Primer probe group and kit for detecting sheep abortion chlamydophila
By designing primer probe sets and kits for Chlamydia abortion, the problem of insufficient sensitivity and stability of existing detection methods is solved, and rapid, accurate and high-sensitivity detection effects are achieved.
Patent Information
- Application Number
- CN202411978713.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-31
- Publication Date
- 2025-03-21
AI Technical Summary
Existing methods for detecting Chlamydia of goat abortion, such as IHA and CFT, have problems with low sensitivity and stability, and cannot distinguish between natural infection and vaccine-induced immune response.
A primer probe set for detection of Chlamydia abortion is designed, including upstream primers, downstream primers and Taqman probes. It can quickly and accurately detect Chlamydia abortion through PCR amplification technology, and provide corresponding kits and detection methods.
Fast, efficient and accurate detection of Chlamydia abortion is achieved, and Chlamydia abortion can be identified from samples as low as 1copies/response, with high sensitivity and specificity and easy operation.
Smart Images
Figure CN119662870A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biological detection technology, in particular to a primer probe set and a kit for detecting Chlamydophila abortus in sheep. Background Art
[0002] Chlamydia is a type of microorganism that is Gram-negative and parasitic in eukaryotic cells. The Chlamydiaceae family can be divided into the Chlamydia genera and the Chlamydophila genera. Chlamydophila abortus is a strain of Chlamydophila abortus that causes goat abortion. It has been called Chlamydophila psittaci serotype I before and is the pathogen of ovine enzootic abortion (OEA). C. abortus is an obligate parasitic pathogen that cannot grow on general culture media and can only survive in chicken embryos or HeLa cells. It needs fresh diseased materials to be isolated and cultured. In addition, C. abortus can cause abortion, stillbirth, weak fetus, etc. in maternal livestock through the placenta. It has local outbreaks in ruminants such as cattle and sheep, and can also cause abortion in pregnant women. Therefore, the detection of Chlamydophila abortus has important public health significance.
[0003] For Chlamydophila abortus in sheep, the commonly used detection methods are: PCR (polymerase chain reaction), qPCR-Taqman probe method, indirect hemagglutination test (IHA) and complement fixation test (CFT), among which the detection of this pathogen mainly relies on IHA test and CFT test. Although these two methods have played a certain role in the diagnosis of Chlamydophila abortus in sheep, their sensitivity and stability need to be further improved. In addition, this method can only detect antibodies to chlamydial disease. If the flock is vaccinated with chlamydia vaccine, it is impossible to judge naturally infected and immune sheep, which has certain limitations.
[0004] In view of this, the present invention is proposed. Summary of the invention
[0005] The purpose of the present invention is to provide a primer probe set and a kit for detecting Chlamydia abortus in sheep. The primer probe set or the kit of the present invention can detect Chlamydia abortus in sheep quickly, efficiently and accurately.
[0006] To achieve the above purpose, the technical solution adopted by the present invention to solve the technical problem is:
[0007] In a first aspect, the present invention provides a primer probe set for detecting Chlamydophila abortus in sheep, comprising an upstream primer, a downstream primer and a Taqman probe;
[0008] The nucleotide sequence of the upstream primer is shown in SEQ ID NO: 1, the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 2, and the nucleotide sequence of the Taqman probe is shown in SEQ ID NO: 3.
[0009] In a second aspect, the present invention provides the use of the above primer probe set in the preparation of a kit for detecting Chlamydia abortus in sheep.
[0010] In a third aspect, the present invention provides a kit for detecting Chlamydophila abortus in sheep, comprising the above-mentioned primer probe set.
[0011] In a fourth aspect, the present invention also provides a method for detecting Chlamydia abortus in sheep, which comprises: using the DNA of the sample to be tested as an amplification template, adopting the above-mentioned primer probe group, establishing a PCR reaction system, and performing PCR amplification; after the reaction is completed, analyzing the test result according to the amplification curve graph and Ct value to obtain the test result.
[0012] The present invention has the following beneficial effects:
[0013] (1) The primer probe set of the present invention can specifically generate corresponding fluorescent amplification signals for the genome of Chlamydia abortus, has strong specificity, and can quickly and accurately detect whether Chlamydia abortus is present in the sample to be tested, and can distinguish it from other common chlamydia such as Chlamydia psittaci, Chlamydia guinea pigs, Chlamydia felis and Chlamydia pneumoniae;
[0014] (2) The detection kit and detection method of the present invention can identify whether there is Chlamydia abortus in the sample to be tested with as low as 1 copy / reaction, and has the characteristic of high sensitivity;
[0015] (3) The detection of Chlamydia abortus by the primer probe set of the present invention is also time-saving and easy to operate. It does not require identification by agarose gel electrophoresis like conventional PCR, but only requires automatic determination of the result based on a threshold value, which is more convenient. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.
[0017] Figure 1 This is a schematic diagram of the primer and probe screening qPCR in Example 1;
[0018] Figure 2 This is the test result of the real sample in Example 2;
[0019] Figure 3 The test results of the specific test in Example 3 are as follows;
[0020] Figure 4 The results of the sensitivity test of Example 4 are shown below:
[0021] Figure 5 The following are the test results of the stability test of Example 5. DETAILED DESCRIPTION
[0022] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.
[0023] Since the current main methods for detecting Chlamydia abortus in sheep - IHA and CFT - have the disadvantages of low sensitivity and stability in the detection of Chlamydia abortus in sheep, in order to further improve the sensitivity and stability of Chlamydia abortus in sheep, the present invention constructs a primer probe set for detecting Chlamydia abortus in sheep based on the qPCR-Taqman probe method. The primer probe set is designed based on the pmpD-specific target region of Chlamydia abortus in sheep, and includes pmpD-F shown in SEQ ID NO.1, pmpD-R shown in SEQ ID NO.2, and pmpD-P shown in SEQ ID NO.3.
[0024] The 5' end of the Taqman probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group. The fluorescent reporter group is selected from any one of FAM, CY5, HEX and CY3, and the fluorescent quencher group is selected from any one of BHQ1, BHQ2, BHQ3 and MGB.
[0025] In some embodiments, the Taqman probe is labeled with FAM at the 5' end and with BHQ1 or BHQ2 at the 3' end.
[0026] Based on the above primer probe set, the present invention can provide a detection kit for ovine Chlamydophila abortus, which contains the above primer probe set and other conventional reagents for PCR amplification, including PCR amplification reagents, positive controls and negative controls.
[0027] Among them, the PCR amplification reagents include Taq hot start enzyme, qPCR reaction buffer, dNTPs and MgCl2, and other commonly used auxiliary reagents. Auxiliary reagents include PCR enhancers and stabilizers. The specific types of PCR enhancers and stabilizers can be adjusted according to actual needs.
[0028] The positive control was a recombinant plasmid containing a nucleotide fragment of the target gene of Chlamydia abortus; the negative control was nuclease-free water.
[0029] In addition to the PCR amplification reagent provided by the present invention, commercial products of the same type can also be used. PCR amplification can be performed by simply adding a specified amount of DNA template and primer probe before amplification. The premise is that the positive control sample must have an amplification curve, and the negative control sample must not have an amplification curve.
[0030] By performing amplification detection on the DNA of the test sample containing or not containing Chlamydia abortus, it was found that the test sample can have an amplification curve only when Chlamydia abortus exists.
[0031] Since the detection primer probe set or the kit can be used to identify whether a sample to be tested contains Chlamydia abortus, the present invention also provides a method for detecting whether a sample to be tested contains Chlamydia abortus.
[0032] The method for detecting whether a sample to be tested contains Chlamydia abortus includes the following steps:
[0033] (1) Extracting genomic DNA from the sample to be tested;
[0034] (2) using the DNA obtained in step (1) as a template and the above-mentioned primer probe set to perform a PCR amplification reaction;
[0035] (3) Analyze the test results according to the amplification curve and Ct value to obtain the test results.
[0036] In the above detection method, the concentrations of the upstream primer and the downstream primer in the PCR amplification reaction system are both 0.1 to 0.8 μM, and the concentration of the Taqman probe is 0.08 to 0.8 μM.
[0037] In some embodiments, the reaction system is: 10 μL of PCR amplification reagent, 0.4 μL of each of 10 μmol / L pmpD-F / pmpD-R / pmpD-P, 2 μL of DNA template, and 20 μL of ddH2O.
[0038] The reaction program of PCR amplification was as follows: pre-denaturation at 94°C for 30 s, 94°C for 5 s, and 60°C for 34 s, for a total of 40 cycles.
[0039] Test result analysis:
[0040] (1) Validity determination: The positive control should show a specific amplification curve and the Ct value should be less than 30, and the negative control should have a NoCt value; otherwise the test results are invalid;
[0041] (2) During sample testing, if the amplification curve is S-shaped and the CT value is ≤38, the sample is positive; if the amplification curve is S-shaped and 38≤CT value≤40, the sample is suspicious and needs to be retested; if the retest result is still 38≤CT value≤40 and the amplification curve is S-shaped, and both negative and positive controls are established, it can be judged as positive, otherwise it is negative; if there is no CT value and it is not detected, it is a negative sample.
[0042] The detection method provided by the present invention can be used to detect Chlamydia abortus in samples, and its minimum detection limit can reach 1 copies / reaction. It can also accurately identify Chlamydia abortus from a variety of Chlamydia with high similarity, and has the characteristics of high sensitivity and strong specificity.
[0043] In addition, for the detection of Chlamydophila abortus in sheep, those skilled in the art can also design other fluorescent probe detection methods based on the target gene in the present invention, such as Molecular Beacons or HyBeacon probes; they can also design other detection methods based on the target gene or primer in the present invention, such as the SYBR Green dye method.
[0044] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.
[0045] Example 1
[0046] This example is the design and screening of primer probes, as follows:
[0047] (1) Primer probe design and plasmid synthesis: The pmpD gene of C. abortus was downloaded, and the sequences of C. abortus and other Chlamydia (such as Chlamydia psittaci, Chlamydia ruminants, Chlamydia guinea pigs, Chlamydia suis, Chlamydia abortus, Chlamydia felis, and Chlamydia pneumoniae) were compared using BLAST and Clustal X software. Primers and probes were designed in the highly specific region of the pmpD gene using the primer design software Primer5. A total of 10 sets of primers and probes were designed for screening. The results were compared based on the fluorescence intensity and CT value ( Figure 1 ) The best combination was selected for subsequent testing, and the best primer-probe combination sequence is shown in Table 1. The fragment where the pmpD target gene is located was synthesized plasmid by Sangon Biotechnology.
[0048] Table 1 Primer probe sequences
[0049]
[0050] (2) Preparation of plasmid standard: centrifuge the plasmid powder at 8000 r / min for 60 s, add 200 μL DNA diluent, and vortex to mix. The initial concentration of the plasmid was determined to be 20 ng / μL. The plasmid stock solution was diluted in a series of gradients, and the diluted plasmid was quantified by digital PCR. The quantitative results are shown in Table 2.
[0051] Table 2 Quantitative results of plasmids
[0052] serial number Dilution multiple Actual copy number (copies / μL) 1 <![CDATA[10 4 ]]> <![CDATA[4.82×10 5 ]]> 2 <![CDATA[10 5 ]]> <![CDATA[4.45×10 4 ]]> 3 <![CDATA[10 6 ]]> <![CDATA[4.64×10 3 ]]> 4 <![CDATA[10 7 ]]> <![CDATA[4.23×10 2 ]]> 5 <![CDATA[10 8 ]]> 40.63
[0053] (3) Reaction system preparation: 2×qPCR SuperMix for qPCR (Sangon Biotechnology, B630015) 10 μL, 10 μmol / L pmpDF / pmpDR / pmpDP 0.4 μL each, positive template was diluted 10 6 Add 2 μL of plasmid DNA, 2 μL of nuclease-free water for negative control, and 6.8 μL of sterile water to make the PCR system 20 μL.
[0054] (4) Reaction procedure: pre-denaturation at 94°C for 30 s, 94°C for 5 s, and 60°C for 34 s, for a total of 40 cycles, and the fluorescence value of the FAM channel was collected.
[0055] The results are as follows Figure 1 As shown, the pair of primer-probe combinations pointed by the red arrow is selected to prepare a kit. The primers and probes in the kit are shown in Table 1. The kit also includes 2×qPCR SuperMix for qPCR, a positive control and a negative control. The positive control is a recombinant plasmid containing a nucleotide fragment of the target gene of Chlamydia abortus; the negative control is nuclease-free water.
[0056] Example 2
[0057] This example is a test kit obtained in Example 1 to verify the detection effect
[0058] (1) Nucleic acid extraction: Nucleic acid was extracted from the samples according to the operating procedures of the Ezup column-based free DNA extraction kit (Sangon Biotechnology, B518270).
[0059] (2) The fluorescent PCR reaction system of the kit is 2×qPCR SuperMix for qPCR 10 μL, 10 μmol / L pmpD-F / pmpD-R / pmpD-P 0.4 μL each, the volume of the template is 2 μL, and 6.8 μL of sterile water is added to make the PCR system 20 μL. The positive control is 1000 copies / μL plasmid DNA in Example 1, and the negative control is nuclease-free water.
[0060] (3) The PCR reaction conditions of the kit were as follows: pre-denaturation at 94°C for 30 s, 94°C for 5 s, and 60°C for 34 s, for a total of 40 cycles, and the fluorescence value of the FAM channel was collected.
[0061] (4) Interpretation of results:
[0062] 1) For validity determination, the positive control should show a specific amplification curve and the Ct value should be less than 30, and the negative control should have no Ct value; otherwise the test results are invalid.
[0063] 2) During sample testing, if the amplification curve is S-shaped and the CT value is ≤38, the sample is positive; if the amplification curve is S-shaped and 38≤CT value≤40, it is a suspicious sample and needs to be retested; if the retest result is still 38≤CT value≤40, and the amplification curve is S-shaped, and both negative and positive controls are established, it can be judged as positive, otherwise it is negative; if there is no CT value and it is not detected, it is a negative sample.
[0064] The results are as follows Figure 2 As shown in the figure, the fluorescence curve of the reaction tube containing the genome of Chlamydia abortus increased (green line), the reaction tube of the positive control showed a specific amplification curve with a Ct value of <30 (red line), and the reaction tube of the negative control showed a smooth straight line (blue line).
[0065] Example 3
[0066] This example is a specific verification of the ovine abortion Chlamydophila fluorescence qPCR detection kit.
[0067] Nucleic acid samples of Chlamydia psittaci, Chlamydia guinea pigs, Chlamydia felis and Chlamydia pneumoniae were extracted as templates, Chlamydia abortus plasmid DNA was used as a positive control, and nuclease-free water was used as a negative control, and amplification was performed using the kit of Example 1 of the present invention.
[0068] The results are as follows Figure 3 As shown, only the positive control containing Chlamydia abortus can be well amplified (green line), and the others did not produce an amplification curve, indicating that the kit has no cross-reaction with Chlamydia psittaci, Chlamydia guinea pigs, Chlamydia felis and Chlamydia pneumoniae, and the kit has a certain degree of conservatism and strong specificity for Chlamydia abortus.
[0069] Example 4
[0070] This example is a sensitivity verification of the ovine abortion Chlamydia fluorescence qPCR detection kit.
[0071] The plasmid reference in Example 1 was diluted with DNA diluent, and its concentration was quantified using a digital PCR instrument (Zhenzhun Biotech). The plasmid was diluted to a quantitative concentration of 100 copies / μL, 50 copies / μL, 10 copies / μL, 5 copies / μL and 1 copy / μL for sensitivity testing.
[0072] Test results such as Figure 4 The sensitivity of detecting Chlamydophila abortus in sheep can reach 1 copy / reaction.
[0073] Example 5
[0074] This example is a stability verification of the ovine abortion Chlamydia fluorescence qPCR detection kit.
[0075] 2 μL of 2 mg / mL hemoglobin and 35 μmol / L total bilirubin were added to the reaction system respectively, and an amplification system without adding interference was used as a control, and amplification was performed using the kit of Example 1 of the present invention.
[0076] Test results such as Figure 5 Compared with the amplification results of the amplification system without adding interference substances: 2mg / mL hemoglobin has no effect on the test results, and 35μmol / L total bilirubin has no effect on the test results. This shows that the kit has good stability.
[0077] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
Claims
1. A primer probe set for detecting Chlamydia abortus in sheep, characterized in that: The primer probe set includes an upstream primer, a downstream primer and a Taqman probe; The nucleotide sequence of the upstream primer is shown in SEQ ID NO: 1, the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 2, and the nucleotide sequence of the Taqman probe is shown in SEQ ID NO:
3.
2. The primer probe set according to claim 1, characterized in that: The 5' end of the Taqman probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group.
3. The primer probe set according to claim 2, characterized in that: The fluorescent reporter group is selected from any one of FAM, CY5, HEX and CY3, and the fluorescent quencher group is selected from any one of BHQ1, BHQ2, BHQ3 and MGB.
4. Use of the primer probe set according to any one of claims 1 to 3 in preparing a kit for detecting Chlamydia abortus in sheep.
5. A kit for detecting Chlamydia abortus in sheep, characterized in that: The kit comprises the primer probe set according to any one of claims 1 to 3.
6. The kit according to claim 5, characterized in that The kit also includes qPCR reaction amplification reagents, positive controls and negative controls.
7. The kit according to claim 6, characterized in that The qPCR reaction amplification reagent comprises Taq hot start enzyme, qPCR reaction buffer and dNTPs; the positive control is a recombinant plasmid comprising a nucleotide fragment of a target gene of Chlamydia abortus; and the negative control is nuclease-free water.
8. A method for detecting Chlamydophila abortus in sheep, characterized in that: include: Using the DNA of the sample to be tested as an amplification template, using the primer probe set described in any one of claims 1 to 3, establishing a PCR reaction system, and performing PCR amplification; After the reaction is completed, the test results are analyzed according to the amplification curve and Ct value to obtain the test results.
9. The method according to claim 8, characterized in that In the reaction system, the concentrations of the upstream primer and the downstream primer are both 0.1-0.8 μM, and the concentration of the Taqman probe is 0.08-0.8 μM.
10. The method according to claim 8, characterized in that The reaction program of PCR amplification was as follows: pre-denaturation at 94°C for 30 s, 94°C for 5 s, and 60°C for 34 s, for a total of 40 cycles.