Primer probe group, kit and method for specifically detecting Gocidium parasites
By designing a primer probe set targeting the 5.8S rRNA gene of the Echinococcus parasite and combining it with a fluorescent probe, high-sensitivity and specific detection of Echinococcus parasites is achieved, solving the problem of low detection sensitivity in the existing technology. The method is suitable for monitoring Echinococcus parasite diseases in aquaculture.
Patent Information
- Application Number
- CN202510927634.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-07
- Publication Date
- 2025-10-17
AI Technical Summary
Existing methods for detecting Coccidia parasites have low sensitivity and lack specific detection targets, resulting in insufficient detection accuracy and sensitivity, making it difficult to effectively monitor Coccidia parasite diseases in aquaculture.
A specific primer-probe set was designed for the 5.8S rRNA gene of the E. coli parasite, combined with a fluorescent probe for real-time fluorescence PCR detection. The set included a forward primer, a reverse primer, and a fluorescent probe labeled with a fluorescent group and a quencher group to improve the specificity and sensitivity of the detection.
The detection sensitivity of E. coccidia parasites reached 3.0×100 copies/μL, with good specificity, repeatability and stability, and is suitable for disease monitoring in aquaculture.
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Figure CN120796535A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of aquatic animal disease detection, and in particular to a primer probe set, a kit and a method for specifically detecting Eococcus parasites. Background Art
[0002] Echinococcus spp. Goussia The parasite belongs to the phylum Protozoa, subphylum Apicomplexa, class Sporozoa, subclass Coccidia, order Eucocidia, suborder Eimeria, and family Barrococcidae. It can parasitize both invertebrates and vertebrates. Studies have reported that C. elegans can cause intestinal coccidiosis, a serious disease in common carp, ornamental carp, and goldfish populations in Europe and North America, with high mortality rates in juveniles and fry. Infected carp experience ion depletion, decreased plasma osmolality, and protein content, and are more susceptible to bacterial invasion. Histopathological findings show that tissues of fish infected with C. elegans parasites exhibit severe intestinal epithelial cell loss, disrupted intestinal architecture, and leukocyte infiltration of the lamina propria. In severely affected tissues, the intestinal epithelium is destroyed, and the lamina propria is covered by large, squamous epithelial-like cells (PMID: 9789977).
[0003] At present, some researchers have used the 18S rDNA The primers designed for the target site can achieve a minimum detection sensitivity of 4.74×10 3 copies / μL (Identification of the pathogen causing intestinal coccidiosis in yellowfin sea bream and establishment of a molecular quantitative detection method). Beyond this, most research on E. coccidia parasites still focuses on classification and evolution, and molecular detection techniques for other targets have not been reported. The specificity and sensitivity of molecular detection methods based on nucleic acid amplification mainly depend on the selection of the corresponding detection target. Currently, PCR detection is one of the most commonly used molecular detection methods, and its primers are designed based on the specific sequence of the target bacterial genomic DNA. Therefore, the detection target and primers are crucial to the accuracy and sensitivity of PCR detection. Therefore, there is an urgent need to systematically and comprehensively explore new targets for specific detection, so as to improve the accuracy and sensitivity of PCR detection technology for E. coccidia parasites. Summary of the Invention
[0004] To overcome the above-mentioned defects and deficiencies in the prior art, the present invention provides a primer probe set, a kit and a method for specifically detecting Echinococcus parasites, aiming to provide a new molecular target and method for detecting Echinococcus parasites.
[0005] A first object of the present invention is to provide a primer probe set for detecting E. coccidia parasites.
[0006] The second object of the present application is to provide the use of the primer probe set as described above in the preparation of a product for detecting parasites of the genus Archosia.
[0007] The third object of the present application is to provide a composition for detecting parasites of the genus Archosia.
[0008] The fourth object of the present application is to provide the use of the composition as described above in the preparation of a product for detecting parasites of the genus Archosia.
[0009] The fifth object of the present application is to provide a kit for detecting parasites of the genus Archosia.
[0010] The sixth object of the present application is to provide a method for detecting parasites of the genus Archosia.
[0011] In order to achieve the above objects, the present application is implemented by the following scheme: The present application is directed to the detection of parasites of the genus Archosia 5.8S rRNA The specific primer probe set is designed for the target gene, and the specific experiment, sensitivity experiment, repeatability experiment and negative stability experiment are carried out by using the primer probe set.
[0012] Therefore, the present application claims the following: A primer probe set for detecting parasites of the genus Archosia, which is composed of a forward primer F, a reverse primer R and a fluorescent probe P, the nucleotide sequence of the forward primer F is shown in SEQ ID NO: 1, the nucleotide sequence of the reverse primer R is shown in SEQ ID NO: 2, and the nucleotide sequence of the fluorescent probe P is shown in SEQ ID NO: 3.
[0013] As an implementable way, the 5' end of the fluorescent probe P is labeled with a fluorescent group FAM, and the 3' end is labeled with a quenching group MGB.
[0014] The use of the primer probe set as described above in the preparation of a product for detecting parasites of the genus Archosia.
[0015] A composition for detecting parasites of the genus Archosia, which comprises the primer probe set as described above.
[0016] The use of the composition as described above in the preparation of a product for detecting parasites of the genus Archosia.
[0017] A kit for detecting parasites of the genus Archosia, which contains the primer probe set as described above.
[0018] Preferably, the kit further comprises a positive control reference and / or a negative control reference.
[0019] More preferably, the positive control reference is plasmid DNA containing the target gene for detection of the parasite of the genus Goussia, and the negative control reference is ddH2O. The target gene is 5.8S rRNA Gene.
[0020] Preferably, the kit further comprises a fluorescent quantitative PCR detection reagent.
[0021] A method for detecting the parasite of the genus Goussia, which utilizes the above-mentioned kit to detect the sample to be tested, and the detection is for the purpose of non-disease treatment diagnosis.
[0022] Compared with the prior art, the present application has the following beneficial effects: The present application discloses a primer probe set, a kit and a method for specifically detecting the parasite of the genus Goussia. 5.8S rRNA The present application is based on the target gene of the parasite of the genus Goussia, and a primer probe set capable of specifically detecting the parasite of the genus Goussia is designed, which is composed of a primer with a nucleotide sequence as shown in SEQ ID NO: 1-2 and a fluorescent probe with a nucleotide sequence as shown in SEQ ID NO: 3. 0 copies / μL, and has good specificity, repeatability and stability. The present application provides a new molecular detection target for detecting the parasite of the genus Goussia, and provides an effective technical means for disease monitoring and detection of the parasite of the genus Goussia in aquaculture, and has a wide application prospect. BRIEF DESCRIPTION OF DRAWINGS
[0023] Figure 1 It is the amplification of the primer probe set of the parasite of the genus Goussia in Example 1.
[0024] Figure 2 It is the specific detection of the primer probe set of the parasite of the genus Goussia, and the negative control is: Aphelenchus spp., Cryptocaryon irritans, Vibrio alginolyticus, Vibrio harveyi, Vibrio parahaemolyticus, Streptococcus agalactiae, Streptococcus iniae; the blank control is: ddH2O.
[0025] Figure 3 It is the sensitivity detection result of the primer probe set of the parasite of the genus Goussia.
[0026] Figure 4 It is the negative stability detection result of the primer probe set of the parasite of the genus Goussia.
[0027] Figure 5 It is the detection result of the parasite of the genus Goussia in 20 actual samples. DETAILED DESCRIPTION
[0028] The application will be further described in conjunction with specific examples, but the examples do not limit the application in any form. Unless otherwise specified, the reagents, methods and equipment used in the application are conventional reagents, methods and equipment in the technical field.
[0029] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.
[0030] Example 1 Influence of primer probe set on detection of parasites of genus Goussia I. Experimental methods 1. Plasmid DNA synthesis The gene sequence designed for the primers of the parasites of genus Goussia was linked to a pUC57 vector to construct a plasmid.
[0031] 2. Design of primers The whole genome sequence of the parasites of genus Goussia was searched from Genbank, and was compared and analyzed by BLAST to select a target gene. 5.8S rRNA The gene was a target gene, and specific primers and probes were designed according to the principle of real-time fluorescent PCR primer probe design and by using PrimerExpress primer design software.
[0032] Primer probe set 1 (Y1) for the parasites of genus Goussia: Forward primer GS-M1-F: 5'-GATTACGTCCCTGCCCTTTGT-3' (SEQ ID NO: 1); Reverse primer GS-M1-R: 5'-ATTCACCGGATCACTCAATCG-3' (SEQ ID NO: 2); Fluorescent probe GS-M1-P: 5'-CACACCGCCCGTCG-3' (SEQ ID NO: 3); The 5' end of the fluorescent probe GS-M1-P was labeled with a fluorescent group FAM, and the 3' end was labeled with a quenching group MGB.
[0033] Primer probe set 2 (Y2) for the parasites of genus Goussia: Forward primer GS-M2-F: 5'-CGATTGAGTGATCCGGTGAA-3' (SEQ ID NO: 4); Reverse primer GS-M2-R: 5'-TTTCCGATCCAGAGCCCTTAC-3' (SEQ ID NO: 5); Fluorescent probe GS-M2-P: 5'-TATTAGGACTGATCTTGTTGCT-3' (SEQ ID NO: 6); The 5' end of the fluorescent probe GS-M2-P is labeled with the fluorescent group FAM, and the 3' end is labeled with the quencher group MGB.
[0034] 3. Perform PCR reaction The reaction system is shown in Table 1.
[0035] Table 1 Real-time fluorescence PCR reaction system
[0036] The reaction program was as follows: decontamination program at 50°C for 5 min, 1 cycle; hoiding stage program at 95°C for 5 min, 1 cycle; cycling stage program at 95°C for 15 s, 60°C for 30 s, 40 cycles, collecting fluorescence signals at 60°C for 30 s, and observing the reaction results using a fluorescence PCR instrument.
[0037] 2. Experimental Results The results are as follows Figure 1 As shown, the Ct value of primer probe set 1 is small and the repeatability is good. There is no nonspecific amplification in the negative result, while primer probe set 2 does not amplify. Therefore, primer probe set 1 is selected as the primer probe set for detecting Eococcus parasites in aquatic animals.
[0038] Example 2 A kit for detecting Eococcus parasites in aquatic animals 1. Composition 1. Primer and probe sets for detecting E. coccidia parasites Forward primer GS-M1-F: 5′-GATTACGTCCCTGCCCTTTGT-3′ (SEQ ID NO: 1); Reverse primer GS-M1-R: 5′-ATTCACCGGATCACTCAATCG-3′ (SEQ ID NO: 2); Fluorescent probe GS-M1-P: 5′-CACACCGCCCGTCG-3′ (SEQ ID NO: 3); The 5' end of the fluorescent probe GS-M1-P is labeled with the fluorescent group FAM, and the 3' end is labeled with the quencher group MGB.
[0039] 2. PCR Reagents PCR reaction premix, positive control reference, negative control reference; The positive control reference material contains E. coccidia parasites 5.8S rRNA The negative control reference was ddH2O.
[0040] 2. Usage The DNA of the sample to be tested is subjected to PCR reaction using the protozoan primer probe set of the genus Gousiekia, and the reaction system is shown in Table 2.
[0041] Table 2 Real-time fluorescent PCR reaction system
[0042] The reaction program is as follows: the decontamination program is 50℃ for 5min, 1 cycle; the Hoiding Stage program is 95℃ for 5min, 1 cycle; the Cycling Stage is 95℃ for 15s, 60℃ for 30s, 40 cycles, and the fluorescence signal is collected at 60℃ for 30s, and the reaction result is observed by using a fluorescent PCR instrument.
[0043] III. Interpretation method If the Ct value of the test sample is ≥40 or no Ct value, the curve is a straight line or a slight oblique line, and there is no “S” type amplification curve, it is determined that the sample does not contain the protozoan of the genus Gousiekia or the content is lower than the detection limit; If the Ct value of the test sample is <40, the curve is an “S” type amplification curve, it is determined that the sample contains the protozoan of the genus Gousiekia.
[0044] Example 3 Specificity experiment 1. Experimental method According to the commercially available DNA extraction kit, the DNA of each sample is extracted, and the purity and concentration of the extracted DNA are determined by using a full-wavelength microspectrophotometer, and the DNA is stored at -20℃ for standby.
[0045] The samples include: Brachionus plicatilis, Cryptocaryon irritans, Vibrio alginolyticus, Vibrio harveyi, Vibrio parahaemolyticus, Streptococcus agalactiae, and Streptococcus iniae.
[0046] The kit of Example 2 is used for PCR amplification.
[0047] 2. Experimental results The specificity experiment results of the primer probe set of the protozoan of the genus Gousiekia show that the sample DNA of Brachionus plicatilis, Cryptocaryon irritans, Vibrio alginolyticus, Vibrio harveyi, Vibrio parahaemolyticus, Streptococcus agalactiae and Streptococcus iniae does not detect the fluorescence signal of amplification Figure 2 , indicating that the established real-time fluorescent PCR detection method has good specificity.
[0048] Example 4 Sensitivity and repeatability experiment 1. Experimental method The synthesized plasmid DNA is quantified on a spectrophotometer, and the plasmid concentration is 100 ng / μL. According to the conversion of DNA concentration into copy number, the DNA copy number is 3.0×10 10The plasmid was serially diluted to 3.0×10 6 copies / μL, 3.0×10 5 copies / μL, 3.0×10 4 copies / μL, 3.0×10 3 copies / μL, 3.0×10 2 copies / μL, 3.0×10 1 copies / μL, 3.0×10 0 copies / μL, 3.0×10 -1 DNA at seven concentrations of copies / μL was used as a template and detected using the kit of Example 2.
[0049] 2. Experimental Results The results showed a typical amplification curve, and the method was effective for the detection of parasites of the genus E. coli. 5.8S rRNA The detection sensitivity of the gene plasmid reached 3.0×10 0 copies / μL( Figure 3 The results of three parallel experiments for each of the seven dilution gradients showed that the coefficient of variation was between 0.15% and 1.36%, indicating that the established real-time fluorescence PCR detection method has good reproducibility (Table 3).
[0050] Table 3 Repeatability of primer-probe sets for E. coli
[0051] Example 5 Negative stability test 1. Experimental Methods A negative stability experiment was performed using ddH2O as a negative control, repeated 20 times, and three positive controls were added for reference.
[0052] 2. Experimental Results The results showed that the established real-time fluorescence PCR detection method had good negative stability and no nonspecific amplification occurred ( Figure 4 ).
[0053] Example 6 Detection of Eococcus parasites in actual samples 1. Experimental Methods DNA was extracted from 20 actual samples and the kit of Example 2 was used to detect whether they were infected with E. coccidia parasites.
[0054] 2. Experimental Results The kit of Example 2 was used to detect the DNA of 20 actual samples extracted. The results are as follows: Figure 5DNA from 3 of the actual samples was detected positive for the presence of Sarcocystis parasites and DNA from 17 of the actual samples was detected negative for the presence of Sarcocystis parasites.
[0055] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications, etc. made without departing from the spirit and principles of the present application should be equivalent replacement manners and should be included in the protection scope of the present application.
Claims
1. A primer probe set for detecting E. coccidia parasites, characterized in that: The primer probe set consists of a forward primer F, a reverse primer R, and a fluorescent probe P. The nucleotide sequence of the forward primer F is shown in SEQ ID NO: 1, the nucleotide sequence of the reverse primer R is shown in SEQ ID NO: 2, and the nucleotide sequence of the fluorescent probe P is shown in SEQ ID NO:
3.
2. The primer probe set according to claim 1, characterized in that The 5' end of the fluorescent probe P is labeled with a fluorescent group FAM, and the 3' end is labeled with a quenching group MGB.
3. Use of the primer probe set according to claim 1 or 2 in the preparation of a product for detecting Eococcus parasites.
4. A composition for detecting E. coccidia parasites, characterized in that The composition comprises the primer probe set according to claim 1 or 2.
5. Use of the composition according to claim 4 in the preparation of a product for detecting Eococcus parasites.
6. A kit for detecting Echinococcus parasites, characterized in that: The kit contains the primer-probe set according to claim 1 or 2.
7. The kit according to claim 6, characterized in that The kit further comprises a positive control reference substance and / or a negative control reference substance.
8. The kit according to claim 7, characterized in that The positive control reference substance is a plasmid DNA containing a target gene for detecting E. coccidia parasites, and the negative control reference substance is ddH2O.
9. The kit according to claim 6, characterized in that The kit also contains a fluorescent quantitative PCR detection reagent.
10. A method for detecting E. coccidia parasites, characterized in that: The test sample is tested using the kit according to any one of claims 6 to 9, wherein the test is for a purpose other than disease treatment and diagnosis.