Absolute fluorescent quantitative PCR (Polymerase Chain Reaction) detection kit for detecting fasciculago fasciculatus

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to an absolute fluorescent quantitative PCR (Polymerase Chain Reaction) detection kit for detecting fasciculago fasciculatus. The invention provides a primer pair for detecting a parasite, namely fasciculago fasciculatus parasitized in Eriocheir sinensis, which is characterized in that an upstream primer and a downstream primer with the amplified fragment size of 257bp are synthesized according to a target gene sequence of the fasciculago fasciculatus. The invention also provides a kit containing the primer pair and used for detecting the fasciculatorula fasciculatorula. The kit provided by the invention can quickly diagnose the content of parasitic fasciculago fasciculatus in the Eriocheir sinensis body through the specific primer of fasciculago fasciculatus in the Eriocheir sinensis body and SYBR Green fluorescent quantitative PCR detection, so that the sensitivity and the specificity of the detection of fasciculago fasciculatus disease of the Eriocheir sinensis are enhanced to a great extent, and missing detection and false detection are avoided.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and specifically relates to an absolute fluorescence quantitative PCR detection kit for detecting clustered crab slaves. Background Art

[0002] In my country's aquatic fishing and breeding industry, Chinese mitten crab is one of the important freshwater economic crabs in my country due to its wide ecological niche and delicious taste. The breeding output in 2023 reached 888,600 tons. However, the parasitism of cluster crab slaves seriously endangers the germplasm resources and industrial development of Chinese mitten crab. Especially in the summer when the disease is most prevalent, a large number of Chinese mitten crabs were found to be parasitized by cluster crab slaves, which made them inedible and infertile, causing serious economic losses.

[0003] The adult Polyascus gregaria parasitizes the Chinese mitten crab. It is divided into two parts: the endosome and the exosome. The endosome is a branched root-like structure that parasitizes the tissues in the host's shell and gradually spreads throughout the host's body. It controls the host's behavior and absorbs nutrients by surrounding the host's hepatopancreas, reproductive system and nervous system. As the parasitic time increases, the endosome will break through the shell under the abdomen of the diseased crab to produce one or more oval exosomes. The plastron of the diseased crab will also become wider to better protect the growth of the exosome. During the parasitic process, the growth, molting and reproductive system development of the diseased crab are inhibited, and the edible value and reproductive ability are lost. When the exosome matures in the late stage of the disease, a large number of Polyascus gregaria larvae will be discharged into the water. The crab larvae will look for new hosts and cause a new round of infection. This poses a huge hidden danger to the Chinese mitten crab farming industry. In addition, the parasitic period of cluster crab parasites is long, and when the internal body is developing and the external body has not yet been formed, the symptoms of the disease are hidden and cannot be distinguished by the naked eye. As a result, Chinese mitten crabs infected with cluster crab parasites are circulated and sold in the market, posing a food safety hazard.

[0004] At present, in the early stage of Chinese mitten crab infection with clustered crab slaves, clustered crab slaves only form root-like endosomes in the diseased crab body, and it is difficult to detect when the spherical crab slave exosomes do not appear in the abdomen. The current conventional detection method is morphological detection or conventional PCR detection. Morphological detection is to check whether the segment width of the abdominal carapace of Chinese mitten crab is widened, but this method is only applicable to the case where the abdomen of the diseased crab becomes feminized due to long-term parasitism of clustered crab slaves. In addition, there are also some healthy crabs that only have symptoms of feminization of the abdomen of male crabs due to abnormal gonadal development, also known as "intersex crabs". Therefore, it is inaccurate to detect whether the crab slave disease is only caused by morphological detection. In addition, the conventional PCR detection method has the defect of low sensitivity, and it is impossible to accurately and quantitatively detect clustered crab slaves and their content in host tissues, and to clarify the degree of disease. The severity of the disease can be determined by inferring the time of illness of the diseased crab based on the growth rate of clustered crab slaves. Clarifying the susceptibility time of crab slave disease is a key condition for early prevention and control of crab slave disease.

[0005] Therefore, there is an urgent need for a primer and a detection method with high specificity and sensitivity for early detection of Chinese mitten crab infection with cluster crab disease. Summary of the invention

[0006] The purpose of the present invention is to solve the above-mentioned technical problems of low detection sensitivity and inability to quantify the early diagnosis technology of cluster crab parasites in Chinese mitten crabs, and to provide a primer pair, absolute fluorescence quantitative PCR detection method and kit for early detection of cluster crab parasites in Chinese mitten crabs.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] On the one hand, the present invention provides a primer pair for detecting Eriocheir sinensis, wherein the primer pair is designed according to the target gene sequence of Eriocheir sinensis, and the target gene sequence is shown in SEQ ID NO.1.

[0009] Preferably, the primer pair sequences are RS16F and RS16R, and their sequences are shown in SEQ ID NO.2 and SEQ ID NO.3 respectively.

[0010] SEQ ID NO.2 is: 5'-GCATTGTTACTTACTCATACTTAGTGCTGGAG-3'

[0011] SEQ ID NO. 3 is: 5'-TATGTGGATGAGGCATCAAAGAAGGAGAT-3'.

[0012] In another aspect, the present invention provides a kit for early detection of Chinese mitten crabs infected with cluster crab disease, the kit comprising a primer pair for cluster crab disease detection.

[0013] Preferably, the kit further comprises a positive standard and / or a negative standard of the Crabella fascicularis plasmid.

[0014] Preferably, the positive standard is a positive plasmid of the target sequence amplified by RS16F / RS16R primers, and the negative standard is ddH2O.

[0015] Preferably, the reaction system of the kit is: the primer pair RS16F and RS16R are each in a volume of 0.4 μL in the reaction system, with a concentration of 10 μM / μL; ddH2O is 7.2 μL; the template is 2 μL; 2×ChamQ Universal SYBR qPCRMasterMix is ​​10.0 μL.

[0016] Preferably, the reaction conditions of the reaction system are pre-denaturation at 95°C for 30s, 1 cycle; denaturation at 95°C for 10s, annealing at 60°C for 30s, and extension at 72°C for 30s, 40 cycles.

[0017] On the other hand, the present invention provides an application in preparing a reagent for detecting clustered crab larvae in Chinese mitten crab.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] (1) Strong specificity: The present invention significantly enhances the specificity of detection by introducing specific amplification primers, thereby ensuring accurate identification of the clustered crab slaves in the Chinese mitten crab.

[0020] (2) High sensitivity: The present invention also combines the SYBR GreenMix system, which greatly improves the sensitivity of detection, so that the presence of cluster crabs can be accurately captured even at low concentrations. For the positive plasmid of the target gene of cluster crab detection, the minimum detection limit can reach 9.1 copies / μL.

[0021] (3) High experimental repeatability: The present invention performs absolute fluorescence quantitative PCR detection on the standard plasmid group and between groups, and the coefficient of variation within the group is less than 1%, and the coefficient of variation between groups is less than 2%, indicating that the SYBR Green fluorescence quantitative PCR method for detecting clustered crab slaves of Chinese mitten crab has good repeatability.

[0022] (4) The detection data obtained by the absolute fluorescence quantitative PCR method of the present invention can accurately determine whether the cluster crab is infected and the infection content, clarify the degree of disease, and provide a technical reference for the prevention and control of cluster crab disease.

[0023] (5) The operation steps are simple and can directly detect cluster crab disease from complex samples, which is easy to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] 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.

[0025] Figure 1 The conventional PCR electrophoresis diagram of the standard plasmids with different dilution gradients in Example 4; wherein: Lane M: DL1000 Marker; Lanes 1-9: 9.1×10 8 -9.1×10 0 Dilution of standard plasmid in different gradients; N: negative control.

[0026] Figure 2 The electrophoresis diagram of specific fragments amplified by conventional PCR in Example 5, wherein: Lane M: DL2000 Marker; Lanes 1-15: 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 suffering from Japanese crab disease; 10: Exosome cDNA of Japanese crab; 11: Hepatopancreas cDNA of healthy Japanese crab; 12: Hepatopancreas cDNA of Chinese mitten crab suffering from cluster crab disease; 13: Exosome cDNA of cluster crab; 14: Hepatopancreas cDNA of healthy Chinese mitten crab; 15: Staphylococcus aureus DNA; N: negative control.

[0027] Figure 3 This is a fluorescence quantitative PCR amplification specificity experiment diagram of Example 5, wherein Figure 2 .

[0028] Figure 4 This is the amplification curve of the recombinant plasmid constructed in Example 4; 8-0 indicated in the figure are: 9.1×10 8 -9.1×10 0 copies / μL gradient of recombinant plasmid.

[0029] Figure 5 A standard curve of the recombinant plasmid was constructed for Example 3.

[0030] Figure 6 The melting curve of the recombinant plasmid constructed for Example 3. DETAILED DESCRIPTION

[0031] 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.

[0032] The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0033] Example 1

[0034] Design of a primer pair for detecting Chinese mitten crab infected with mitten crab disease

[0035] 1. RNA Extraction and Reverse Transcription to cDNA

[0036] RNA was extracted from the hepatopancreas of Chinese mitten crabs suffering from cluster crab disease in strict accordance with the instructions of the marine animal tissue genomic RNA extraction kit. The extracted total RNA was reverse transcribed according to the instructions of HiScript III RT SuperMix for qPCR (+gDNAwiper). The cDNA concentration was determined by ultramicrospectrophotometer and then stored at -20°C.

[0037] 2. Primer Design and Synthesis

[0038] According to the gene sequence, primers were designed using Primer 5.0 (as shown in Table 1), and the length of the amplified fragment was 257 bp, which was synthesized by Shanghai Biotechnology Co., Ltd.

[0039] Table 1 List of PCR primers for identification of Crabella fasciatus

[0040]

[0041] 3. Preparation of Plasmid Standards

[0042] 3.1 Conventional PCR amplification

[0043] The cDNA samples extracted from the hepatopancreas of Chinese mitten crabs infected with cluster crab disease were used as templates for amplification using RS16F / RS16R primers.

[0044] The reaction system was: 2×PCR Mix 5 μL, specific primer pair (concentration 1 nmol / L) 0.8 μL, crab hepatopancreas cDNA 1 μL, ddH2O 3.2 μL.

[0045] The reaction program was: 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 were performed for 35 cycles.

[0046] 3.2 Purification and recovery of DNA fragments

[0047] The above PCR amplification product was transferred into the well of 2% agarose gel, and the electrophoresis instrument was adjusted to 150V and run for 25min. After the electrophoresis, under ultraviolet light, the gel with the strong positive target band at 257bp was quickly cut with a clean scalpel, and the gel containing the target band was placed in a 1.5ml centrifuge tube. Subsequently, the DNA was recovered using FastPureGelDNAExtractionMiniKit. At this time, care should be taken to cut off the gel without the target DNA part as much as possible to improve the DNA recovery rate.

[0048] 3.3 Vector Recombination

[0049] The recovered DNA fragments were connected to 1 μL pMD-19-T vector, and then 1 μL of recovered DNA sample and 3 μL ddH2O were added according to the instructions of the kit (PMDTM19-T Vector Cloning Kit) to form a total volume of 5 μL. The prepared mixed reaction solution was added with an equal amount of 5 μL Solution I, and the total connection system was 10 μL, which was placed at 4°C overnight.

[0050] 3.4 Screening of strains - transformation of ligation products

[0051] 3.4.1 Add 10 μL of the ligation solution containing the pMD-19-T vector to 50 μL of DH5α competent cells, transfer to a 1.5 ml centrifuge tube, and place on ice for 30 minutes;

[0052] 3.4. Heat in a 242℃ water bath for 45 seconds, then transfer to ice and place for 1 minute;

[0053] 3.4.3 Add 890 μL LB medium to each tube, mix well, place in a shaker, and culture at 37°C for 60 min to culture the bacterial solution;

[0054] 3.4.4 Pipette 100 μL of bacterial solution and apply it to AMP-LB solid medium containing 40 μL of X-Gal and 5 μL of IPTG staining solution, seal the plate with sealing film, place the plate upright at room temperature for 1 hour, then invert it and place it in a 37°C constant temperature incubator overnight (12-16 hours);

[0055] 3.4.5 Extraction of recombinant plasmid

[0056] Pick a single white positive colony from the plate, mix it with 100 μL ddH2O, and take 1 μL as a DNA sample for sequencing identification. The purpose is to confirm whether the inserted fragment of the recombinant plasmid is consistent with the designed primer amplification fragment (257 bp). If it is consistent, it means that the construction is successful, and ensure that the extracted plasmid is not a false positive, so as to extract the recombinant plasmid with the correct sequence. The remaining bacterial liquid is placed in a centrifuge tube containing 5 ml AMP-LB liquid, placed in a shaker and cultured at 37°C and 180 rpm for about 4-5 hours until the liquid is turbid and hyphae are seen. The recombinant plasmid is extracted using FastPurePlasmidMiniKit for amplified hyphae and stored at -20°C for later use.

[0057] 4. Calculate the copy number of recombinant plasmid

[0058] The copy number of the clustered crab slave plasmid was calculated and the concentration of the constructed recombinant plasmid was measured using an ultramicrospectrophotometer, which was 287.9 ​​ng / μL.

[0059] According to the formula copies / μL = [initial concentration (ng / μL) × 10-9] × (6.02 × 1023) / [(number of bases in the vector fragment + number of bases in the target gene) × 660], the number of plasmid copies was calculated as the plasmid standard. The number of recombinant plasmid copies was 9.1 × 10 10 copies / μL.

[0060] Example 2

[0061] Absolute fluorescence quantitative PCR for detecting Chinese mitten crab infected with mitten crab disease

[0062] Using the standard plasmid as a template, upstream and downstream primers were added to the reaction system and amplified on an Applied Biosystems QuantStudio 3 real-time quantitative PCR instrument.

[0063] The reaction system is: 20μL, 2×ChamQ Universal SYBR qPCR MasterMix 10.0μL, upstream and downstream primers 0.4μL each, ddH2O 7.2μL, template 2μL; when adding samples, first mix the primers and ddH2O evenly and dispense them into the reaction tube, then add the evenly mixed template and 2×ChamQ Universal SYBR qPCR MasterMix, and centrifuge before loading on the machine;

[0064] The reaction conditions were: pre-denaturation at 95°C for 30 s, 1 cycle; denaturation at 95°C for 10 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s, 40 cycles.

[0065] Example 3

[0066] Drawing of standard curve of fluorescence quantitative PCR

[0067] The recombinant plasmid was diluted 10-fold and 9.1×10 8 -9.1×10 0 The recombinant plasmid with a gradient of copies / μL was used as the standard, and each concentration was tested 4 times to obtain a standard curve (such as Figure 5 The logarithmic value of the initial template concentration and the Ct value showed a linear relationship, Ct = -3.115logx + 36.278, the amplification efficiency was 109.414%, and the correlation coefficient R2 was 0.999 (see Figure 5 Table 2 Sensitivity test results of clustered crab slave fluorescence quantitative PCR); Melting curve of amplified product ( Figure 6 ) has a single peak, the peak positions are highly overlapped, and the melting temperature (Tm value) is 85°C, indicating that there is no nonspecific amplification and primer dimer generation during the amplification process of this method.

[0068] Table 2

[0069]

[0070] Example 4

[0071] Fluorescence quantitative PCR sensitivity test

[0072] 9.1×10 of the standard plasmid was diluted 10-fold 8 -9.1×10 0 copies / μL as template for fluorescence quantitative PCR. 0 The Ct value was 33.225, which is the lowest detection concentration (e.g. Figure 4 and Figure 5 The sensitivity of the template is 100 times higher than that of conventional PCR. 2 When the strip brightness is darker (such as Figure 1 shown).

[0073] Example 5

[0074] Fluorescence quantitative PCR specific detection

[0075] The specificity was detected by 1. Enterocytophthora (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 infected with Japanese crab disease; 10. Exosome cDNA of Japanese crab infected with Japanese crab disease; 11. Hepatopancreas cDNA of healthy Japanese crab; 12. Hepatopancreas cDNA of Chinese mitten crab infected with cluster crab disease; 13. Exosome cDNA of Chinese mitten crab infected with cluster crab disease; 14. Hepatopancreas cDNA of healthy Chinese mitten crab; 15. Staphylococcus aureus DNA.

[0076] The above genetic material was used to perform conventional PCR (RS16F / RS16R) reaction, and agarose gel electrophoresis analysis was performed using a 2% gel plate (eg Figure 2 The results showed that only the hepatopancreas cDNA of Chinese mitten crab infected with cluster crabs and the exosomes of cluster crabs showed bright and single target bands. At the same time, the fluorescence quantitative PCR (RS16F / RS16R) test was used to verify again. The samples were also the above 15 samples, and ddH2O was used as a template for negative control. The amplification curve (as shown in Figure 3 As shown in the figure, only the hepatopancreas cDNA and exosomal cDNA of Chinese mitten crab suffering from cluster crab disease showed amplification curves, and no fluorescent signals were detected in non-target genes.

[0077] Example 6

[0078] Fluorescence quantitative PCR repeatability analysis

[0079] 10-fold serial dilution of standard plasmid 9.1×10 8 -9.1×10 0 The template was 400 copies / μL, and each template was set with 4 replicate template concentrations. The mean and variance between the 4 parallels of each gradient were counted to detect the intra-group repetition; the above standard plasmid was tested by fluorescence quantitative PCR 3 times under the same conditions to detect the inter-group repetition. According to the formula, coefficient of variation = (SD÷MN) × 100% (SD: standard deviation, MN: Ct average), the intra-group coefficient of variation was less than 1%, indicating that the intra-group repetition was very good. The coefficient of variation between each group was less than 2%, indicating that the method had good repetition, thus ensuring the stability and reliability of the test results (Table 3).

[0080] Table 3

[0081]

[0082]

[0083] In summary, it can be seen that the specific primers introduced in the present invention greatly improve the sensitivity of detection, and the minimum detection limit can reach 9.1 copies / μL. The present invention uses absolute fluorescence quantitative PCR detection, and the coefficient of variation within the group is less than 1%, and the coefficient of variation between groups is less than 2%, indicating that the SYBR Green fluorescence quantitative PCR method for detecting the content of clustered crab slugs in Chinese mitten crabs has good repeatability.

[0084] 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

1. A primer pair for detecting clustered crab slaves, characterized in that: The primer pair is designed according to the target gene sequence of Crab Stomach, and the target gene sequence is shown in SEQ ID NO.

1.

2. The primer pair according to claim 1, characterized in that The primer pair sequences are RS16F and RS16R, and their sequences are shown in SEQ ID NO.2 and SEQ ID NO.3 respectively.

3. A kit, characterized in that: The kit comprises the primer pair for detecting clustered crab slaves as described in any one of claims 1-2.

4. The kit according to claim 3, characterized in that The kit also includes a positive standard and / or a negative standard for the cynomolgus Stomach plasmid.

5. The kit according to claim 4, characterized in that The positive standard is a positive plasmid of the target sequence amplified by RS16F / RS16R primers, and the negative standard is ddH2O.

6. The kit according to claim 3, characterized in that The reaction system of the kit is as follows: the volume of the primer pair RS16F and RS16R described in claim 2 in the reaction system is 0.4 μL each, and the concentration is 10 μM / μL; ddH2O is 7.2 μL; the template is 2 μL; 2×ChamQ Universal SYBR qPCR MasterMix is ​​10.0 μL.

7. The kit according to claim 3, characterized in that The reaction conditions of the reaction system are: pre-denaturation at 95°C for 30s, 1 cycle; denaturation at 95°C for 10s, annealing at 60°C for 30s, and extension at 72°C for 30s, 40 cycles.

8. Use of the primer pair according to claims 1-2 or the kit according to claims 4-7 in preparing a reagent for detecting clustered crab disease.

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