Primer, probe and kit for constant-temperature fluorescence rapid detection of mycoplasma bovis

Abstract

The invention relates to the technical field of biological virus detection, in particular to a primer, a probe and a kit for constant-temperature fluorescence rapid detection of mycoplasma bovis. Based on a multi-enzyme constant-temperature rapid amplification technology and in combination with real-time fluorescence detection, the constant-temperature fluorescence rapid detection kit is established, the reaction is completed within 20 minutes under the constant-temperature condition of 39 DEG C, the sensitivity reaches 0.1 fg / mu L, standard strains of escherichia coli, salmonella and common golden yellow coccus and streptococcus and escherichia coli separated from cattle farms are not detected, and the specificity is high; the positive plasmid sample is repeatedly detected for 6 times, the results are consistent, and the coefficient of variation is 2.93%. The kit can be applied to rapid diagnosis and on-site monitoring of mycoplasma bovis, and provides technical support for epidemic situation control and epidemiological research.

Description

Technical Field

[0001] The present application relates to the technical field of biological virus detection, and in particular to primers, probes, and a kit for constant-temperature fluorescence rapid detection of bovine Mycoplasma. Background Art

[0002] Mycoplasma bovis (M. bovis) is one of the most common pathogens causing bovine respiratory disease complex (BRDC). It can also cause arthritis, mastitis, conjunctivitis, reproductive system problems, and transport stress syndrome in cattle. In severe cases, it can lead to infertility and even miscarriage. Furthermore, M. bovis infection can induce a state of host immunosuppression, significantly increasing the risk of secondary infection with opportunistic pathogens (including hemolytic Pasteurella multocida, members of the Enterobacteriaceae family, and Streptococcus pyogenes). It is estimated that annual losses due to M. bovis in the UK amount to €192 million, and global losses due to M. bovis are estimated to exceed $3 billion annually. In 1983, Li Jishen of my country first isolated M. bovis from dairy cows with mastitis. In 2008, Xin Jiuqing et al. successfully isolated and cultured M. bovis from the respiratory tract and lungs of infected cows, attracting the attention of researchers. In 2011, Shen Qiang and colleagues from the Shanghai Fengxian District Animal Disease Prevention and Control Center tested 190 cattle samples in Shanghai for antibodies to Mycoplasma bovis, finding a 42.1% antibody-positive rate. In 2019, Zhao Jinyu and colleagues conducted PCR testing for Mycoplasma bovis in four regions of southern Xinjiang, reporting an overall positive rate of 22.86%. By 2021, Mycoplasma bovis had spread to nearly every province and autonomous region in my country. As a globally distributed pathogen, establishing a rapid diagnostic system is crucial for controlling the disease.

[0003] Pathogen isolation and culture is the gold standard for detecting and identifying Mycoplasma bovis. However, the isolation process is highly susceptible to contamination, is time-consuming, and requires specialized culture conditions, delaying disease detection and treatment, making it unsuitable for diagnosis. Serological diagnosis is generally used for initial diagnosis in cattle, but antibodies can only be detected 6–10 days after infection. Molecular biological testing is relatively rapid, convenient, and highly sensitive. In 2011, Bai Zhidi developed a loop-mediated isothermal amplification (LAMP) assay targeting the uvrC gene of M. bovis, which can quickly and efficiently distinguish M. bovis from Maga. In 2016, Li Jian developed a TaqMan real-time fluorescence quantitative PCR assay targeting the oppD / F genes of M. bovis. Lai et al. developed a multiplex PCR method that can simultaneously detect seven common ruminant mycoplasmas, including M. bovis. D. Jaramillo et al. developed a qPCR method to detect M. bovis in frozen semen imported from cattle into New Zealand.

[0004] Guizhou's beef cattle industry is rapidly developing, and the frequent cross-regional transportation of live cattle has increased the risk of mycoplasma transmission. Traditional isolation, culture, and serological testing are time-consuming and do not meet the requirements for rapid detection. Therefore, the development of rapid diagnostic methods based on molecular nucleic acid amplification technology has become a key technological breakthrough in the industry. Multienzyme isothermal rapid amplification (MIRA) is a novel nucleic acid amplification reaction involving reverse transcriptase, DNA polymerase, enzyme-linked catalyst, and DNA helicase. The entire reaction can be completed at 25-42°C in 5-30 minutes and can be used with various brands of fluorescent quantitative PCR instruments and isothermal fluorescence amplifiers.

[0005] Based on this, the current key research direction is to find primers that are sensitive and specific to bovine Mycoplasma, establish a multi-enzyme constant temperature rapid amplification method for bovine Mycoplasma, screen pathogens early, block the transmission chain, and provide key technical support for cattle disease prevention and control. Summary of the Invention

[0006] In order to solve the above-mentioned technical problems existing in the prior art, the present application provides primers, probes and kits for the constant temperature fluorescence rapid detection of Mycoplasma bovis, which are specifically achieved through the following technical solutions:

[0007] A primer set for constant temperature fluorescence rapid detection of Mycoplasma bovis, comprising any one or more of the following nine combinations of three pairs of primers:

[0008]

[0009]

[0010] Furthermore, the primer combination is: M.bovisF3+M.bovisR2 or M.bovisF3+M.bovisR3, preferably: M.bovisF3+M.bovisR2.

[0011] A probe for constant temperature fluorescence rapid detection of Mycoplasma bovis, comprising the following probe sequence:

[0012]

[0013] Among them, FAM is a fluorescent group used to emit fluorescent signals, which can be collected by fluorescent quantitative PCR instruments; THF (tetrahydrofuran) is the recognition site of nuclease exonuclease, and BHQ is a quenching group that is paired with FAM.

[0014] Principle: The probe sequence does not overlap with the specific primer recognition site and is 46-52 nt in length. The sequence avoids palindromes, internal secondary structures, and continuous repeated bases. The probe has four modification sites: a dSpacer (tetrahydrofuran, THF) is labeled in the middle position ≥35 nt from the 5' end, which serves as the recognition site for the nuclease; a fluorescent group is labeled upstream of the THF site, and a quenching group is labeled downstream, with the two groups separated by 2-4 nt; the THF is ≥15 nt from the 3' end, and the 3' end is labeled with a modification group, such as an amine group, a phosphate group, or a C3-Spacer.

[0015] A constant-temperature fluorescence rapid detection test for Mycoplasma bovis based on its principle: The probe sequence does not overlap with the specific primer recognition site, is 46-52 nt in length, and avoids palindromic sequences, internal secondary structures, and continuous repeated bases. The probe has four modification sites: a dSpacer (tetrahydrofuran, THF) is labeled in the middle position ≥35 nt from the 5' end, serving as a recognition site for the exonuclease; a fluorescent group is labeled upstream of the THF site, and a quenching group is labeled downstream, with the two groups spaced 2-4 nt apart; the THF is ≥15 nt from the 3' end, and the 3' end is labeled with a modification group, such as an amine group, a phosphate group, or a C3-Spacer. A kit containing the aforementioned primer set is provided.

[0016] Furthermore, the kit contains primers whose primer combination is: M.bovisF3+M.bovisR2.

[0017] Furthermore, the kit also contains the probe for constant-temperature fluorescence rapid detection of Mycoplasma bovis according to claim 4.

[0018] Furthermore, the specific composition of the kit is as follows: ① ABuffer 1.6mL; ② BBuffer 150μL; ③ specific primer mixture 250μL, concentration is 10μM; ④ probe mixture 50μL; ⑤ positive control: pMD-18T-D positive standard 300μL, concentration is 100fg / μL; ⑥ ultrapure water 1000μL.

[0019] Note: All the above reagents were used in dry powder reaction tubes. The dry powder reaction tubes were purchased from Amp Future Changzhou Biotechnology Co., Ltd. (Cat. No. WLE8202KIT). The components of the dry powder reaction tubes mainly include recombinase, polymerase, single-strand binding protein, helicase, and other buffer auxiliary materials.

[0020] Furthermore, the reaction system of the kit was configured according to the instructions of the DNA isothermal rapid amplification fluorescent kit, 50 μL: 29.4 μL of Abuffer, 2.0 μL of upstream and downstream primers (10 μM), 0.6 μL of probe, 2.5 μL of B buffer, 5 μL of template, and ddH2O was used to make up to 50 μL. ddH2O was used as a negative control.

[0021] Furthermore, the amplification program of the kit is: 39° C., 30 s, 40 cycles.

[0022] Compared with the existing technology, the technical effects created by this application are embodied in:

[0023] (1) This application designed three pairs of primers, screened for the most sensitive and specific primers, and established a multi-enzyme constant-temperature rapid amplification method for Mycoplasma bovis. This method has high sensitivity (up to 0.1 fg / μL), good reproducibility, and a shelf life of up to one year.

[0024] (2) The real-time fluorescence multi-enzyme constant temperature amplification technology (MIRA) of the present application uses a rapid nucleic acid release agent to extract nucleic acids. It is simple to operate and can obtain the DNA template for amplification in just one step (usually 5 minutes). By integrating constant temperature amplification and fluorescence signal capture systems, it breaks through the dependence of traditional nucleic acid detection on precision instruments. This technology only requires a constant temperature reaction at 39°C for 20 minutes, and the results can be interpreted through a portable fluorescence reading device. Its sensitivity can detect 0.1fg / μL of target nucleic acid. It can meet the real-time detection needs of farms, slaughterhouses and other scenarios, and provide an innovative technical path for the real-time diagnosis of pathogens in the grassroots veterinary system. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 For primer F3-R2 specific detection.

[0026] Figure 2 For primer F3-R3 specific detection.

[0027] Figure 3 This is a test to test the sensitivity of the reaction system.

[0028] Figure 4 To test the repeatability of the reaction system. DETAILED DESCRIPTION

[0029] The technical solution of the present application is further defined below in conjunction with specific implementation methods, but the scope of protection required is not limited to the description.

[0030] Development of a constant temperature fluorescence rapid detection kit for Mycoplasma bovis

[0031] 1 Materials and Methods

[0032] 1.1 Strains

[0033] Escherichia coli 25922 standard strain, Salmonella 13076 standard strain, Staphylococcus aureus The 6538 standard strain was preserved by the Guizhou Provincial Animal Husbandry and Veterinary Research Institute, and Escherichia coli EC1, Streptococcus str1, and Salmonella Salmon1 were isolated from cattle farms in Guizhou Province.

[0034] 1.2 Main Reagents

[0035] Takara MiniBEST Universal RNA Extraction Kit and EZNATM Gel Extraction Kit were purchased from Omega; ampicillin, pMD18-T Vector, DH-5α competent cells, a common plasmid mini-extraction kit, and deionized water were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; Rapid Nucleic Acid Releaser (DNA Type)-II and DNA Constant Temperature Rapid Amplification Kit (Fluorescence Type) were purchased from Anpu Future Changzhou Biotechnology Co., Ltd.

[0036] 1.3 Main Equipment

[0037] QuantStudio TM 6and Real-Time PCR Systems (Thermo Fisher Scientific (China) Co., Ltd., Catalog No. 4485691); NanoDrop Eight Microvolume UV-Vis Spectrophotometer (Thermo Fisher Scientific (China) Co., Ltd., Catalog No. 912A1101).

[0038] 1.4 Primer design and screening

[0039] Three pairs of primers were designed using Oligo 6.0 software based on the opp D / F gene sequence of M. bovis in GenBank. The specificity of these primers was verified using http: / / www.ncbi.nlm.nih.gov / BLAST / . Probe sequences were designed based on the fluorescent probe. Primer and probe sequences were synthesized by Guangzhou Aikerui Biotechnology Co., Ltd. The primer and probe sequences are shown in the table below.

[0040]

[0041] 1.5 Nucleic acid extraction

[0042] Place the nasal swab sample in an EP tube containing a release agent, vortex or pipette to mix, and then incubate at 40°C for 5 minutes. The supernatant of the lysate can be used as a template.

[0043] 1.6 Construction of pMD18-T-oppD positive plasmid

[0044] Using opp sample DNA as a template, the primers designed in this experiment were added for PCR amplification. PCR products with the expected band size and good specificity were selected for identification by 2% agarose gel electrophoresis. The target fragment was recovered using a gel recovery kit, and the target fragment was ligated with the pMD18-T cloning vector and transformed into competent cells DH-5α. The recombinant plasmid was named pMD18-TD. The constructed recombinant plasmid was sent to Shanghai Bioengineering Co., Ltd. for sequencing. The recombinant plasmid identified as positive by sequencing was expanded and cultured, and pMD18-TD was extracted using a plasmid extraction kit. Its concentration was determined using an ultra-micro nucleic acid protein analyzer and used as the recombinant plasmid standard for subsequent reactions.

[0045] 1.7 Reaction conditions

[0046] The reaction system was prepared according to the instructions of the DNA isothermal rapid amplification fluorescence kit, 50 μL: 29.4 μL of Abuffer, 2.0 μL of upstream and downstream primers (10 μM), 0.6 μL of probe, 2.5 μL of B buffer, and 5 μL of template. The volume was made up to 50 μL with ddH2O, and ddH2O was used as a negative control.

[0047] Amplification program: 39°C, 30 s, 40 cycles.

[0048] 1.8 Primer screening

[0049] The constructed recombinant plasmids were diluted to 10 -3 and 10 -5 , using two different template concentrations and based on the TT values ​​of the reaction results, detect the primers with the best sensitivity when using three primer pairs and nine combinations. The amplification system and procedure are shown in 1.7.

[0050] 1.9 Specificity

[0051] Escherichia coli 25922 standard strain, Salmonella 13076 standard strain, Staphylococcus aureus Sixteen bacterial nucleic acids, including 6538 standard strain, Escherichia coli EC1, Streptococcus str1, and Salmonella Salmon1, were diluted to 10 ng / μL as templates. The constructed OPP recombinant plasmid was diluted to 10 pg / μL as a positive control, and ddH2O was used as a negative control to test the specificity of the method.

[0052] 1.10 Sensitivity

[0053] After determining the nucleic acid concentration of the constructed recombinant plasmid pMD18-TD, the DNA was diluted five times in a 10-fold gradient and then used as a template for PCR reactions. ddH2O was used as a negative control. Three replicates were performed at each dilution factor to test the sensitivity of the established method.

[0054] 1.11 Repeatability

[0055] When the recombinant plasmid was diluted to a concentration of 100 fg / μL, the amplification reaction was repeated several times to test the reliability of the results.

[0056] 1.12 Kit Assembly and Shelf Life Testing

[0057] The components of the kit were stored at room temperature, 4°C and -20°C, respectively. Three time gradients of 1 week, 6 months and 1 year were set to detect positive plasmids.

[0058] 2 Results

[0059] 2.1 Primer combination screening

[0060] By diluting the M. bovis plasmid to 10 -3 with 10 -5 Two screenings of nine primer-probe combinations at different concentrations were performed. Based on the minimum TT values ​​of the reactions of different primer combinations, two primer-probe combinations with better performance were selected: M.bovisF3+Probe 1+M.bovisR2 and M.bovisF3+Probe 1+M.bovisR3. The results are shown in Tables 1 and 2. These two pairs of primer-probe combinations were subsequently used for specificity screening experiments.

[0061] Table 1 Plasmid concentration dilution 10 -3 TT value of each primer reaction

[0062]

[0063] Table 2 Plasmid concentration dilution 10 -5 TT value of each primer reaction

[0064]

[0065] 2.2 Specificity

[0066] Results: F3-R2 can specifically distinguish all samples, while F3-R3 has poor specificity and can detect non-specific samples, so the F3-R3 combination was eliminated. The results are shown in Figure 1 and Figure 2 , F3-R2 continues to perform subsequent sensitivity testing.

[0067] 2.3 Sensitivity

[0068] The concentration of the recombinant plasmid was 100 fg / μL, and the template concentrations after dilution were 10 fg / μL, 1 fg / μL, 0.1 fg / μL, and 0.01 fg / μL, respectively. The sensitivity of the F3-R2 primer was 0.1 fg / μL.

[0069] 2.4 Repeatability

[0070] The established constant temperature fluorescence method was used to repeatedly detect the recombinant plasmid 100 fg / μL sample 6 times to test the reliability of the results. The results are shown in Table 3. The coefficient of variation was 2.93%, which was less than 5% and met the requirements.

[0071] Table 3 Reaction system repeatability test

[0072]

[0073] 2.5 Kit Assembly and Validity Period Testing

[0074] Based on the test results, a constant-temperature fluorescence rapid detection kit for bovine Mycoplasma was established. Each kit can be used to detect 48 samples. The kit composition is as follows: ① A Buffer 1.6 mL; ② B Buffer 150 μL; ③ specific primer mixture 250 μL, concentration of 10 μM; ④ probe mixture 50 μL; ⑤ positive control: pMD-18T-D positive standard 300 μL, concentration of 100 fg / μL; ⑥ ultrapure water 1000 μL.

[0075] The kit components were stored at room temperature, 4°C, and -20°C, respectively. Positive strains were tested over three time gradients: one week, six months, and one year. Storage at 4°C for one year reduced the positive copy number by 100-fold, while storage at -20°C for one month, three months, six months, and one year had no effect. Therefore, the shelf life of the kit is one year.

[0076] 3 Discussion and Analysis

[0077] Mycoplasma bovis, a cross-species pathogen, has a host range encompassing major bovine species, including beef cattle, dairy cows, water buffalo, and yaks. There have even been reports of Mycoplasma bovis being detected in the human oral cavity. With the rise of the beef cattle industry and the introduction of new breeds, particularly during stressful conditions such as cold seasons and cross-regional transportation, morbidity and mortality rates in livestock herds have increased significantly. Studies have found that Mycoplasma bovis can persist in infected cattle for extended periods. Existing antibiotic treatments struggle to completely eliminate the pathogen, and the lack of an effective vaccine makes infected cattle a persistent source of infection. Therefore, timely diagnosis of the pathogen and early isolation and prevention are key to controlling the disease.

[0078] Isolation and culture of the pathogen is the gold standard for detecting the presence of Mycoplasma bovis, but isolation is difficult and time-consuming, making it unsuitable for rapid diagnosis of Mycoplasma bovis infection. While ELISA and colloidal gold assays offer high specificity and sensitivity, they can lead to false positives due to cross-reactivity or nonspecific binding, and antibody production is delayed (approximately two weeks after infection). Immunohistochemistry, a technique that combines immunology with histochemistry, accurately determines pathogen infection and clearly demonstrates the localization and distribution of pathogens within tissues, organs, and cells. Nunoya used immunohistochemistry to examine lung tissue from artificially challenged calves and found Mycoplasma bovis on the surface and within bronchiolar epithelial cells in pneumonia lesions, as well as in bronchiolar phagocytes. However, test results are significantly affected by sample freshness, fixation method, and tissue processing techniques, requiring strict control of staining conditions, specialized equipment, and experienced operators. Furthermore, immunohistochemistry detects antigens rather than live pathogens, and may not accurately reflect the current infection status.

[0079] Innovations in molecular biology techniques, particularly PCR-based derivatives (such as multiplex amplification, real-time fluorescence quantification, random amplified polymorphic DNA analysis, and restriction fragment length polymorphism detection), have significantly improved the diagnostic efficacy of bovine respiratory diseases. While real-time fluorescence quantification methods can detect nucleic acids at the nanogram level, repeated opening of the container can lead to aerosol dispersion of the amplified product. The 4-6 hours required from nucleic acid extraction to data analysis make them inefficient.

[0080] A comparative analysis of isothermal amplification technology systems reveals that recombinase polymerase amplification (RPA) technology, due to its reliance on foreign patents, suffers from high reagent costs and long supply chain cycles. While domestically developed recombinase-mediated amplification (RAA) technology has achieved technological localization, its reaction stability is susceptible to interference from the sample matrix, and its production process lacks standardization. In contrast, my country's independently developed multi-enzyme isothermal amplification (MIRA) technology significantly improves inhibitor resistance and batch stability by optimizing the enzyme source combination and reaction system. This technology not only enables large-scale standardized production of reagents but also compresses the detection process to under 20 minutes. It also utilizes a portable fluorescence detector to visualize results, completely eliminating the need for specialized laboratory environments and providing a breakthrough solution for immediate pathogen diagnosis at the grassroots level and in the field. MIRA technology has been applied to the diagnosis of various epidemic diseases and the detection of pathogens, but its use in MIRA detection of Mycoplasma bovis has yet to be reported.

[0081] The real-time fluorescent multi-enzyme constant temperature amplification technology (MIRA) constructed in this study uses a rapid nucleic acid release agent to extract nucleic acids. It is simple to operate and can obtain the DNA template for amplification in just one step (usually 5 minutes). By integrating constant temperature amplification and fluorescence signal capture systems, it breaks through the dependence of traditional nucleic acid detection on precision instruments. This technology only requires a constant temperature reaction at 42°C for 20 minutes, and the results can be interpreted through a portable fluorescence reading device. Its sensitivity can detect 0.1fg / μL of target nucleic acid. It can meet the real-time detection needs of farms, slaughterhouses and other scenarios, and provides an innovative technical path for the immediate diagnosis of pathogens in the grassroots veterinary system.

[0082] Finally, it should be noted that the above embodiments are merely representative examples of this application. Obviously, the technical solutions of this application are not limited to the above embodiments and are subject to numerous variations. All variations that can be directly derived or conceived by a person of ordinary skill in the art from the disclosure of this application should be considered within the scope of protection of this application.

Claims

1. A primer set for constant temperature fluorescence rapid detection of Mycoplasma bovis, characterized in that: Contains any one or more of the following 9 combinations of 3 pairs of primers: 。 2. The primer set for constant temperature fluorescence rapid detection of Mycoplasma bovis according to claim 1, characterized in that: The primer combination is: M.bovisF3+M.bovisR2 or M.bovisF3+M.bovisR3.

3. The primer set for constant temperature fluorescence rapid detection of Mycoplasma bovis according to claim 2, characterized in that: The primer combination is: M.bovisF3+M.bovisR2.

4. A probe for constant temperature fluorescence rapid detection of Mycoplasma bovis, characterized in that: Contains the following probe sequences: 。 5. A constant temperature fluorescence rapid detection kit for Mycoplasma bovis, characterized in that: Contains the primer set according to claim 1.

6. The Mycoplasma bovis constant temperature fluorescence rapid detection kit according to claim 5, characterized in that The primer combination contains primers: M.bovisF3+M.bovisR2.

7. The Mycoplasma bovis constant temperature fluorescence rapid detection kit according to claim 5, characterized in that The invention also contains the probe for constant temperature fluorescence rapid detection of Mycoplasma bovis according to claim 4.

8. The Mycoplasma bovis constant temperature fluorescence rapid detection kit according to claim 5, characterized in that The specific components of the kit are as follows: ①A Buffer 1.6mL; ②B Buffer 150μL; ③Specific primer mixture 250μL, concentration is 10μM; ④Probe mixture 50μL; ⑤Positive control: pMD-18T-D positive standard 300μL, concentration is 100fg / μL; ⑥Ultrapure water 1000μL.

9. The Mycoplasma bovis constant temperature fluorescence rapid detection kit according to claim 5, characterized in that The reaction system was prepared according to the instructions of the DNA isothermal rapid amplification fluorescence kit, 50 μL: 29.4 μL of Abuffer, 2.0 μL of upstream and downstream primers (10 μM), 0.6 μL of probe, 2.5 μL of B buffer, 5 μL of positive control, and ddH2O was used to make up to 50 μL. At the same time, ddH2O was used as a negative control.

10. The Mycoplasma bovis constant temperature fluorescence rapid detection kit according to claim 5, characterized in that: The amplification program is: 39°C, 30s, 40 cycles.

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