A TaqMan probe-based dual-fluorescence quantitative PCR primer and probe set, detection method, and application for detecting MG and MS.

Abstract

This invention discloses a TaqMan probe-based dual-fluorescence quantitative PCR primer and probe set, detection method, and application for detecting myocardial infarction (MG) and metabolic syndrome (MS). The primer and probe set of this invention includes primers for detecting MG and primers for detecting MS; the primers for detecting MG are shown in SEQ ID NO.1 and SEQ ID NO.2; the primers for detecting MS are shown in SEQ ID NO.3 and SEQ ID NO.4. The specific primer set and TaqMan probe of this invention enable rapid diagnosis of diseases caused by MG and MS, which is more efficient and faster than single-detection methods.

Description

Technical Field

[0001] This invention relates to the field of detection, and in particular to a TaqMan probe dual-fluorescent quantitative PCR primer and probe set, detection method and application for detecting MG and MS. Background Technology

[0002] Mycoplasma gallisepticum (MG), also known as chicken septicemic mycoplasma, primarily causes chronic respiratory disease in chickens, characterized by runny nose, cough, conjunctivitis, and air sacculitis. Mycoplasma synoviae (MS) can infect the synovial bursa membrane and tendon sheath of chicken joints, causing exudative synovitis, air sacculitis, genital tract inflammation, tenosynovitis, and swelling of the joint appendages and footpads. MG and MS infections are widespread worldwide, seriously harming the poultry industry. MG and MS are prone to co-infection with Escherichia coli (E. coli), Newcastle disease virus (NDV), and infectious bronchitis virus (IBV), and the respiratory symptoms and pathological changes they cause are similar, making direct diagnosis based on clinical symptoms alone difficult. Therefore, in actual diagnosis, it is usually necessary to combine pathogen isolation, serological or molecular biological detection for accurate diagnosis.

[0003] Laboratory detection methods for MS and MG mainly include bacterial isolation and identification, serological testing, and PCR identification. Bacterial isolation and identification is the gold standard for mycoplasma diagnosis; however, it suffers from a long processing time and a high risk of false negatives, failing to meet the needs of rapid diagnosis. Serological testing can detect large numbers of samples and is simple to perform, but it is prone to cross-reactivity and has relatively poor sensitivity. PCR identification includes conventional PCR and quantitative real-time PCR. Conventional PCR has higher sensitivity and specificity than other traditional methods, but it still suffers from a long testing process, is time-consuming, and has relatively low sensitivity. Especially in the early stages of the disease, when the bacterial load in the body is relatively low, conventional multiplex PCR may miss detections, failing to accurately diagnose early infection.

[0004] Multiplex PCR is a PCR technique that simultaneously amplifies different regions of a template or multiple target fragments in a single reaction. It is characterized by its speed, high accuracy, and good specificity. TaqMan probe-based real-time quantitative PCR cleverly utilizes the advantages of efficient DNA amplification in PCR, the high specificity of TaqMan probes with DNA templates, and the sensitivity and quantitative analysis capabilities of spectral techniques. It overcomes the shortcomings of conventional PCR qualitative detection, such as poor specificity and low sensitivity, and is currently used in the detection of various bacterial and viral diseases.

[0005] Callison et al. established a qPCR detection method using the MG pMGA gene with a single probe, achieving a sensitivity of 25 plasmid copies. Raviv et al. used MS ISR... b A single-probe qPCR detection method was established using the MG 16S rRNA and MS 16S rRNA genes, with sensitivities of 27 and 28 DNA copies / reaction, respectively. Sprygin et al. established a dual-probe qPCR detection method using the MG mgc2 and MS vlha genes, with sensitivities of 34 and 29 DNA copies / reaction, respectively. Summary of the Invention

[0006] Objective of the Invention: To more sensitively detect both MG and MS simultaneously, this invention provides a TaqMan probe-based dual real-time PCR primer and probe set, detection method, and applications for detecting MG and MS. Based on the MG0586 and MS0151 gene sequences registered in GenBank, this invention designs specific primer sets and TaqMan probes. The detection method using this probe set is more efficient and faster than single detection methods, enabling rapid diagnosis of diseases caused by MG and MS.

[0007] Technical solution: The TaqMan probe dual real-time PCR primer and probe set for detecting MG described in this invention includes primers for detecting MG and a TaqMan probe. The nucleotide sequences of the primers for detecting MG are shown in SEQ ID NO.1 and SEQ ID NO.2; the nucleotide sequence of the TaqMan probe is shown in SEQ ID NO.5.

[0008] In one embodiment of the present invention, the fluorescent group and the quenching group of the TaqMan probe are the FAM group and the BHQ1 group, respectively.

[0009] The TaqMan probe dual real-time PCR primer and probe set for detecting MS according to the present invention includes primers for detecting MS and TaqMan probes; the nucleotide sequences of the primers for detecting MS are shown in SEQ ID NO.3 and SEQ ID NO.4; the nucleotide sequence of the TaqMan probes is shown in SEQ ID NO.6.

[0010] In one embodiment of the present invention, the fluorescent group and the quenching group of the TaqMan probe are the JOE group and the BHQ1 group, respectively.

[0011] The TaqMan probe dual real-time PCR primer and probe set for detecting MG and MS described in this invention includes primers for detecting MG and primers for detecting MS; the primers for detecting MG are shown in SEQ ID NO.1 and SEQ ID NO.2; the primers for detecting MS are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0012] As one embodiment of the present invention, the primer and probe set of the present invention further includes a TaqMan probe for detecting MG and a TaqMan probe for detecting MS; the sequence of the TaqMan probe for detecting MG is shown in SEQ ID NO.5, wherein the fluorescein group and the quencher group are FAM group and BHQ1 group, respectively; the sequence of the TaqMan probe for detecting MS is shown in SEQ ID NO.6, wherein the fluorescein group and the quencher group are JOE group and BHQ1 group, respectively.

[0013] The method for detecting MG and MS based on TaqMan probe dual real-time PCR described in this invention has the following reaction system: 10 μL Premix Ex Taq (Probe qPCR), 1 μL each of forward and reverse primers (10 μmol / L, SEQ ID NO. 1-4), 0.5 μL each of probe (10 μmol / L, SEQ ID NO. 5-6), 2 μL each of template, and ddH2O to a final volume of 20 μL.

[0014] As one embodiment of the present invention, the amplification method is as follows: one cycle is 95℃ for 10s and 55℃ for 30s, and 45 cycles are performed.

[0015] As one embodiment of the present invention, the following pretreatment is performed before amplification: digestion at 37°C for 2 min; pre-denaturation at 95°C for 5 min.

[0016] The method for detecting MG and MS based on TaqMan probe dual-fluorescence quantitative PCR described in this invention has the following reaction system:

[0017] Premix Ex Taq (Probe qPCR) 10 μL; Primer system: MG0586F (SEQ ID NO.1): 10 μmol / L, 1 μL; MG0586R (SEQ ID NO.2): 10 μmol / L, 1 μL; MS0151F (SEQ ID NO.3): 10 μmol / L, 1 μL; MS0151R (SEQ ID NO.4): 10 μmol / L, 1 μL; Probe system: MG0586 probe (SEQ ID NO.5): 10 μmol / L, 0.5 μL; MS0151 probe (SEQ ID NO.6): 10 μmol / L, 0.5 μL; Template (MG): 2 μL; Template (MS): 2 μL; ddH2O to 20 μL.

[0018] The amplification program was as follows: digestion at 37℃ for 2 min; pre-denaturation at 95℃ for 5 min; amplification at 95℃ for 10 s; and amplification at 55℃ for 30 s for 45 cycles.

[0019] Unless otherwise stated, "%" in this invention refers to mass percentage concentration.

[0020] Beneficial effects: (1) This invention establishes a primer and probe set for TaqMan probe real-time fluorescence quantitative PCR that can simultaneously detect two pathogens, MG and MS. The detection limit for pure bacterial cultures is within 3 DNA copies / reaction range; (2) The detection method established by the specific primers and probes of this invention targeting highly conserved sequences of MG and MS can detect MG and MS with a sensitivity of 3 DNA copies, which is more sensitive than ordinary PCR and 1000 times more sensitive than ordinary PCR. It can simultaneously detect and quantify MG and MS, and can complete the detection of pathogens in one reaction. Moreover, this detection method can be used for single or double reactions depending on the actual situation; (3) The detection method of the present invention is more efficient and faster than the single detection method, and has a wide range of application value for the rapid diagnosis of diseases caused by MG and MS, as well as the safety monitoring of poultry flocks; (4) The detection method of the present invention has a short reaction time, and the detection results can be obtained in 1 hour. Moreover, the fluorescence signal can be directly observed in real time through a computer connected to the fluorescence quantitative PCR amplification instrument. Compared with ordinary multiplex PCR, this reaction does not require electrophoresis, ultraviolet light observation, etc., saving time and making the operation simple. It is more important for monitoring a large number of samples during sudden outbreaks. Attached Figure Description

[0021] Figure 1 Figure A shows the standard curves for dual-fluorescence quantitative PCR; Figure B shows the standard curve for MG genomic DNA and Figure C shows the standard curve for MS genomic DNA.

[0022] Figure 2Figure A shows the amplification curves of the primers and probes; Figure A shows the amplification curves of the MG0586 primer and probe (amplification curves of each isolate for the MG primer and probe), 1-6: MG(R low , FBH, 08F8, SS, 013, SGN); 7-19: MS (WVU 1853 Genomic DNA of JS1, HB1, SH1, SD1), MI, E. coli, SP, SA, cDNA of IBV, NDV, AIV, and negative control (ddH2O) amplification curves; Figure B shows the amplification curves of MS0151 primer and probe (amplification curves of primers and probes for each isolate), 1-5: MS (WVU) 1853 , JS1, HB1, SH1, SD1); 6-19: MG(R low Genomic DNA of FBH, 08F8, SS, 013, SGN), MI, E. coli, SP, SA, cDNA of IBV, NDV, AIV, and negative control (ddH2O) amplification curves.

[0023] Figure 3 Figure A shows the sensitivity detection results of the dual quantitative PCR system; Figure A shows the sensitivity detection results of dual quantitative PCR for MG genomic DNA, where 0–9 in Figure A represent 2.7 × 10⁻⁹ ppm respectively. 8 2.7×10 7 2.7×10 6 2.7×10 5 2.7×10 4 2.7×10 3 2.7×10 2 2.7×10 1 2.7×10 0 2.7×10 -1 Copy of MG R low Genomic amplification curve; 10: negative control (ddH2O) amplification curve; Figure B shows the sensitivity of dual-fluorescence quantitative PCR for MS genomic DNA detection, where 0–9 in Figure B represent 2.5 × 10⁻⁶ cells respectively. 8 2.5×10 7 2.5×10 6 2.5×10 5 2.5×10 4 2.5×10 3 2.5×10 2 2.5×10 1 2.5×10 0 2.5×10 -1 Copy of MS WVU 1853 Genome amplification curve; 10: Amplification curve of negative control (ddH2O).

[0024] Figure 4 The results represent the sensitivity detection results of conventional doublet PCR; where M is the DNA molecular weight standard (DL2000); 1–7 are 2.7 × 10⁻⁶ respectively. 7 2.7×10 6 2.7×10 5 2.7×10 4 2.7×10 3 2.7×10 2 2.7×10 1 Copy of MGR low Genome, 2.5 × 10 7 2.5×10 6 2.5×10 5 2.5×10 4 2.5×10 3 2.5×10 2 2.5×10 1 Copy of MS WVU 1853 Genome amplification bands. Detailed Implementation

[0025] I. Materials and Methods

[0026] 1.1 Microbial strains

[0027] Mycoplasma synovitis (MS) WVU 1853 Mycoplasma gallisepticum (MG)R low Mycoplasma Iowa (MI) strain 695, Staphylococcus aureus (SA) strain CVCC4098, and Salmonella pullorum (SP) strain CVCC519 were purchased from the China Institute of Veterinary Drug Control.

[0028] Escherichia coli (E. coli) O78 serotype strain E937, MS isolates JS1, HB1, SH1, and SD1 were isolated and preserved by the Animal Bacterial Infectious Diseases Team of the Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences. MG isolates FBH, 08F8, SS, 013, and SGN were kindly provided by Professor Sui Zhaofeng of Shandong Vocational College of Animal Husbandry and Veterinary Medicine. Newcastle disease virus (NDV) LaSota strain and avian influenza virus (AIV) CVCC AV1534 strain (H9N2) were purchased from the China Institute of Veterinary Drug Control. Infectious bronchitis virus (IBV) M41 strain was kindly provided by Professor Ding Chan's research group at the Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0029] 1.2 Reagents and Instruments

[0030] Bacterial genomic DNA extraction kit, viral DNA / RNA extraction kit, and tissue genomic DNA extraction kit were purchased from Beijing Tiangen Biotech Co., Ltd.; oral swab genomic DNA rapid extraction kit was purchased from Beijing Adley Biotechnology Co., Ltd.; HiFiScript 1st Strand cDNA Synthesis Kit was purchased from Beijing Convair Biotechnology Co., Ltd.; AceQ Universal U+Probe Master Mix V2 and 2×Rapid MasterMix were purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0031] Mycoplasma culture medium basal was purchased from Qingdao Haibo Biotechnology Co., Ltd.; horse serum and porcine serum were purchased from Hyclone, USA; nicotinamide adenine dinucleotide (NAD) was purchased from Roche; Tryptone, Yeast extract, and agar were purchased from Oxoid. Mycoplasma liquid complete culture medium: 26.4 g of mycoplasma basal culture medium was dissolved in 800 mL of deionized water, autoclaved for 15 min, cooled to room temperature, and 10% horse serum was aseptically added for MG culture (MS was cultured with 10% porcine serum and 0.01% NAD). LB liquid culture medium: 8 g of Tryptone, 4 g of Yeast extract, and 8 g of NaCl were dissolved in 800 mL of deionized water, autoclaved, and used for later use.

[0032] Main instruments: Real-time PCR instrument (Eppendorf); Micro Spectrophotometer (NanoDrop).

[0033] 1.3 Primer and Probe Design

[0034] Homology alignment analysis was performed on all MG and MS gene sequences included in GenBank to identify their conserved genes. The conserved MG gene MG0586 (GenBank accession number AE015450.2) and the conserved MS gene MS0151 (GenBank accession number CP011096.1) were selected, and specific primers and fluorescent probes for each gene were designed using Primer 5.0 software (Table 1), which were then sent to Shanghai Ruimian Biotechnology Co., Ltd. for synthesis.

[0035] Table 1 Primer and probe sequences

[0036]

[0037]

[0038] 1.4 Nucleic acid extraction and template preparation

[0039] Different MG isolates (R) low FBH, 08F8, SS, 013, SGN), different MS isolates (WVU) 1853 Bacterial mycoplasma (Synthetic molecule JS1, HB1, SH1, SD1) and MI were inoculated into mycoplasma culture medium at a 5% transfer ratio and cultured at 37°C in a 5% (v / v) CO2 incubator for 24 to 48 hours. After the culture medium changed from red to orange-yellow, genomic DNA was extracted using a bacterial genomic DNA extraction kit, following the instructions. E. coli, SP, and SA were inoculated into LB liquid medium at a 1% transfer ratio and cultured at 37°C in a shaker at 220 rpm for 12 hours. Genomic DNA was then extracted using a bacterial genomic DNA extraction kit, following the instructions.

[0040] IBV, NDV, and AIV were inoculated into 9-10 day old chicken embryos. After 48 hours, the allantoic fluid of the chicken embryos was collected, centrifuged for 10 minutes (4℃, 8000 rpm), filtered through a 0.22 μm filter for sterilization, and viral RNA was extracted using a viral DNA / RNA extraction kit and reverse transcribed into cDNA.

[0041] Preparation of clinical sample templates: Chicken throat swabs and lung tissue were collected; genomic DNA was extracted from throat swab samples using an oral swab genomic DNA rapid extraction kit, and genomic DNA was extracted from lung tissue samples after grinding using a tissue genomic DNA extraction kit. The extraction methods were all performed in accordance with the instructions.

[0042] The concentration of the extracted genomic DNA was determined using a micro spectrophotometer and stored at -20°C for later use.

[0043] 1.5 Preparation of Genomic Standards

[0044] Extract MG R low strains and MS WVU 1853 Genomic DNA from the strain was serially diluted 10-fold using ddH2O to prepare genomic standards. MG R low Genome diluted to 2.7 × 10⁻⁶ -1 ~2.7×10 8 Copy / reaction, MS WVU 1853 Genome diluted to 2.5 × 10⁻⁶ -1 ~2.5×10 8 Copy / react, store at -20℃ for later use.

[0045] 1.6 Establishment of a dual-fluorescence quantitative PCR reaction system

[0046] First, using a standard system: 10 μL of AceQ Universal U+Probe Master Mix V2, 0.4 μL each of forward and reverse primers (10 μmol / L), 0.2 μL each of probe (10 μmol / L), 2 μL each of template, and ddH2O to a final volume of 20 μL. Amplification was performed using a real-time PCR instrument according to the following program: digestion at 37℃ for 2 min (this step eliminates the influence of amplification products on qPCR); pre-denaturation at 95℃ for 5 min, followed by amplification at 95℃ for 10 s and 60℃ for 30 s for 45 cycles.

[0047] Annealing temperatures were optimized at six gradients: 53℃, 55℃, 57℃, 59℃, 61℃, and 63℃. After determining the optimal annealing temperature, primer and probe concentrations were further optimized by diluting both primers and probes to 10 μm / L. Different combinations of primer and probe amounts were used, as shown in Table 2. The cycle threshold (Ct) for each concentration gradient was compared, and the lowest Ct value was selected as the optimal primer, probe concentration, and annealing temperature. Fluorescence signal detection was performed at the end of each extension cycle. The selected fluorescence channels were FAM and JOE. Table 2 shows the primer and probe amounts.

[0048]

[0049] 1.7 Establishment of the Standard Curve

[0050] MG R low MS WVU 1853 The genome was serially diluted 10-fold. Using the diluted genome as a template, quantitative real-time PCR amplification was performed under optimized reaction conditions. The results were analyzed using Excel software to determine the relationship between copy number and Ct value. A standard curve was plotted with copy number on the x-axis and Ct value on the y-axis.

[0051] 1.8 Conservatism and Specificity of Dual-Fluorescence Quantitative PCR Detection Method

[0052] Using MG isolates (R) low FBH, 08F8, SS, 013, SGN), MS isolates (WVU) 1853 Genomic DNA from strains JS1, HB1, SH1, SD1), MI, E. coli, SP, and SA, and cDNA from IBV, NDV, and AIV were used as templates. An optimized real-time PCR reaction system was used for amplification to verify the conservation and specificity of the method.

[0053] 1.9 Sensitivity of Dual Real-Time PCR Detection Method

[0054] MG R low The genome has a size of 2.7 × 10⁻⁶.-1 Copy ~ 2.7×10 8 Copy, MS WVU 1853 The genome is 2.5 × 10 -1 Copy ~ 2.5 × 10 8 Using copies as templates, amplification was performed in an optimized real-time PCR reaction system. The relationship between template concentration and Ct value was analyzed using Excel software. A standard curve was plotted with copy number on the x-axis and Ct value on the y-axis. The detection sensitivity of the real-time PCR detection system was evaluated using trend lines and correlation coefficients. Simultaneously, the template was subjected to conventional MG / MS duplex PCR detection. The reaction system and procedure were: 10 μL of 2×Taq Master Mix, 1 μL each of forward and reverse primers (10 μmol / L), and MG R... low MS WVU 1853 2 μL of each genome sample was added, and ddH2O was added to bring the volume to 20 μL. Program: 95℃ pre-denaturation for 4 min, denaturation at 95℃ for 30 s, annealing at 56℃ for 30 s, extension at 72℃ for 45 s, and final extension at 72℃ for 10 min for 30 cycles.

[0055] 1.10 Reproducibility of the dual-fluorescence quantitative PCR detection method

[0056] MG R low MS WVU 1853 The genome sample was diluted to 3.9 × 10⁻⁶. 1 Copy ~ 3.9×10 3 For each dilution, three parallel reaction tubes were prepared and amplified using an optimized quantitative real-time PCR reaction system. The standard deviation and coefficient of variation (CV) of the Ct values ​​were calculated to perform intra-group repeatability tests. Template samples from the above dilution gradients were stored at -20℃ for 1 month, 2 months, and 3 months, respectively, and then subjected to quantitative real-time PCR amplification. The standard deviation and coefficient of variation (CV) of the Ct values ​​between each group were calculated to evaluate inter-group repeatability.

[0057] 1.11 Clinical Sample Testing

[0058] The established dual-fluorescence quantitative PCR method was used to extract genomes from 40 lung tissue samples and 50 throat swab samples from Suqin green-shelled chickens submitted by a chicken farm in Jiangsu Province, and the results were compared with those from conventional PCR methods.

[0059] II. Results

[0060] 2.1 Optimization of the reaction system and establishment of the standard curve

[0061] The optimized reaction system for quantitative real-time PCR was 20 μL, including 10 μL of AceQ Universal U+Probe Master Mix V2, 1 μL each of forward and reverse primers (10 μmol / L), 0.5 μL each of probe (10 μmol / L), 2 μL each of template, and ddH2O to a final volume of 20 μL. Amplification was performed using a quantitative real-time PCR instrument according to the following program: digestion at 37℃ for 2 min (this step eliminates the influence of amplification products on qPCR); pre-denaturation at 95℃ for 5 min, amplification at 95℃ for 10 s, and amplification at 55℃ for 30 s for 45 cycles. Based on the copy number in the reaction system and the amplified Ct value, the standard curves were calculated as follows: y MG = -3.7595l x + 40.355; y MS = -3.6953l x + 42.263. (x is the genomic DNA copy number, y is the Ct value) The correlation coefficients were 0.9971 and 0.9945, respectively, both greater than 0.99, indicating a good linear relationship (see results). Figure 1 ).

[0062] 2.2 Reaction Specificity

[0063] The established system was used for amplification, with the templates being MG isolate R. low FBH, 08F8, SS, 013 and SGN, MS isolate WVU 1853 Genomic DNA of strains JS1, HB1, SH1, and SD1, MI, E. coli, SP, and SA, as well as cDNA of IBV, NDV, and AIV, and a negative control (ddH2O), were collected. Results are shown in [link to results]. Figure 2 MG0586 amplification of various MG isolates ( Figure 2 Figure A), MS0151 amplification of each isolate ( Figure 2 (Figure B) The genome showed an S-shaped amplification curve after amplification, while other pathogen genomes and negative controls did not show an S-shaped amplification curve, indicating that the established dual-fluorescence quantitative PCR detection method has very high specificity.

[0064] 2.3 Reaction Sensitivity

[0065] MG R low The genome has a size of 2.7 × 10⁻⁶. -1 Copy ~ 2.7×10 8 Copy, MS WVU 1853 The genome is 2.5 × 10 -1 Copy / ~2.5×10 8 Using copies as templates for quantitative real-time PCR amplification, the Ct value increased with decreasing concentration, indicating a good linear relationship between Ct and concentration within the above dilution range. Results are shown in [Figure number missing]. Figure 3Within the Ct value range of <40, the minimum genomic DNA amount required for detecting MG and MS by this quantitative real-time PCR was 3 DNA copies / reaction. The above template was then subjected to conventional duplex PCR detection, and the results are shown below. Figure 4 The detection sensitivity of conventional PCR for both MG and MS is within 3 × 10⁻⁶. 3 For every DNA copy / reaction, the sensitivity of the present invention's real-time PCR method is 1000 times that of conventional PCR.

[0066] 2.4 Reaction repeatability

[0067] MG R low MS WVU 1853 After the genome was preserved for different periods, the coefficient of variation of the detected Ct values ​​was less than 5% (Table 3), indicating that the method has good stability and reproducibility.

[0068] Table 3 Coefficient of Variation

[0069]

[0070]

[0071] 2.5 Detection of clinical samples

[0072] The established dual-quantitative real-time PCR method was used to detect genomic DNA in 40 lung tissue samples and genomic DNA in 50 animal throat swab samples. The results were compared with those of dual conventional PCR (Table 4). Statistical analysis (Table 5) showed that the positive rate of MG in lung tissue samples was 42.5%, and the positive rate of MS was 35.0%, with mixed infection accounting for 15.0%. In throat swabs, the positive rate of MG was 76.0%, and the positive rate of MS was 10.0%, with mixed infection accounting for 8.0%. Dual conventional PCR results showed that only MG was positive in lung tissue samples (27.5%), with no positive results for MS; in throat swabs, the positive rate of MG was 54.0%, and the positive rate of MS was 2.0%, with no mixed infection results. This indicates that this method can be applied to the detection of clinical samples, and the positive detection rate is significantly higher than that of dual conventional PCR.

[0073] Table 4 Clinical Sample Detection Results

[0074]

[0075]

[0076]

[0077]

[0078] Table 5. Statistics on the positive rate of clinical samples

[0079]

[0080] The results above demonstrate that the dual quantitative PCR reaction established in this invention has a sensitivity within the range of 3 DNA copies, which is more sensitive than all currently established dual quantitative PCR methods. Therefore, this method is suitable for the clinical differential diagnosis of early MG and MS infections, and is of great significance for early detection, early diagnosis, early control, and reducing morbidity and mortality.

[0081] In summary, the MG and MS dual-fluorescence quantitative PCR based on TaqMan probes established in this invention has the characteristics of being rapid, sensitive, specific, accurate, quantitative, and real-time. It is of great significance for early and rapid diagnosis of infection by these two pathogens, effective prevention and control of disease outbreaks, prevention of pathogen spread, and promotion of the healthy development of the poultry industry. sequence list <110> Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences Yangzhou Youjiachuang Biotechnology Co., Ltd. <120> A TaqMan probe-based dual-fluorescence quantitative PCR primer and probe set, detection method, and application for detecting MG and MS. <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> twenty two <212> DNA <213> Artificial Sequence <400> 1 gacccaagta agcaagcaat tc 22 <210> 2 <211> twenty one <212> DNA <213> Artificial Sequence <400> 2 cgcatcacga gtagcagtat c 21 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <400> 3 gtactagatg ggttgattca agttat 26 <210> 4 <211> twenty two <212> DNA <213> Artificial Sequence <400> 4 attcagctgc tgtttgtgaa at 22 <210> 5 <211> 29 <212> DNA <213> Artificial Sequence <400> 5 accagtgcaa gagataagtt agatgctgc 29 <210> 6 <211> twenty four <212> DNA <213> Artificial Sequence <400> 6 agctgctgac gaaatgatag cgca 24

Claims

1. A TaqMan probe-based dual-fluorescence quantitative PCR primer and probe set for detecting MG and MS, characterized in that, The device includes primers for detecting MG and primers for detecting MS; the primers for detecting MG are shown in SEQ ID NO.1 and SEQ ID NO.2; the primers for detecting MS are shown in SEQ ID NO.3 and SEQ ID NO.4; it also includes TaqMan probes for detecting MG and TaqMan probes for detecting MS; the TaqMan probe sequence for detecting MG is shown in SEQ ID NO.5; the TaqMan probe sequence for detecting MS is shown in SEQ ID NO.

6.

2. The TaqMan probe dual-fluorescent quantitative PCR primer and probe set for detecting MG and MS according to claim 1, characterized in that, The fluorophore and quencher of the TaqMan probe used to detect MG are FAM and BHQ1 groups, respectively; the fluorophore and quencher of the TaqMan probe used to detect MS are JOE and BHQ1 groups, respectively.

3. A method for detecting MG and MS using TaqMan probe-based dual real-time PCR for non-disease diagnostic purposes, characterized in that, The device includes primers for detecting myoglobulin (MG) and for detecting stromal inflammatory syndrome (MS); the primers for detecting MG are shown in SEQ ID NO. 1 and SEQ ID NO. 2; the primers for detecting MS are shown in SEQ ID NO. 3 and SEQ ID NO. 4; it also includes TaqMan probes for detecting MG and for detecting MS; the TaqMan probe sequence for detecting MG is shown in SEQ ID NO. 5; the TaqMan probe sequence for detecting MS is shown in SEQ ID NO. 6; The reaction system was as follows: Premix Ex Taq: 10 μL, forward and reverse primers: 1 μL each, probe: 0.5 μL each, template: 2 μL each, and ddH2O added to a final volume of 20 μL.

4. The method according to claim 3, characterized in that, The amplification method is as follows: 45 cycles are performed, consisting of 95℃ for 10s and 55℃ for 30s.

5. The method according to claim 4, characterized in that, The following pretreatment was performed before amplification: digestion at 37℃ for 2 min; pre-denaturation at 95℃ for 5 min.

6. The application of the primer-probe set as described in claim 1 in the detection of MG and MS for non-disease diagnostic purposes.

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