A RAA primer pair and probe combination for detecting Mycoplasma hyopneumoniae and a kit thereof

Abstract

The present invention provides an RAA primer pair and probe combination and a kit for detecting Mycoplasma hyopneumoniae. The primer pair comprises an upstream primer with a sequence of SEQ ID NO: 1, a downstream primer with a sequence of SEQ ID NO: 2, and a probe with a sequence of SEQ ID NO: 3. The RAA primer pair and probe for detecting Mycoplasma hyopneumoniae provided by the present invention can detect Mycoplasma hyopneumoniae with specificity and high sensitivity. The detection kit using the primer pair and probe can be visually determined by a portable blue light instrument, thus truly realizing portable visual rapid nucleic acid detection. The kit is simple, rapid, sensitive, and has good specificity, and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene detection of animal diseases, and specifically relates to a RAA primer pair and probe combination and a kit for detecting Mycoplasma hyopneumoniae. That is, a primer pair and probe for rapid detection of Mycoplasma hyopneumoniae by recombinase-mediated isothermal amplification (RAA), as well as a kit using the primer pair and probe. Background Art

[0002] Mycoplasma pneumonia of swine (MPS), also known as swine enzootic pneumonia and swine panting disease, is a chronic respiratory infectious disease of swine caused by Mycoplasma hyopneumoniae (Mhp), with high morbidity and low mortality. MPS exclusively infects pigs, regardless of breed, age, or sex. Suckling pigs and weaned piglets are particularly susceptible, with high morbidity and mortality rates. Late-gestation and lactating sows are also susceptible. Fattening pigs are less susceptible, often presenting with chronic and latent infections. The prevalence and presence of MPS have long plagued the healthy development of the swine industry worldwide, causing significant economic losses to the global pig industry. Currently, the commonly used methods for the prevention and control of MPS are to improve animal husbandry management and establish a comprehensive biosafety system. Therefore, early diagnosis and real-time monitoring of this pathogen are crucial for the prevention and control of the disease and are of great significance to the development and sustainable health of the swine industry.

[0003] Recombinase-mediated isothermal amplification (RAA) is a novel isothermal in vitro nucleic acid amplification technology. By utilizing recombinase, single-strand DNA binding protein (SSB), and DNA polymerase, RAA enables rapid nucleic acid amplification at a constant temperature of 37-42°C. This technology is suitable for rapid diagnosis in clinical settings. It is an isothermal amplification technology with independent intellectual property rights in my country and has promising applications in the field of nucleic acid testing.

[0004] However, RAA technology places stringent requirements on probe and primer design. Primers are a key factor in determining the sensitivity of the RAA reaction. Inappropriate primer pairs can severely reduce sensitivity, leading to missed detection of clinically positive samples. Probes, on the other hand, are crucial for the specificity of the RAA reaction. Inappropriate probes can produce nonspecific fluorescence, resulting in false positives; and inappropriate probes can also lead to missed detection of clinically positive samples. Therefore, the design of primers and probes is a major limiting factor in the clinical application of RAA technology.

[0005] Notably, by designing exo probes and incorporating the FAM fluorescent group into the reaction system, amplification products can be visualized using a portable blue-light instrument at the corresponding excitation wavelength. This technology offers advantages such as low cost, high sensitivity, good reproducibility, simple operation, and visual results. It is suitable for rapid nucleic acid testing, particularly in resource-limited settings, at the grassroots level, and for point-of-care testing. Summary of the Invention

[0006] The purpose of the present invention is to provide an RAA primer pair and probe combination and a kit for detecting Mycoplasma hyopneumoniae, so as to achieve simple, rapid, sensitive and specific detection of Mhp.

[0007] The present invention first provides an RAA primer pair and probe for detecting Mycoplasma hyopneumoniae, the sequence information of which is as follows:

[0008] Mhp-p46-F: 5′-TTTGTAACAATTACTGGACTTACTAAAATTGTATT-3′ (SEQ ID NO: 1);

[0009] Mhp-p46-R: 5′-AGTTGCTGTTGAAGTTCTTCGGGTTTTAAT-3′ (SEQ ID NO: 2);

[0010] The sequence of Mhp-p46-Probe is as follows:

[0011] 5′-GGTAGCTTTGCTTTTAGTTCGTTTTCGACTTTTGATCTAGATGCTTTA-3′ (SEQ ID NO: 3);

[0012] The probe Mhp-p46-Probe is labeled with a FAM luminescent group at the 31st base T from the 5' end, the 32nd base T is replaced by tetrahydrofuran (THF), the 33rd base T is labeled with a BHQ1 quenching group, and the 3' end is modified with a C3-spacer blocking agent.

[0013] The primer pair and probe provided by the present invention can be used to prepare a kit for detecting Mycoplasma hyopneumoniae;

[0014] In another aspect, the present invention provides a RAA kit for detecting Mycoplasma hyopneumoniae, wherein the kit comprises the above-mentioned RAA primer pair and probe.

[0015] The kit also comprises a reagent system suitable for recombinase-mediated isothermal amplification reaction and a fluorescence-based reaction unit.

[0016] The reagent system includes a reaction buffer, nuclease-free water and magnesium acetate.

[0017] The fluorescence basic reaction unit comprises a single-stranded DNA binding protein, a recombinase and a polymerase.

[0018] The RAA primer pair and probe for detecting Mycoplasma hyopneumoniae provided by the present invention can detect Mycoplasma hyopneumoniae specifically and with high sensitivity. The detection kit using the primer pair and the probe can be visually judged with the naked eye through a portable blue light instrument, and can truly realize portable visual rapid nucleic acid detection. It has the characteristics of simplicity, rapidity, sensitive response, and good specificity, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 :Comparison and analysis diagram of Mycoplasma hyopneumoniae,

[0020] Figure 2 :Comparison and analysis of Mycoplasma hyopneumoniae and Mycoplasma floccosum,

[0021] Figure 3 : Screening diagram of Mycoplasma hyopneumoniae RAA primers, wherein (A): primer first round screening pattern diagram; (B): first round downstream primer screening result diagram; (C): first round upstream primer screening result diagram; (D): primer first round screening pattern diagram; (E): second round downstream primer screening result diagram; (F): second round upstream primer screening result diagram; (G): primer third round screening pattern diagram; (H): third round downstream primer screening result diagram; (I): third round upstream primer screening result diagram.

[0022] Figure 4 : Specificity validation diagram of the RAA method for Mycoplasma hyopneumoniae, where (A): real-time fluorescence Mhp-RAA amplification results, (B): Mhp-RAA visualization results; in the picture, 1: Mycoplasma hyopneumoniae, 2: Streptococcus suis, 3: Klebsiella pneumoniae, 4: Glasserella suis, 5: Actinobacillus pleuropneumoniae, 6: Staphylococcus aureus, 7: Pseudomonas aeruginosa, 8: Salmonella, 9: Escherichia coli, 10: negative control group.

[0023] Figure 5 :Mycoplasma hyopneumoniae RAA sensitivity validation diagram, (A): Mhp-qPCR sensitivity test results and statistical analysis results; (B): Real-time fluorescence Mhp-RAA sensitivity test results and statistical analysis results; (C): Visualization Mhp-RAA sensitivity test results and statistical analysis results; 1:10 6 copies / μL APC, 2:10 5 copies / μL APC, 3:10 4 copies / μL APC, 4:10 3copies / μL APC, 5:10 2 copies / μL APC, 6:10 1 copies / μL APC, 7:10 0 copies / μL APC, 8: negative control. DETAILED DESCRIPTION

[0024] The present invention is described in detail below with reference to the embodiments and accompanying drawings.

[0025] Example 1: Design of primers and probes

[0026] The present invention compares the representative reference strains of Mycoplasma hyopneumoniae in Genbank and selects the conserved region in the Mhp-p46 gene as the target ( Figure 1 Since the p46 gene of Mycoplasma hyopneumoniae is highly similar to the p46 gene of Mycoplasma suis, the p46 gene sequence of Mycoplasma suis was compared ( Figure 2 The probe was designed by selecting regions with significant differences between the two. Through rigorous analysis of the target sequence, a region that meets both amplification requirements and maintains both conservation and specificity was selected as the target, and the exo probe Mhp-p46-Probe (5′-GGTAGCTTTGCTTTTAGTTCGTT TTCGACTTTTGATCTAGATGCTTTA-3′) was designed.

[0027] The probe Mhp-p46-Probe is labeled with a FAM luminescent group at the 31st base T from the 5′ end, the 32nd base T is replaced by tetrahydrofuran (THF), the 33rd base T is labeled with a BHQ1 quenching group, and the 3′ end is modified with a C3-spacer blocking group.

[0028] 5′-GGTAGCTTTGCTTTTAGTTCGTTTTCGACT(FAM-dT)(THF)(BHQ1-dT)GATCTAGATGCTTTA[C3-Spacer]-3′.

[0029] Subsequently, in order to obtain RAA primer pairs for Mycoplasma hyopneumoniae, we designed and screened them. In addition to conventional RAA primer design principles, the 3′ end of the candidate primers must be conservative, avoiding palindromic sequences and stem-loop structures, and not containing five or more repeat sequences.

[0030] According to the above principles, the present invention designed 34 candidate primers and carried out three rounds of primer screening.

[0031] To ensure that each screening primer can react simultaneously, add magnesium acetate B buffer to the lid of the reaction tube during screening and then centrifuge to allow the reactions in each reaction tube to proceed simultaneously. The specific system (25μL) includes:

[0032]

[0033] Using Bole CFX96 TM The experiment was performed using a Touch real-time fluorescence quantitative PCR instrument. The fluorescence detection program was set as follows: constant temperature at 42°C, FAM channel fluorescence values ​​were collected every 1 min, and a total of 30 cycles.

[0034] The screening criteria for each screening is to select candidate primers corresponding to the curve with strong total fluorescence signal intensity and short amplification peak starting time, ensuring that the primers screened not only have higher sensitivity but can also complete detection in a shorter time.

[0035] First, the first round of screening is to roughly determine the location of the primers. The length of the first round of screening candidate primers is 30nt. The present invention designed 5 upstream (F1216-1245, F1206-1235, F1172-1202, F1158-1187, F1132-1162) and 5 downstream candidate primers (R958-987, R992-1021, R1005-1034, R1046-1075, R1060-1089) covering the entire target region ( Figure 3 A). First, the upstream primer was fixed, and all downstream primers R958-987, R992-1021, R1005-1034, R1046-1075, and R1060-1089 were screened. Then, the optimal first-round downstream primer R1046-1075 was obtained. Then, this downstream primer was used to screen all upstream primers F1216-1245, F1206-1235, F1172-1202, F1158-1187, and F1132-1162, and finally the optimal first-round primer pair F1206-1235, R1046-1075 ( Figure 3 B, Figure 3 C).

[0036] Since the positions of the primers have been roughly determined in the first round of screening, the purpose of the second round of screening in the present invention is to accurately determine the positions of the primers. The optimal primers obtained in the first round of screening were used as the standard, the primer length was kept unchanged at 30 nt, and the positions were continuously moved forward or backward by 1-3 nt to design 8 second-round upstream primers (F1211-1240, F1210-1239, F1208-1237, F1207-1236, F1206-1235, F1203-1232, F1202-1231, F1200-1229) and 8 downstream primers (R1044-1073, R1046-1075, R1047-1076, R1048-1077, R1053-1082, R1054-1083, R1055-1084, R1056-1085) ( Figure 3 D) First, the upstream primer F1206-1235 was fixed, and all downstream primers R1044-1073, R1046-1075, R1047-1076, R1048-1077, R1053-1082, R1054-1083, R1055-1084, and R1056-1085 were screened. Then the optimal second round downstream primer R1053-1082 was obtained, and then all upstream primers were screened using this downstream primer F1211-1240, F1210-1239, F1208-1237, F1207-1236, F1206-1235, F1203-1232, F1202-1231, F1200-1229, and the optimal second round primer pair F1203-1232, R1053-1082 ( Figure 3 E, Figure 3 F).

[0037] Now that the positions of the primer pairs have been determined, the purpose of the third round of screening is to further determine the length of the primers. The method used is to fix the position of the best primers in the second round and gradually increase the bases at the 3' end. The primer length range is 30-36 nt. Six upstream primers (F1203-1232, F1202-1232, F1201-1232, F1200-1232, F1199-1232, F1198-1232) and six downstream primers (R1053-1082, R1053-1083, R1053-1084, R1053-1085, R1053-1086, R1053-1087) were designed. Figure 3G). First, the upstream primer F1203-1232 was fixed, and all downstream primers R1053-1082, R1053-1083, R1053-1084, R1053-1085, R1053-1086, and R1053-1087 were screened. The optimal third-round downstream primer R1053-1082 was then obtained. This downstream primer was then used to screen all upstream primers F1203-1232, F1202-1232, F1201-1232, F1200-1232, F1199-1232, and F1198-1232, resulting in the optimal upstream primer F1198-1232.

[0038] After three rounds of screening of 34 candidate primers, the optimal primer pair for RAA reaction was finally obtained: F1198-1232, R1053-1082 (Mhp-p46-F, Mhp-p46-R) ( Figure 3 H, Figure 3 I). Since the screening criteria for each screening are to select candidate primers corresponding to the curve with strong total fluorescence signal intensity and short amplification peak starting time, Mhp-p46-F and Mhp-p46-R not only have higher sensitivity but also can complete detection in a shorter time.

[0039] The sequences of the upstream primer Mhp-p46-F and the downstream primer Mhp-p46-R are as follows:

[0040] Mhp-p46-F: 5′-TTTGTAACAATTACTGGACTTACTAAAATTGTATT-3′;

[0041] Mhp-p46-R: 5′-AGTTGCTGTTGAAGTTCTTCGGGTTTTAAT-3′.

[0042] Example 2: Specificity detection of screened primer probes

[0043] In order to verify the specificity of the present invention, in this example, nucleic acids of Mycoplasma hyopneumoniae, Streptococcus suis, Klebsiella pneumoniae, Glaseria suis, Actinobacillus pleuropneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhimurium and Escherichia coli were extracted respectively as reaction templates according to the instructions of the DNA extraction reagent, and a negative control was set.

[0044] The specific system (25 μL) for the Mycoplasma hyopneumoniae fluorescent RAA specificity assay includes:

[0045]

[0046]

[0047] Using Bole CFX96 TM The experiment was performed using a Touch real-time fluorescence quantitative PCR instrument. The fluorescence detection program was set as follows: constant temperature at 42°C, FAM channel fluorescence values ​​were collected every 1 min, and a total of 30 cycles.

[0048] Figure 4 The results in Figure A show that the experimental group corresponding to Mycoplasma hyopneumoniae DNA showed a specific fluorescence detection curve, while other bacterial pathogens and the negative control group did not show specific amplification curves. Figure 4 The results in Figure B show that the test group corresponding to Mycoplasma hyopneumoniae DNA exhibited specific green fluorescence, while the visualization results for other bacterial pathogens and the negative control were colorless. These results demonstrate that the primer pair and probe combination used in the present invention can achieve specific detection of Mycoplasma hyopneumoniae without cross-reaction with other related bacterial pathogens, demonstrating good specificity.

[0049] Example 3: Verification of RAA sensitivity of Mycoplasma hyopneumoniae

[0050] In order to test the sensitivity of the selected primer pairs and probe combinations, 10 6 -10 0 The positive standard plasmid pEASY-Blunt-Mhp-p46 was constructed by adding 10 copies / μL of Mhp-p46 positive standard plasmid (artificial positive control, APC) and performing RAA nucleic acid amplification according to the above-mentioned sample addition method. 6 copies / μL and then diluted 10-fold to 10 5 copies / μL, 10 4 copies / μL, 10 3 copies / μL, 10 2 copies / μL, 10 1 copies / μL, 10 0The RAA nucleic acid amplification was performed according to the aforementioned loading method and compared with the qPCR method in the published literature (Marois, C., Dory, D., Fablet, C., Madec, F., Kobisch, M., 2010. Development of a quantitative Real-Time TaqMan PCR assay for determination of the minimal dose of Mycoplasma hyopneumoniae strain 116 required to induce pneumonia in SPF pigs. J Appl Microbiol 108, 1523-1533.). The test results are shown in Figure 2. Figure 5 shown.

[0051] like Figure 5 As shown in Figures AC, the RAA primer and probe combination designed in the present invention has a strong sensitivity. The detection limit of Mhp-qPCR is 10 copies / μL, while the real-time fluorescence Mhp-RAA detection method and the visual Mhp-RAA detection method established in the present invention can also detect 10 copies / μL. Using SPSS software to calculate and analyze eight replicates, the sensitivity of the Mhp-qPCR detection method reached 29 copies / μL (95% confidence interval), while the sensitivity of the real-time fluorescence Mhp-RAA detection method reached 18 copies / μL (95% confidence interval). Notably, the sensitivity of the visual Mhp-RAA detection method also reached 29 copies / μL (95% confidence interval).

[0052] Example 4: Preparation of a kit for detecting Mycoplasma hyopneumoniae using a real-time fluorescence RAA method

[0053] This embodiment provides a fluorescent RAA kit for detecting Mycoplasma hyopneumoniae. The kit comprises a reagent system suitable for recombinase-mediated isothermal amplification reaction and a basic reaction unit.

[0054] Wherein, the reagent system specifically includes reaction buffer, nuclease-free water, and magnesium acetate;

[0055] The basic reaction unit specifically comprises a single-stranded DNA binding protein, a recombinase and a polymerase, and the basic reaction unit exists in the form of a lyophilized powder.

[0056] The specific system (25 μL) of the fluorescent RAA kit for detecting Mycoplasma hyopneumoniae includes:

[0057]

[0058] The operation method of the real-time fluorescence RAA detection method for detecting Mycoplasma hyopneumoniae using the above-mentioned detection kit specifically includes the following steps:

[0059] (1) Extracting DNA from the sample to be tested by conventional methods;

[0060] (2) Using the extracted DNA as a template, RAA amplification was performed at a constant temperature of 42°C for 30 min;

[0061] (3) Result determination.

[0062] The reaction conditions of the above RAA detection method are constant temperature amplification for 30 minutes at 42° C. The extracted DNA is used as a template, and the RAA primers and probes for detecting Mycoplasma hyopneumoniae in the kit and corresponding reagents are added to form a RAA reaction system for amplification.

[0063] Example 5: Detection and verification of clinical samples

[0064] A total of 108 clinical samples were collected and the 108 clinical samples to be tested were extracted according to the instructions of the DNA extraction reagent, and the extracted DNA was stored at -20°C.

[0065] The test kit of Example 4 was used for detection, and the clinical sample was detected by the qPCR method in the published literature for comparison. The test results are shown in Table 1 below.

[0066] Table 1: Test results of clinical samples

[0067]

[0068] Results showed that both real-time fluorescence Mhp-RAA and Mhp-qPCR detected positive results in 22 of the 108 clinical samples, with a concordance rate of 98%. Visual Mhp-RAA also detected positive results in 21 samples, with a concordance rate of 99%. The Kappa values ​​for real-time fluorescence and visual Mhp-RAA versus Mhp-qPCR were 0.943 (P < 0.001) and 0.971 (P < 0.001), respectively.

[0069] In summary, the present invention designs a real-time fluorescent RAA primer pair and probe for the Mhp-p46 gene, and screens out upstream and downstream primers Mhp-p46-F, Mhp-p46-R and probe Mhp-p46-Probe suitable for rapid RAA detection. The real-time fluorescent RAA kit for detecting Mycoplasma hyopneumoniae based on the above primer pair and probe of the present invention only requires a 30-minute reaction at a constant temperature of 42°C to complete. The entire reaction process has low requirements for instrumentation, is time-consuming, and simple to operate. The results can be visualized with the naked eye, making it more suitable for grassroots on-site diagnosis and immediate testing. It truly realizes portable rapid nucleic acid detection and can provide technical support for the rapid diagnosis, real-time monitoring, and prevention and control of Mycoplasma hyopneumoniae.

Claims

1. A RAA primer pair and probe for detecting Mycoplasma hyopneumoniae, characterized in that: The primer pair has an upstream primer sequence of SEQ ID NO: 1, a downstream primer sequence of SEQ ID NO: 2, and a probe sequence of SEQ ID NO: 3; The 31st base T of the probe is labeled with a FAM luminescent group from the 5' end, the 32nd base T is replaced by tetrahydrofuran (THF), the 33rd base T is labeled with a BHQ1 quenching group, and the 3' end is modified with a C3-spacer blocking agent.

2. Use of the primer pair and probe according to claim 1 in preparing a kit for detecting Mycoplasma hyopneumoniae.

3. The use according to claim 2, characterized in that The kit is a RAA kit.

4. A RAA kit for detecting Mycoplasma hyopneumoniae, characterized in that: The kit comprises the primer pair and probe according to claim 1.

5. The RAA kit according to claim 4, wherein The kit also comprises a reagent system suitable for recombinase-mediated isothermal amplification reaction and a fluorescence-based reaction unit.

6. The RAA kit according to claim 5, wherein The reagent system comprises a reaction buffer, nuclease-free water and magnesium acetate.

7. The RAA kit according to claim 5, wherein The fluorescent basic reaction unit comprises single-stranded DNA binding protein, recombinase and polymerase.

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