Fluorescent PCR (Polymerase Chain Reaction) primer, probe and detection kit for detecting mycoplasma putrefaciens
By designing fluorescent PCR primers and probe compositions and detection kits for detecting Mycoplasma fertilized, the problem of long-term detection and low sensitivity in the prior art is solved, and rapid and high-sensitivity detection of Mycoplasma fertilized is achieved, supporting clinical diagnosis and vaccine manufacturing.
Patent Information
- Application Number
- CN202411775711.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-05
- Publication Date
- 2025-04-01
AI Technical Summary
The methods used in the prior art for detecting Mycoplasma corrupt have problems such as long time and low sensitivity, and lack of high sensitivity detection methods.
A set of fluorescent PCR primer and probe compositions for detecting Mycoplasma spoilage are designed, and the corresponding detection kits are used to achieve rapid and high-sensitivity detection of Mycoplasma spoilage based on the reference genome reported by the NCBI database.
It has achieved rapid and high-sensitivity detection of Mycoplasma corrupt, which can provide accurate detection support in clinical diagnosis and vaccine manufacturing, and effectively prevent and control the occurrence of epidemics.
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Figure CN119736418A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of animal disease veterinary detection in the field of biological detection technology, and specifically relates to a fluorescent PCR primer, a probe and a detection kit for detecting Mycoplasma putrefaciens. Background Art
[0002] Contagious agalactia syndrome (CA) is a mycoplasma disease of small ruminants with significant consequences. The syndrome includes mastitis and agalactia, which may be accompanied by conjunctivitis and polyarthritis. Mycoplasma putrefaciens (Mput) is one of the pathogens of contagious agalactia syndrome, but so far, it has received little attention and its virulence properties are not fully understood. The species was confirmed as a new species in 1974, but so far, the identification of Mputa still relies on classical isolation and identification techniques, which makes clinical detection very limited. For example, both in vitro culture and serological methods can be used to detect Mputa. In vitro culture is a classic detection method, but it is time-consuming, while serological methods have low sensitivity. At present, there is no method with higher sensitivity for detecting Mputa, so it is necessary to develop a real-time fluorescent PCR primer and TaqMan probe for Mputa. Summary of the invention
[0003] In view of the problems existing in the prior art, the present invention designs a set of fluorescent PCR primers and probe compositions and a detection kit for detecting Mycoplasma putrefaciens based on the Mycoplasma putrefaciens reference genome reported in the NCBI database, so as to realize rapid and high-sensitivity detection of Mycoplasma putrefaciens.
[0004] In the first aspect, the present invention proposes a fluorescent PCR primer and probe combination for detecting Mycoplasma putrefaciens, which includes an upstream primer with a nucleotide sequence as shown in SED ID NO: 4, a downstream primer with a nucleotide sequence as shown in SED ID NO: 6, and a probe with a nucleotide sequence as shown in SED ID NO: 5.
[0005] The 5' end of the probe is labeled with a fluorescent reporter group, the 3' end is labeled with a fluorescent quencher group, and the 3' end of the probe is phosphorylated; optionally, the fluorescent reporter group is selected from one of FAM, HEX, JOE, TET, CY3, CY5, ROX, and Texas, and the fluorescent quencher group is selected from one of TAMRA, BHQ1, BHQ2, BHQ3, MGB, and DABCYL.
[0006] The primer and probe combination can have good specificity for Mycoplasma putrefaciens, and has high detection sensitivity, convenience and rapidity.
[0007] In the second aspect, the present invention further proposes the use of the primer and probe combination in the preparation of a kit for detecting Mycoplasma putrefaciens.
[0008] Furthermore, the present invention provides a kit containing the primer and probe combination.
[0009] Furthermore, the 25 μL reaction system for the fluorescent PCR detection comprises:
[0010] HieffUnicon Universal TaqMan multiplex qPCRmaster mix 10-15μL;
[0011]
[0012] The balance was DEPC-treated water.
[0013] In a third aspect, the present invention further proposes a method for detecting Mycoplasma putrefaciens using a kit, comprising: extracting the target genomic DNA of Mycoplasma putrefaciens, using the extracted genomic DNA as a template, and performing fluorescent PCR detection under the guidance of the primer and probe combination; using the obtained Ct value and the specific amplification curve that appears to perform qualitative detection of Mycoplasma putrefaciens, and / or calculating the copy number of Mycoplasma putrefaciens contained in the sample to be tested according to the standard curve and the obtained Ct value, so as to achieve quantitative detection.
[0014] Preferably, the reaction conditions for fluorescent PCR detection are: pre-denaturation at 95°C for 5 minutes; PCR amplification at 95°C for 15 seconds, extension at 60°C for 30 seconds, and 45 cycles.
[0015] Furthermore, the template is a standard of a recombinant plasmid containing the arcB gene of Mycoplasma putrefaciens, and the concentration of the standard is selected from 1×10 10 , 1×10 9 , 1×10 8 , 1×10 7 , and 1×10 6 copies / μL.
[0016] Furthermore, the standard was diluted 10-fold to a concentration of not less than 1×10 1 copies / μL, and obtain standard samples. Using the standard samples as templates, after fluorescence PCR detection, the concentration Log values of each standard sample are plotted against their corresponding Ct values to obtain a standard curve.
[0017] Furthermore, if a specific amplification curve appears in the sample to be tested and the Ct value is ≤38, the result is judged to be positive for Mycoplasma putrefaciens nucleic acid; if a specific amplification curve appears in the sample to be tested and 38<Ct value≤40, the result is judged to be suspicious for Mycoplasma putrefaciens nucleic acid and re-testing is required; after re-testing, if the Ct value is ≤40 and a specific amplification curve appears, the result is judged to be positive, otherwise it is negative; if the sample to be tested has no Ct value or no specific amplification curve, the result is judged to be negative for Mycoplasma putrefaciens nucleic acid.
[0018] The fluorescent PCR primers, probes and detection kit for detecting Mycoplasma putrefaciens provided based on the above technical solution can implement rapid and highly sensitive detection of Mycoplasma putrefaciens, and can provide a strong basis for the investigation of the main epidemic situation of pathogens, the purification of epidemics, the screening of strains and quality monitoring, and effectively prevent and control the occurrence of epidemics. The kit and detection method of the present invention are simple to operate, have strong specificity, high sensitivity and good repeatability, can realize the qualitative detection of Mycoplasma putrefaciens, and will play an important role in the early technical support such as accurate detection of Mycoplasma putrefaciens in diseased samples or cultures in clinical diagnosis and vaccine manufacturing (application for non-diagnostic purposes), and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 The results of the fluorescent PCR primer and probe screening for Mycoplasma putrefaciens; from left to right are groups 1, 2, and 3 of primer and probe.
[0020] Figure 2 The results of constructing the fluorescence PCR standard curve of Mycoplasma putrefaciens; Figure A shows the detection standard 1×10 7 -1×10 1 Amplification curve of 1×10 copies / μL. Figure B is the detection standard 1×10 7 -1×10 1 Standard curve of copies / μL.
[0021] Figure 3 This is the amplification curve of the lowest detection line of Mycoplasma putrefaciens fluorescent PCR.
[0022] Figure 4 This is the amplification curve of the fluorescent PCR specific detection of Mycoplasma putrefaciens.
[0023] Figure 5 This is the amplification curve of the repeatability detection of Mycoplasma putrefaciens by fluorescent PCR. DETAILED DESCRIPTION
[0024] The content of the present invention is described in detail below with reference to specific embodiments and drawings.
[0025] The methods used in the following examples are all conventional methods unless otherwise specified. For specific steps, please refer to: "Molecular Cloning Laboratory Manual" (Sambrook, J., Russell, David W., MolecμLar Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor).
[0026] The methods for obtaining various biological materials described in the examples are only provided as an experimental method to achieve the specific disclosed purpose, and should not be construed as limiting the sources of the biological materials of the present invention. In fact, the sources of the biological materials used are extensive, and any biological material that can be obtained without violating laws and moral ethics can be replaced and used according to the instructions in the examples.
[0027] All primers and probes used were synthesized using existing techniques.
[0028] The embodiments are implemented on the premise of the technical solution of the present invention, and provide detailed implementation methods and specific operation processes. The embodiments will help to understand the present invention, but should not be used as a limitation to the contents of the present invention.
[0029] Example 1. Design of primers and probes for fluorescent PCR detection of Mycoplasma putrefaciens
[0030] 1.1. Strains
[0031] The Mycoplasma putrefaciens used in the examples was isolated from clinical milk samples from an infected sheep farm and was transported to Jinyu Baoling Biological Pharmaceutical Co., Ltd. for storage after approval of the highly pathogenic microorganism transportation procedures.
[0032] 1.2. Extraction of genomic DNA
[0033] Using Axyprep TM The Body FluidViral DNA / DNAMiniprep Kit (AXYGEN) was used to extract nucleic acid from the inactivated samples provided by Jinyu Baoling Biological Pharmaceutical Co., Ltd. The extracted nucleic acid was stored at -20°C for future use.
[0034] 1.3 Design and screening of primers and probes
[0035] Starting from the reference genome of Mycoplasma putrefaciens retrieved from the NCBI nucleic acid database GenBank (http: / / www.ncbi.nlm.nih.gov) (Table 1), the arcB full gene nucleotide reference sequence was obtained, its specific conserved region was determined, and according to the primer and TaqMan probe design principles, a total of multiple sets of fluorescent PCR primers and TaqMan probes for detecting Mycoplasma putrefaciens were designed. The following Table 2 lists three sets of primers and probes for detecting Mycoplasma putrefaciens (as shown in Group 1, Group 2, and Group 3 in Table 2 below). To prevent extension during PCR amplification, the 3' end of the probe has been phosphorylated. The 5' end of the TaqMan probe is labeled with a fluorescent reporter group, the 3' end is labeled with a fluorescent quencher group, and the 3' end of the TaqMan probe has been phosphorylated; optionally, the fluorescent reporter group is selected from FAM, HEX, JOE, TET, CY3, CY5, ROX, Texas, and the fluorescent quencher group is selected from TAMRA, BHQ (BHQ1, BHQ2, BHQ3), MGB, DABCYL.
[0036] The nucleic acid of Mycoplasma putrefaciens extracted by method 1.2 was diluted 5 times in a 10-fold gradient, and the nucleic acid of the 5 dilutions was amplified by fluorescent PCR using 3 sets of primers and probes respectively. Figure 1 Panels AC show the fluorescent PCR results of primer probes of groups 1-3. By comparing the Ct values, curve shapes, fluorescence intensities, etc. of the same template detected by different primer probe groups, it was determined that the primer probes of group 2 (Figure B) were PCR primers and TaqMan probes that can be used for high-sensitivity detection of Mycoplasma putrefaciens.
[0037] Table 1: Mycoplasma putrefaciens reference strain information
[0038]
[0039] Table 2: Primer and probe information
[0040]
[0041] Example 2: Fluorescence PCR Detection Method for Mycoplasma putrefaciens
[0042] 2.1. Extraction of genomic DNA
[0043] AxyPrep Bacterial Genomic DNA Miniprep Kit (Corning Life Sciences (Wujiang) Co., Ltd.) was used to extract nucleic acids from samples infected with Mycoplasma putrefaciens. For tissue samples, an appropriate amount was added to PBS buffer and mixed. After being fully ground with a mortar, the tissue suspension was transferred to a sterile centrifuge tube and centrifuged to extract the supernatant. For oral or nasal swab samples, they were infiltrated with PBS buffer and centrifuged to extract the supernatant.
[0044] 2.2. Establishment of standard curve
[0045] 2.2.1. PCR amplification of target sequence
[0046] Using the upstream primer Mput-F2 and the downstream primer Mput-R2 in Table 2 in the above Example 1, the genomic DNA of Mycoplasma putrefaciens extracted by method 2.1 was amplified by PCR to obtain the target gene fragment of Mycoplasma putrefaciens. The PCR reaction system is shown in Table 3 below. The PCR reaction conditions are: pre-denaturation at 95°C for 5 min; PCR amplification at 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, for 35 cycles; and final extension at 72°C for 7 min.
[0047] Table 3: Mycoplasma putrefaciens PCR reaction system
[0048]
[0049] 2.2.2 Preparation of Standards
[0050] Purify and recover the target gene fragment of Mycoplasma putrefaciens amplified in step 2.2.1 above, and connect it to the pMD19-T vector (purchased from TakaRa) to construct a recombinant plasmid, transfer it into JM109 competent cells (purchased from TakaRa) for cloning, and screen the positive recombinant plasmids and send them to Beijing Biotech Co., Ltd. for sequencing. After the sequencing sequence is correct, the recombinant plasmid pMD19-T-arcB carrying the target gene of Mycoplasma putrefaciens is obtained. Use an ultra-micro nucleic acid protein analyzer to measure the concentration according to the formula "copies / μl = (6.02×10 23 copies / mol×concentration ng / μl) / (DNA base number×660×10 9 ng / mol)” to calculate the copy number of the plasmid and dilute it with TE buffer to 1×10 10 copies / μl or lower dilution (e.g. 1×10 9 , 1×10 8 , 1×10 7 , 1×10 6 copies / μL, etc.), as a standard for Mycoplasma putrefaciens, and stored below -15°C.
[0051] 2.2.3. Establishment of standard curve
[0052] The Mycoplasma putrefaciens standard prepared in step 2.2.2 was diluted 10-fold to 1 × 10 1 copies / μl, take 1×10 7 , 1×10 6 , 1×10 5 , 1×10 4, 1×10 3 , 1×10 2 , 1×10 1 The dilution of 100 copies / μL was used as the standard sample (template), and fluorescent PCR detection was carried out under the guidance of the primers and TaqMan probe of Group 2 in Example 1. The reaction system of fluorescent PCR includes: 12.5μl of fluorescent PCR premix HieffUnicon Universal TaqMan mμltiplex qPCRmastermix, 1μl of upstream and downstream primers (10μmol / L), 0.5μl of probe (10μmol / L), 5μl of template, and DEPC-treated water was used to make up to 25μl. The reaction conditions of fluorescent PCR are: pre-denaturation: 95℃5min; PCR amplification conditions: 95℃15s, 60℃30s, 45 cycles. The real-time fluorescent PCR detection results of the standard sample of Mycoplasma putrefaciens are shown in the figure. Figure 2 As shown in the AB panels, panel A is the amplification curve, and from left to right it corresponds to 1×10 7 , 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 , 1×10 2 , 1×10 1 The standard samples of each concentration are 1000 copies / μL. It can be seen that the amplification curves of the standard samples of each concentration are all smooth "S" shapes, and the curve shape is good; Panel B is a graph of the concentration Log value (X axis) of each standard sample and its corresponding Ct value (Y axis) to draw the standard curve. The curve correlation coefficient R 2 =1, the amplification efficiency E is 104.65%, and the linear equation is y=-3.22X+39.56, which meets the requirements. Therefore, the standard curve can be used for fluorescent PCR quantitative detection of Mycoplasma putrefaciens.
[0053] 2.3 Fluorescence PCR amplification
[0054] The extracted genomic DNA of the sample to be tested was used as a template, the prepared Mycoplasma putrefaciens standard sample of appropriate concentration was used as a positive control, and DEPC-treated water was used as a negative control. According to the real-time fluorescence PCR test results, the sample to be tested was judged, and the copy number of the Mycoplasma putrefaciens nucleic acid-positive sample was quantified.
[0055] The specific detection method includes the following steps:
[0056] (1) Using the genomic DNA extracted from the sample to be tested as a template, fluorescent PCR detection was performed under the guidance of the primer probe of Group 2 in Table 1. The detection system of 25 μL fluorescent PCR included: 12.5 μL of fluorescent PCR premix HieffUnicon UniversalTaqMan mμltiplex qPCR master mix, 1 μL of each upstream and downstream primers (10 μmol / L), 0.5 μL of probe (10 μmol / L), 5 μL of template, and DEPC-treated water was used to make up to 25 μL. The reaction conditions of fluorescent PCR were: pre-denaturation, 95°C for 5 min; PCR amplification conditions, 95°C for 15 s, 60°C for 30 s, and 45 cycles. Fluorescent signal detection was performed at the end of annealing of each cycle.
[0057] (2) The obtained Ct value and the specific amplification curve are used to achieve qualitative detection of Mycoplasma putrefaciens. Then, according to the standard curve generated by method 2.2.3, the copy number of Mycoplasma putrefaciens nucleic acid contained in the sample to be tested is obtained to achieve accurate quantification.
[0058] 2.4. Result determination
[0059] (1) If a specific amplification curve appears in the fluorescence channel of the sample to be tested and the Ct value is ≤38, the result is judged as positive for Mycoplasma putrefaciens nucleic acid.
[0060] (2) If a specific amplification curve appears in the fluorescence channel of the sample to be tested and the Ct value is 38<Ct value≤40, the result is judged as suspicious; if the suspicious sample is positive or suspicious after re-testing, the result is judged as positive, otherwise it is negative.
[0061] (3) If the sample to be tested has no Ct value or no specific amplification curve, the result is judged to be negative for Mycoplasma putrefaciens nucleic acid.
[0062] Example 3: Evaluation of the fluorescent PCR detection method for Mycoplasma putrefaciens
[0063] 3.1. Minimum detection limit detection
[0064] The standard sample with a concentration of 100 copies / μL was diluted to 10 copies / μL and 5 copies / μL using TE buffer. These three concentrations of standard samples were used as templates. Under the guidance of the primers of Group 2 and the TaqMan probe in Example 1, 20 repeated tests were performed for each test. The fluorescent PCR reaction system and reaction conditions were the same as those in step 2.2.3 of Example 2 to evaluate the minimum detection limit of this method.
[0065] Test results such as Figure 3As shown in Table 4 below, it can be seen that in the 20 repeated tests of the standard samples with concentrations of 100 copies / μL and 10 copies / μL by the fluorescent PCR method provided by the present invention, the specific amplification curve appeared in all 20 test results, and the Ct value was ≤38, which was determined to be positive for the nucleic acid of Mycoplasma putrefaciens, so it can be confirmed that the minimum detection limit of the method is 10 copies / μL. It can also be seen from Table 4 that the detection rate of the method of the present invention for the standard sample of 5 copies / μL can reach 15 / 20 (75%), which also proves that the Mycoplasma putrefaciens fluorescent PCR detection method provided by the present invention has high sensitivity.
[0066] Table 4: Results of the minimum detection limit of Mycoplasma putrefaciens fluorescence PCR
[0067]
[0068]
[0069] 3.2 Specificity detection
[0070] Genomic DNA was extracted from the inactivated Mycoplasma ovis (Mo), Mycoplasma caprine pneumonia (Mccp), Mycoplasma mycoides subspecies caprine (Mmc), Mycoplasma hyopneumoniae (Mhp), Mycoplasma bovis (M.bovis), and Mycoplasma putrefaciens (Mput) provided by Jinyu Baoling Biological Pharmaceutical Co., Ltd., and fluorescent PCR detection was performed under the guidance of the primers and probes shown in Group 2 of Example 1, using DEPC-treated water as a negative control. The PCR reaction system and reaction conditions referred to Method 2.2.3 in Example 2.
[0071] Specific test results such as Figure 4 As shown, only Mycoplasma putrefaciens showed a specific amplification curve, and the Ct value was 26.21, and the result was determined to be Mycoplasma putrefaciens nucleic acid positive; while the other samples did not show specific amplification curves and Ct values, and the results were determined to be Mycoplasma putrefaciens nucleic acid negative. The results show that the method of the present invention can specifically detect Mycoplasma putrefaciens, and has no cross reaction with the other five pathogens, and has strong specificity.
[0072] 3.3 Repeatability test
[0073] The concentration used in Example 2 was 1×10 5 , 1×10 3 , 1×10 1 The standard sample with 100 copies / μL was used as a template, and the fluorescence PCR detection was repeated 4 times. The reaction system and reaction conditions were referred to method 2.2.3 in Example 2. The average, standard deviation and coefficient of variation of each template Ct value were calculated to analyze and evaluate the repeatability of the method.
[0074] Test results such as Figure 5 As shown in Table 5 below, it can be seen that for the standard sample of the same concentration, the amplification curves of the four repeated tests are relatively close, there is no obvious difference in the number of cycles, and the coefficient of variation CV values of the number of cycles detected for standard samples of different concentrations are all less than 1%, indicating that the fluorescent PCR method for detecting Mycoplasma putrefaciens provided by the present invention has good repeatability.
[0075] Table 5: Results of fluorescence PCR repeatability test of Mycoplasma putrefaciens
[0076]
[0077] Example 4. Mycoplasma putrefaciens fluorescence PCR detection kit
[0078] The fluorescent PCR detection kit for Mycoplasma putrefaciens provided in this embodiment mainly includes: primer probes: primers (Mput-F2, Mput-R2) and TaqMan probe (Mput-P2) for fluorescent PCR detection of Mycoplasma putrefaciens shown in Group 2 in Example 1.
[0079] Further, for the convenience of detection, the fluorescent PCR detection kit for Mycoplasma putrefaciens provided in this embodiment may include:
[0080] (1) Fluorescent PCR premix: For example, a premix for a 25 μL fluorescent PCR detection system, comprising: HieffUnicon Universal TaqMan multiplex qPCR master mix, 10-15 μL; 10 μM Mput-F2, 0.25-1.0 μL; 10 μM Mput-R2, 0.25-1.0 μL; 10 μM Mput-P2, 0.4-1.0 μL.
[0081] (3) Controls: For the convenience of detection, the kit may also include a positive control and a negative control. The positive control may be a recombinant plasmid pMD19-T-arcB containing the Mput target gene, such as that prepared in Example 2 above. The negative control is a reaction system that does not contain Mycoplasma putrefaciens or its genomic DNA, such as DEPC-treated water (double distilled water, sterile deionized water, etc.). DEPC-treated water is used to make up the fluorescent PCR premix to 25uL.
[0082] (4) Instructions: To facilitate detection, the kit may also include the instructions obtained in Example 2, which may include the PCR reaction procedure: pre-denaturation, 95°C for 5 min; PCR amplification conditions, 95°C for 15 s, 60°C for 30 s, 45 cycles. Fluorescence signal detection is performed at the end of annealing in each cycle.
[0083] Example 5: Fluorescence PCR detection of Mycoplasma putrefaciens in clinical samples
[0084] The kit of Example 4 was used (wherein the detection system of 25 μL fluorescent PCR includes: Hieff Unicon Universal TaqMan mμltiplex qPCR master mix 12.5 μL, 10 μM Mput-F2 1.0 μL, 10 μM Mput-R2 1.0 μL, 10 μM Mput-P2 0.5 μL, template 5 μL, and DEPC-treated water was used to make up to 25 μL.) 88 clinical samples provided by the National Engineering Laboratory of Jinyu Baoling Biological Pharmaceutical Co., Ltd. were tested, and the samples to be tested were numbered 1-88. The test results are shown in Table 6 below, of which 70 were positive for Mycoplasma putrefaciens nucleic acid and 18 were negative for Mycoplasma putrefaciens nucleic acid. It can be seen that the kit for fluorescent PCR detection of Mycoplasma putrefaciens provided by the present invention can realize the detection of clinical samples, and can provide a strong basis for the screening and quality control of raw material strains in the subsequent production process.
[0085] Table 6: Results of fluorescence PCR detection of Mycoplasma putrefaciens in 88 clinical samples
[0086]
[0087]
[0088] The fluorescent PCR primers, probes and detection kit for detecting Mycoplasma putrefaciens provided based on the above technical solution can implement rapid and highly sensitive detection of Mycoplasma putrefaciens, and can provide a strong basis for the investigation of the main epidemic situation of pathogens, the purification of epidemics, the screening of strains and quality monitoring, and effectively prevent and control the occurrence of epidemics. The kit and detection method of the present invention are simple to operate, have strong specificity, high sensitivity and good repeatability, can realize the qualitative detection of Mycoplasma putrefaciens, and will play an important role in the early technical support such as accurate detection of Mycoplasma putrefaciens in diseased samples or cultures in clinical diagnosis and vaccine manufacturing (application for non-diagnostic purposes), and have broad application prospects.
[0089] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall belong to the content of the present invention.
Claims
1. A fluorescent PCR primer and probe combination for detecting Mycoplasma putrefaciens, characterized in that: It comprises an upstream primer having a nucleotide sequence as shown in SED ID NO:4, a downstream primer having a nucleotide sequence as shown in SED ID NO:6, and a probe having a nucleotide sequence as shown in SED ID NO:
5.
2. The primer and probe combination according to claim 1, characterized in that The 5' end of the probe is labeled with a fluorescent reporter group, the 3' end is labeled with a fluorescent quencher group, and the 3' end of the probe is phosphorylated; optionally, the fluorescent reporter group is selected from one of FAM, HEX, JOE, TET, CY3, CY5, ROX, and Texas, and the fluorescent quencher group is selected from one of TAMRA, BHQ1, BHQ2, BHQ3, MGB, and DABCYL.
3. Use of the primer and probe combination as described in claim 1 or 2 in the preparation of a kit for detecting Mycoplasma putrefaciens.
4. A kit comprising the primer and probe combination as claimed in claim 1 or 2.
5. The kit according to claim 4, characterized in that The 25 μL reaction system for the fluorescent PCR detection comprises:
6. A method for detecting Mycoplasma putrefaciens using the kit as claimed in claim 5, characterized in that: Extract the target genomic DNA of Mycoplasma putrefaciens, use the extracted genomic DNA as a template, and perform fluorescent PCR detection under the guidance of the primer and probe combination; use the obtained Ct value and the specific amplification curve to qualitatively detect Mycoplasma putrefaciens, and / or calculate the copy number of Mycoplasma putrefaciens contained in the sample to be tested according to the standard curve and the obtained Ct value to achieve quantitative detection.
7. The method for detecting Mycoplasma putrefaciens according to claim 6, characterized in that: The reaction conditions for fluorescent PCR detection were as follows: pre-denaturation at 95°C for 5 min; PCR amplification was performed with denaturation at 95°C for 15 seconds and extension at 60°C for 30 seconds for 45 cycles.
8. The method according to claim 6, characterized in that The template is a standard of a recombinant plasmid containing the arcB gene of Mycoplasma putrefaciens, and the concentration of the standard is selected from 1×10 10 , 1×10 9 , 1×10 8 , 1×10 7 , and 1×10 6 copies / μL.
9. The method for detecting Mycoplasma putrefaciens according to claim 8, characterized in that: The standard was diluted 10-fold to a concentration of no less than 1×10 1 copies / μL, and obtain standard samples. Using the standard samples as templates, after fluorescence PCR detection, the concentration Log values of each standard sample are plotted against their corresponding Ct values to obtain a standard curve.
10. The method for detecting Mycoplasma putrefaciens according to claim 9, characterized in that: If the sample to be tested shows a specific amplification curve and the Ct value is ≤38, the result is judged as positive for Mycoplasma putrefaciens nucleic acid; If the sample to be tested shows a specific amplification curve and 38<Ct value≤40, the result is judged as suspicious Mycoplasma putrefaciens nucleic acid and needs to be retested; after retesting, if the Ct value is ≤40 and a specific amplification curve appears, the result is judged as positive, otherwise it is negative; If the sample to be tested has no Ct value or no specific amplification curve, the result is judged to be negative for Mycoplasma putrefaciens nucleic acid.