, and then extracted from the laboratory and reverse transcribed into cDNA for purification.

1.2 Kit

DNA extraction kit was purchased from Tiangen (Beijing) Biochemical Technology Co., Ltd., PCR Mix and PCR product recovery kit were purchased from Nanjing Novozyme Biotechnology Co., Ltd.; plasmid extraction kits were purchased from Dingguo Biotechnology Co., Ltd.

1.3 Extraction of DNA from crab nudibranchs and purification of target gene fragments

According to the instructions of each kit, extract, recover, purify and extract the plasmid.

1.4 Primer design

According to the genome sequence of Japanese crab slave in the transcriptome, primers were designed using Primer 5.0 software. Two pairs of primers were designed: the first specific primer pair ND4-F/ND4-R and the second specific primer pair ND5-F/ND5-R for the diagnosis of Japanese crab slave specific nucleic acid. The sequences are shown in Table 1.

Table 1 List of PCR primers for identification of Crabworm japonicus

1.5 Amplification experiment

PCR amplification was performed using the hepatopancreas tissue samples of Japanese crabs with crab disease as templates. The reaction system was: 2×PCRMix 5μL, specific primer pair (nmol/L) 0.8μL, dd water 3.2μL.

The PCR amplification program is as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s; annealing at 55°C for 30 s; extension at 72°C for 30 s; final extension at 72°C for 7 min; denaturation, annealing and extension for 35 cycles. After the above PCR reaction is completed, 5 μL of the PCR product is analyzed by 2% agarose gel electrophoresis.

The PCR amplification results of the primers for the ND4 gene of Japanese crab are shown in Figure 1. Among them: Lane M is a DNA marker; Lane P is a positive control; Lane N is a negative control; Lanes 1-2 are the amplification results of the hepatopancreas tissue samples of Japanese crabs with crab disease;

The PCR amplification results of the primers for the ND5 gene of Japanese crab are shown in Figure 2. Among them: Lane M is a DNA marker; Lane P is a positive control; Lane N is a negative control; Lanes 1-2 are the amplification results of the hepatopancreas tissue samples of Japanese crabs with crab disease;

Example 2

Annealing temperature of primers for PCR amplification of Crabella japonica

PCR amplification was performed using the nucleic acid of crab nucleus of Japanese crab as a template. The reaction system was: 2×PCR Mix 5 μL, specific primer pair (concentration 1 nmol/L) 0.8 μL, crab hepatopancreas cDNA of Japanese crab 1 μL, and dd water 3.2 μL.

The annealing temperature of the primers was determined. According to the reference temperature after the primers were synthesized, 51°C, 53°C, 55°C, 57°C, and 59°C were selected for PCR amplification. The PCR amplification program was as follows: pre-denaturation at 95°C for 5min; denaturation at 95°C for 30S; annealing at 51-59°C for 30s; extension at 72°C for 30S; final extension at 72°C for 7min; denaturation, annealing, and extension were 35 cycles. After the above PCR reaction was completed, 5 μL of the PCR product was taken for 2% agarose gel electrophoresis analysis.

The results of the first specific primer pair ND4-F/ND4-R are shown in Figure 3, and the results of the second specific primer pair ND5-F/ND5-R are shown in Figure 4. The results show that a single bright band is obtained at different annealing temperatures, and the position is consistent with the expected size. Therefore, the PCR annealing temperature of the two pairs of primers can be 51°C--59°C.

Example 3

Sensitivity Identification of Specific Primer Pairs for Crab Crab Stomach

The PCR product of ND4-F/R in Example 1 was excised from the gel, and the extracted plasmid was recovered and the concentration was measured. The plasmid copy number was calculated according to the DNA copy number calculation formula.

Copy number = (6.02 × 10 ¹⁴ × concentration ng/μL) / (DNA length × 660)

The Japanese crab nudibranch ND4 plasmid was diluted to about 16.251×10 ⁹ , 16.251×10 ⁸ , 16.251×10 ⁷ , 16.251×10 ⁶ , 16.251×10 ⁵ , 16.251×10 ⁴ , 16.251×10 ³ , 16.251×10 ² , 16.251×10 ¹ , 16.251×10 ⁰ /μL. 1 μL was taken as a template, and ND4-F/R was amplified by PCR using the first specific primer of the present invention. The reaction conditions were the same as those in Example 2, and the annealing temperature was 55° C. After the reaction, 5 μL of the PCR product was taken for 2% agarose gel electrophoresis analysis.

The experimental results are shown in Figure 5. The copy numbers of lanes 1-10 are 16.251×10 ⁹ , 16.251×10 ⁸ , 16.251×10 ⁷ , 16.251×10 ⁶ , 16.251×10 ⁵ , 16.251×10 ⁴ , 16.251×10 ³ , 16.251×10 ² , 16.251×10 ¹ , and 16.251×10 ⁰ /μL, respectively. After PCR amplification, the electrophoresis results showed that the minimum detection limit was 16.251×10 ⁰ /μL.

The sensitivity identification method of the second specific primer pair ND5-F/R is the same as that of the first specific primer pair ND4-F/R except for the primers. The experimental results are shown in Figure 6. The ND5 plasmid copy numbers corresponding to lanes 1-10 are 7.759×10 ⁹ , 7.759×10 ⁸ , 7.759×10 ⁷ , 7.759×10 ⁶ , 7.759×10 ⁵ , 7.759×10 ⁴ , 7.759×10 ³ , 7.759×10 ² , 7.759×10 ¹ , and 7.759×10 ^0. After PCR amplification, the electrophoresis results showed that the minimum detection limit was 7.759×10 ⁰ /μL.

Example 4

Identification of the specificity of the primer pair ND4-F/R of Crab Crab

The DNA of enterocytosis hepatocystis (EHP), microsporidia DNA, rotifer microsporidia DNA, yeast DNA, white spot syndrome virus (WSSV) DNA, saccharomyces cerevisiae DNA, Candida albicans DNA, Artemia DNA, hepatopancreas cDNA of Japanese crab with crab disease, cDNA of Japanese crab exosomes with crab disease, cDNA of healthy Japanese crab, hepatopancreas cDNA of river crab with crab disease, cDNA of river crab exosomes with crab disease, cDNA of healthy river crab, hepatopancreas cDNA of narrow mitten crab with crab disease, cDNA of narrow mitten crab exosomes with crab disease were used as templates, and PCR amplification was performed using the first specific primer to the ND4-F/R primer, and the reaction conditions were the same as those in Example 2, the annealing temperature was 55° C., and the template was 1 μL. After the end, 5 μL of PCR products were taken for 2% agarose gel electrophoresis analysis.

The results are shown in Figure 7. Except for the hepatopancreas and exosomes of Japanese crabs, no specific target bands were obtained with several other common diseased crabs, yeasts, and bacteria. This shows that the first specific primer has good specificity for ND4-F/R.

In Figure 7: Lane M is a DNA marker; Lane P is a positive control; Lane N is a negative control; 1: Enterocytosis hepatocellularis (EHP) DNA; 2: Microsporidia DNA; 3: Rotifer Microsporidia DNA; 4: Metschnikowia bicuspids DNA; 5: White spot syndrome virus (WSSV) DNA; 6: Saccharomyces cerevisiae DNA; 7: Candida albicans DNA; 8: Artemia DNA; 9: Hepatopancreas cDNA of Japanese crab with crab disease; 10: Exosome cDNA of Japanese crab with crab disease; 11: Hepatopancreas cDNA of healthy Japanese crab; 12: Hepatopancreas cDNA of river crab with crab disease; 13: Exosome cDNA of river crab with crab disease; 14: Hepatopancreas cDNA of healthy river crab; 15: Hepatopancreas cDNA of narrow-grass mitten crab with crab disease; 16: Exosome cDNA of narrow-grass mitten crab with crab disease.

The specific identification method of the second specific primer pair ND5-F/R is the same as the first specific primer pair ND4-F/R except for the primers. The results are shown in Figure 8. Except for the hepatopancreas and exosomes of Japanese crabs, no specific target bands were obtained with several other common diseased crabs, yeasts, and bacteria. This shows that the second specific primer pair ND5-F/R has good specificity.

In Figure 8: Lane M is a DNA marker; Lane P is a positive control; Lane N is a negative control; 1: Enterocytosis hepatocellularis (EHP) DNA; 2: Microsporidia DNA; 3: Rotifer Microsporidia DNA; 4: Metschnikowia bicuspids DNA; 5: White spot syndrome virus (WSSV) DNA; 6: Saccharomyces cerevisiae DNA; 7: Candida albicans DNA; 8: Artemia DNA; 9: Hepatopancreas cDNA of Japanese crab with crab disease; 10: Exosome cDNA of Japanese crab with crab disease; 11: Hepatopancreas cDNA of healthy Japanese crab; 12: Hepatopancreas cDNA of river crab with crab disease; 13: Exosome cDNA of river crab with crab disease; 14: Hepatopancreas cDNA of healthy river crab; 15: Hepatopancreas cDNA of narrow-grass mitten crab with crab disease; 16: Exosome cDNA of narrow-grass mitten crab with crab disease.

Example 5

PCR diagnosis of clinical samples of ND4-F/R and ND5-F/R using specific primers of Crab Crab Stomach

Using ND4-F/R and ND5-F/R as primers, PCR diagnostic identification was carried out on some samples infected with Japanese crab slaves collected in 2023.

The clinical samples collected at different times were identified using the Japanese crab disease specific PCR detection method in the above embodiment of the present invention. The results are shown in Figures 9 and 10. The detection rate of Japanese crab disease detected by the method in the above embodiment of the present invention is 100%.

FIG9 is the result of the first pair of specific primers for Japanese crab disease ND4-F/R clinical samples, wherein: lane M is DNA marker; lane P is a positive control; lane N is a negative control; lanes 1-9 are the amplification results of the hepatopancreas tissue of diseased Japanese crabs;

FIG10 is the result of the clinical sample of the second pair of specific primers ND5-F/R for Japanese crab disease of the present invention, wherein: lane M is a DNA marker; lane P is a positive control; lane N is a negative control; lanes 1-9 are the amplification results of the hepatopancreas tissue of diseased Japanese crabs;

The amplified products identified as positive by the specific PCR of the Japanese crab disease established in the present invention in the hepatopancreas tissue of the Japanese crab were sequenced and analyzed, and the sequence analysis confirmed that they were all sequences of the Japanese crab.

From the above examples, it can be seen that the specific primer pairs ND4-F/R and ND5-F/R for Japanese crab disease provided by the present invention have no cross-reaction with other common crab diseases and bacteria, have good specificity, and high detection sensitivity, making the detection method of Japanese crab disease simpler and the diagnosis more accurate.

Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims (8)

1. A primer pair for detecting Japanese crab disease, characterized in that the primer pair detects the Japanese crab disease ND4 gene and/or ND5 gene, the nucleotide sequence of the ND4 gene is shown in SEQ ID NO.1, and the nucleotide sequence of the ND5 gene is shown in SEQ ID NO.2; The primer pair for detecting the ND4 gene of Crabella japonica is ND4-F and ND4-R, and the sequences thereof are shown in SEQ ID NO.3 and 4; The primer pair for detecting the ND5 gene of Crabella japonica is ND5-F and ND5-R, and the sequences thereof are shown in SEQ ID NOs. 5 and 6. 2. A PCR system for detecting Japanese crab disease, characterized in that the PCR system comprises the primer pair according to claim 1. 3. The PCR system according to claim 2, characterized in that the PCR system also includes PCRMix and dd water. 4. The PCR system according to claim 2, characterized in that the PCR system also includes a positive control sample and a negative control sample. 5. The PCR system according to claim 2, characterized in that the annealing temperature in the PCR reaction of the PCR system is 51°C-59°C. 6 . The PCR system according to claim 5 , wherein the annealing temperature in the PCR reaction of the PCR system is 55° C. 7. The PCR system according to claim 2 is characterized in that the PCR reaction amplification program of the PCR system is: preliminary denaturation at 95°C for 5 min; denaturation at 95°C for 30 s; annealing at 51°C-59°C for 30 s; extension at 72°C for 30 s; final extension at 72°C for 7 min; wherein the denaturation, annealing and extension are 35 cycles. 8. Use of the primer pair according to claim 1 or the PCR system according to claims 2-7 in preparing a reagent for detecting Japanese crab disease.