Primer probe combination, kit and method for detecting Mycoplasma crocodile and Mycoplasma alligator by real-time fluorescence quantitative PCR

Abstract

The present invention provides a primer-probe combination, a kit, and a method for detecting Mycoplasma alligatoris and Mycoplasma crocodyli using real-time fluorescence quantitative PCR. The nucleotide sequence of the primer-probe combination is shown in SEQ ID NOs: 1-3. The kit and detection method provided by the present invention can detect Mycoplasma alligatoris and Mycoplasma crocodyli in test samples. The kit and detection method have the characteristics of rapid detection, high sensitivity, and good specificity. These methods can have a positive impact on the prevention and control of mycoplasma diseases in crocodiles and alligators, as well as the development of related vaccines and drugs, and are conducive to the development of the crocodile and alligator breeding industries.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection, and particularly relates to a primer-probe combination, a kit and a method for detecting Mycoplasma crocodile and Mycoplasma alligator by real-time fluorescence quantitative PCR. Background Art

[0002] Mycoplasmas are bacteria that lack cell walls and are currently considered the smallest known cells. Reptile infections caused by various mycoplasmas are commonly known as mycoplasmosis. Common examples include upper respiratory tract disease (URTD) in turtles and tortoises, tracheal and pneumonia in Burmese pythons, and arthritis in crocodiles.

[0003] The mycoplasmas that cause mycoplasmosis in alligators are Mycoplasma alligatoris and Mycoplasma crocodyli. Mycoplasma crocodyli is associated with polyarthritis in Nile crocodiles (Crocodylus niloticus). It can infect Nile crocodiles of all ages, with clinical symptoms including polyarthritis, lameness, and sometimes pneumonia. It is less virulent. Mycoplasma alligatoris has been shown to cause pneumonia, polyserositis, and polyarthritis in American alligators (Alligator mississippiensis) and broad-nosed caimans (Caiman latirostris). It is highly virulent and one of the few mycoplasmas that can be fatal.

[0004] China's crocodile farming industry has grown rapidly in recent years. According to the "2024-2030 China Crocodile Farming Industry Market Status Survey and Development Trend Analysis Report" published by Market Research Online, as of 2018, there were 36,000 crocodile farmers in China, farming 145 million mu (approximately 1.6 hectares) of land and raising 390 million crocodiles, accounting for over 70% of the global total, making China the world's largest crocodile farming country. Consequently, the healthy farming of crocodiles is gaining increasing attention, and prevention and regular testing are essential for the prevention of mycoplasma, which can cause fatal crocodile mycoplasma disease.

[0005] In existing technologies, the detection of Mycoplasma crocodile or Mycoplasma alligatorium is often done through PCR, which is cumbersome, time-consuming, and poorly sensitive. However, no products are currently available that utilize real-time fluorescence PCR to detect Mycoplasma crocodile or Mycoplasma alligatorium. Real-time fluorescence quantitative PCR is a method that uses fluorescent chemicals to measure the total amount of product after each polymerase chain reaction (PCR) cycle in a DNA amplification reaction. Compared to conventional detection technologies, this method not only shortens detection time and has higher sensitivity, but also saves manpower and material resources, providing a reliable basis for rapid diagnosis of pathogens.

[0006] Therefore, there is an urgent need in the art for a detection reagent and method for Mycoplasma crocodile and Mycoplasma alligator that is time-saving, specific, and sensitive, so as to facilitate the development of the crocodile and alligator breeding industries. Summary of the Invention

[0007] To solve the above technical problems, the present invention provides a primer-probe combination for detecting Mycoplasma crocodile and Mycoplasma alligatorii by real-time fluorescence quantitative PCR, characterized in that the primer-probe combination comprises a forward primer, a reverse primer, and a probe, and the nucleotide sequences of the forward primer, reverse primer, and probe are as follows:

[0008] Forward primer: 5'-ATCATGCCTCTTACGAGA -3', as shown in SEQ ID NO: 1;

[0009] Reverse primer: 5'-AAGTTTTTTGAGATTTGCTTAAC -3', as shown in SEQ ID NO: 2;

[0010] Probe: 5'-CACGTGCTACAATGGGAAGTAC-3', as shown in SEQ ID NO: 3, wherein the 5' end of the probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group.

[0011] Specifically, the fluorescent reporter group is selected from any one of FAM, VIC, JOE, TET, CY3, CY5, ROX, Texas RED or LCRED460, and the fluorescent quencher group is selected from any one of BHQ1, BHQ2, BHQ3 or MGB.

[0012] A second aspect of the present invention provides a use of the aforementioned primer-probe combination for detecting Mycoplasma crocodile and Mycoplasma alligator by real-time fluorescence quantitative PCR in the preparation of a kit for detecting Mycoplasma crocodile and Mycoplasma alligator.

[0013] A third aspect of the present invention provides a kit for detecting Mycoplasma crocodile and Mycoplasma alligator based on real-time fluorescence quantitative PCR technology, the kit comprising a PCR amplification reagent, a negative control, and a positive control, the PCR amplification reagent comprising the primer probe combination for detecting Mycoplasma crocodile and Mycoplasma alligator according to any one of claims 1 or 2.

[0014] Specifically, the PCR amplification reagent also includes DNA polymerase, Mg + and dNTPs.

[0015] Specifically, in the primer-probe combination for detecting Mycoplasma crocodile and Mycoplasma alligatorii by real-time fluorescence quantitative PCR, the concentrations of the forward primer, reverse primer, and probe are all 5-10 μM.

[0016] Specifically, the negative control is ultrapure water, and the positive control is a plasmid containing the gene sequences of Mycoplasma crocodile and Mycoplasma alligator.

[0017] A fourth aspect of the present invention provides a real-time fluorescence quantitative PCR method for detecting Mycoplasma crocodile and Mycoplasma alligator for non-diagnostic purposes. The method is based on the aforementioned kit for detecting Mycoplasma crocodile and Mycoplasma alligator based on real-time fluorescence quantitative PCR technology, and comprises the steps of:

[0018] 1) Extract total DNA from the sample to be tested;

[0019] 2) preparing a reaction system, wherein the reaction system includes the aforementioned PCR amplification reagents;

[0020] 3) Perform real-time fluorescence quantitative PCR using the extracted total DNA of the sample to be tested as a template and adding negative and positive controls as quality control templates;

[0021] 4) After the reaction is complete, the test results are determined based on the Ct value.

[0022] Specifically, the procedure of the real-time fluorescence quantitative PCR reaction is:

[0023] Step 1: 94°C, 5 min;

[0024] Step2: 94℃, 30s, 50℃, 30s;

[0025] Step 2 executes 40 cycles.

[0026] Beneficial effects:

[0027] The primer-probe combination for real-time fluorescence quantitative PCR detection of Mycoplasma crocodile and Mycoplasma alligator provided in the present application can quickly and accurately detect Mycoplasma crocodile and Mycoplasma alligator in the test sample, has no cross-reaction with other pathogens, can effectively distinguish other mycoplasmas, has high sensitivity, good specificity, and high practicality, and the detection limit can reach 1000 copies / mL. Specific detection can be performed in the early stage of pathogen infection, which is conducive to further good application in the timely detection and prevention of Mycoplasma crocodile and Mycoplasma alligator. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a graph showing the results of detecting plasmid linearity in Example 1.

[0029] Figure 2This is a standard curve diagram for detecting the linearity of the plasmid in Example 1.

[0030] Figure 3 This is the result diagram of the positive sample detected in Example 3. DETAILED DESCRIPTION

[0031] The following is a clear and complete description of the technical solution of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0032] Example 1

[0033] This example illustrates the design and screening process of primer-probe combinations for detecting Mycoplasma crocodile and Mycoplasma alligator.

[0034] Based on GenBank nucleotide sequences, M. alligatoris (accession number NR 118838.1) and M. crocodyli (accession number NR 074301.1) were selected for bioinformatics analysis. Conserved regions were selected as amplification targets, and mycoplasma plasmids were synthesized. Three pairs of primers and probes were designed using Primer Premier 5.0 and oligo 7 for plasmid amplification. After experimental screening, one primer pair and one probe were selected, including a forward primer, a reverse primer, and a probe. The nucleotide sequences are as follows:

[0035] Forward primer (named M.ca F1): 5′-ATCATGCCTCTTACGAGA -3′, as shown in SEQ ID NO: 1;

[0036] Reverse primer (named M.ca R1): 5′-AAGTTTTTTGAGATTTGCTTAAC -3′, as shown in SEQ ID NO: 2;

[0037] Probe (named M.ca PF1): 5′-CACGTGCTACAATGGGAAGTAC-3′, as shown in SEQ ID NO:3.

[0038] The 5' end of the probe is labeled with a fluorescent reporter group FAM, and the 3' end of the probe is labeled with a fluorescent quencher group MGB. The amplification length of the forward primer, reverse primer, and probe is 93 bp.

[0039] The specific screening process is as follows: the sequences of the three pairs of primer probes are shown in Table 1. Each primer probe was diluted to a concentration of 10 μM, and the PCR amplification reagent was prepared according to Table 2. The synthesized Mycoplasma crocodile plasmid was diluted to 1×10 4 Copies / mL and 1×10 8 Copies / mL. Add 5 μL of each concentration of plasmid to each T for amplification.

[0040] Table 1 Primer and probe sequence information

[0041]

[0042] Among them, the nucleotide sequences of M.ca F2, M.ca R2, M.ca PF2 and M.ca PF3 are shown in SEQ ID NOs: 4-7.

[0043] Table 2 PCR amplification system

[0044] Components concentration 1T dosage <![CDATA[ddH2O]]> / 7.5 μL Buffer Mix 4 5μL M.ca F 10 μM 0.5μL M.ca R 10 μM 0.5μL M.ca PF 10 μM 0.5μL

[0045] Test results such as Figure 1-2 As shown in Table 3, the T1 combination has the best comprehensive performance in fluorescence intensity, sensitivity and Ct value, so F1 / R1 / PF1 was selected as the final primer-probe combination. The primer-probe combination and the detection method provided in this application have an amplification efficiency of 99.548%, R 2 =1.0000, good amplification.

[0046] Table 3 Linearity test results

[0047]

[0048] Example 2

[0049] This example shows the kit and method for detecting Mycoplasma crocodile and Mycoplasma alligator disclosed in this application:

[0050] A kit for detecting Mycoplasma crocodile and Mycoplasma alligator using real-time fluorescence quantitative PCR technology includes PCR amplification reagents, negative controls, and positive controls. The PCR amplification reagents include the primer-probe combination shown in SEQ ID NOs: 1-3 and 4× Buffer Mix (DNA polymerase, Mg+, dNTPs). The positive control is a synthetic plasmid containing the target fragments of M. crocodile and M. alligator. The negative control is ultrapure water. The concentrations of the forward primer, reverse primer, and probe are all 10 μM. The PCR amplification system is shown in Table 2. The specific steps are as follows:

[0051] 1) Extract nucleic acid from the sample to be tested: Pipette 200 μL of fecal sample into the lysis buffer of the Gangzhu Medical Nucleic Acid Extraction Reagent (Cartridge) and use the Gangzhu Medical Nucleic Acid Extractor to extract nucleic acid from the fecal sample of the crocodile or alligator to be tested.

[0052] 2) Prepare PCR amplification reagents according to Table 2. Pipette 5 μL of the test sample nucleic acid, positive control, and negative control into the PCR amplification reagents. Perform the real-time fluorescence quantitative PCR reaction according to the following procedure:

[0053] Step 1: 94°C, 5 min;

[0054] Step2: 94℃, 30s, 50℃, 30s;

[0055] Step 2 executes 40 cycles.

[0056] 3) After the reaction is complete, the test results are determined based on the Ct value.

[0057] When making a judgment, the fluorescence curve above the threshold should have an obvious S-shaped curve, otherwise the experiment will be considered invalid and errors in instruments, reagents, amplification conditions, etc. should be checked.

[0058] When using the method provided by the present invention to detect Mycoplasma crocodiliae, the judgment criteria are as follows:

[0059] If the negative control shows no amplification and the positive control Ct value is less than 32, the sample with a Ct value of 38 or less is considered positive. If the Ct value of the sample is 38 or less and is less than or equal to 40, retesting is recommended. If the retest result shows a Ct value of 40 or less, it is considered positive; otherwise, it is considered negative.

[0060] Test results such as Figure 3 As shown in Table 4, it can be seen that the primer-probe combination, detection kit and detection method provided by the present application can efficiently and accurately detect Mycoplasma crocodile and Mycoplasma alligator in the test sample.

[0061] Table 4 Sample test results

[0062] sample Samples to be tested Positive control Negative control Ct value 35.43 29.98 -

[0063] Example 3

[0064] This example tests the specificity and sensitivity of the kit and detection method prepared by the primer-probe combination disclosed in this application.

[0065] 1. Specificity detection

[0066] Mycoplasma crocodile and Mycoplasma alligatorii belong to the Mycoplasma family, Mycoplasma genus, which includes Mycoplasma bovis, Mycoplasma felis, Mycoplasma canis, Mycoplasma serpentis, and Mycoplasma chelonae. Clinical samples of these mycoplasmas, as well as samples of various types from healthy crocodiles and alligators, were collected for specificity testing. PCR amplification reagents were prepared according to Table 2, and 5 μL of sample was added to each T. Amplification was performed according to the conditions of Example 2, repeated five times. The results are shown in Table 5.

[0067] Table 5 Specificity test results

[0068]

[0069] As shown in Table 4, the primer-probe combination and the detection method provided by the present application have good specificity, and no other mycoplasmas of the same genus and alligator samples of various types that are known to be negative were detected.

[0070] 2. Repeatability test

[0071] The synthesized mycoplasma plasmid was diluted to 1×10 5 Copies / mL and 1×10 7 Prepare PCR amplification reagents according to Table 2. Pipette 5 μL of each concentration into the PCR amplification reagents for amplification, repeating 10 times.

[0072] Table 6 Repeatability test results

[0073]

[0074] As shown in Table 6, the primer-probe combination and detection method provided by this application are 1×10 5 Copies / mL and 1×10 7 The CV values ​​of the two plasmid concentrations of copies / mL were 0.6% and 0.9%, respectively, and the CV was less than 1%, indicating good reproducibility.

[0075] 3. Sensitivity test

[0076] The synthesized mycoplasma plasmid was diluted to 1×10 3 Copies / mL and 5×10 2 Prepare PCR amplification reagents according to Table 2. Pipette 5 μL of each concentration into the PCR amplification reagents for amplification, and repeat 20 times.

[0077] Table 7 Sensitivity test results

[0078]

[0079] As shown in Table 7 above, the primer-probe combination and detection method provided by this application are 1×103 The detection rate of copies / mL concentration was 100%, and the detection rate of 5×10 2 The detection rate of copies / mL is 55%. Therefore, the sensitivity of this application is 1×10 3 Copies / mL.

[0080] In summary, the primer-probe combination and detection method provided in this application have a detection limit of up to 1000 copies / mL, and have the characteristics of short detection time, strong detection specificity, good repeatability and high sensitivity.

[0081] In summary, the above embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A primer-probe combination for detecting Mycoplasma crocodile and Mycoplasma alligatorii by real-time fluorescence quantitative PCR, characterized in that: The primer-probe combination includes a forward primer, a reverse primer and a probe, and the nucleotide sequences of the forward primer, the reverse primer and the probe are as follows: Forward primer: 5'-ATCATGCCTCTTACGAGA -3', as shown in SEQ ID NO: 1; Reverse primer: 5'-AAGTTTTTTGAGATTTGCTTAAC -3', as shown in SEQ ID NO: 2; Probe: 5'-CACGTGCTACAATGGGAAGTAC-3', as shown in SEQ ID NO: 3, wherein the 5' end of the probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group.

2. The primer-probe combination for detecting Mycoplasma crocodile and Mycoplasma alligatorii by real-time fluorescence quantitative PCR according to claim 1, characterized in that: The fluorescent reporter group is selected from any one of FAM, VIC, JOE, TET, CY3, CY5, ROX, Texas RED or LC RED460, and the fluorescent quencher group is selected from any one of BHQ1, BHQ2, BHQ3 or MGB.

3. Use of the primer-probe combination for detecting Mycoplasma crocodile and Mycoplasma alligatorii by real-time fluorescence quantitative PCR according to any one of claims 1 or 2 in the preparation of a kit for detecting Mycoplasma crocodile and Mycoplasma alligatorii.

4. A kit for detecting Mycoplasma crocodile and Mycoplasma alligatorii based on real-time fluorescence quantitative PCR technology, characterized in that: The kit includes a PCR amplification reagent, a negative control, and a positive control. The PCR amplification reagent includes the primer-probe combination for detecting Mycoplasma crocodile and Mycoplasma alligatorii by real-time fluorescence quantitative PCR according to any one of claims 1 or 2.

5. The kit for detecting Mycoplasma crocodile and Mycoplasma alligatorii based on real-time fluorescence quantitative PCR technology according to claim 4, characterized in that: The PCR amplification reagent also includes DNA polymerase, Mg + and dNTPs.

6. The kit for detecting Mycoplasma crocodile and Mycoplasma alligatorii based on real-time fluorescence quantitative PCR technology according to claim 4, characterized in that: In the primer-probe combination for detecting Mycoplasma crocodile and Mycoplasma alligatorii by real-time fluorescence quantitative PCR, the concentrations of the forward primer, reverse primer, and probe are all 5-10 μM.

7. The kit for detecting Mycoplasma crocodile and Mycoplasma alligatorii based on real-time fluorescence quantitative PCR technology according to claim 4, characterized in that: The negative control is ultrapure water, and the positive control is a plasmid containing the gene sequences of Mycoplasma crocodile and Mycoplasma alligator.

8. A real-time fluorescence quantitative PCR method for detecting Mycoplasma crocodile and Mycoplasma alligator for non-diagnostic purposes, characterized in that: The method is based on the kit for detecting Mycoplasma crocodile and Mycoplasma alligatorii based on real-time fluorescence quantitative PCR technology according to any one of claims 4 to 7, comprising the steps of: 1) Extract total DNA from the sample to be tested; 2) preparing a reaction system, wherein the reaction system comprises the PCR amplification reagent according to any one of claims 4 to 7; 3) Perform real-time fluorescence quantitative PCR using the extracted total DNA of the sample to be tested as a template and adding negative and positive controls as quality control templates; 4) After the reaction is complete, the test results are determined based on the Ct value.

9. The real-time fluorescence quantitative PCR method for detecting Mycoplasma crocodile and Mycoplasma alligator for non-diagnostic purposes according to claim 8, characterized in that: The procedure of the real-time fluorescence quantitative PCR reaction is: Step 1: 94°C, 5 min; Step2: 94℃, 30s, 50℃, 30s; Step 2 executes 40 cycles.

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