CN116640867B - A primer pair and probe combination for detecting Streptococcus dysgalactiae by RAA-LFD and its application - Google Patents

Abstract

The invention provides a primer pair and probe combination for detecting streptococcus dysgalactiae by RAA-LFD and application thereof, belonging to the technical field of pathogenic bacteria detection. In the primer pair and the probe for detecting streptococcus agalactiae, the upstream primer sequence is shown as SEQ ID NO: 1-3, the sequence of the downstream primer is shown as SEQ ID NO: 4-5, the probe sequence is shown as SEQ ID NO:6 is shown in the figure; the primer pair and the probe combination can be used for accurately detecting the streptococcus agalactiae, the minimum detection limit of the primer pair and the probe combination on the streptococcus agalactiae is 1.002 multiplied by 10 ² pg/mu L, and the primer pair and the probe combination have the advantages of short detection time, easy achievement of reaction temperature, strong specificity, high sensitivity, high detection result coincidence rate and the like, have low technical requirements on equipment and personnel operation, and are convenient for carrying out the detection of the streptococcus agalactiae under the condition of lacking laboratory detection equipment.

Description

A primer pair and probe combination for detecting Streptococcus dysgalactiae by RAA-LFD and its application

Technical Field

The invention belongs to the technical field of pathogenic bacteria detection, and specifically relates to a primer pair and a probe combination for detecting Streptococcus dysgalactiae by RAA-LFD and an application thereof.

Background Art

Streptococcus dysgalactiae can not only cause mastitis, polyarthritis, sepsis and other diseases in a variety of animals such as cattle, pigs and sheep, but can also infect humans, causing bacteremia, endocarditis, meningitis, septic arthritis, respiratory and skin infections and other diseases. Streptococcus dysgalactiae is an emerging pathogen in aquaculture. It can cause serious diseases in many farmed fish species, including channel catfish, greater amberjack (Seriola dumerili), yellowtail amberjack (Seriola quinqueradiata), yellowtail amberjack (Seriola lalandi), pomfret (Trachinotus blochii), white snapper (Lutjanus stellatus), hybrid red tilapia (Oreochromis sp.), Nile tilapia (Oreochromis niloticus), mullet (Mugil cephalus), cobia (Rachycentron canadum) and grey mullet (Liza haematocheila) as well as different species of farmed sturgeon (Acipenser baerii, Acipenser schrenckii and Acipenser gueldenstaedti), posing a huge threat to aquaculture.

The gene sequence of immunogenic secreted protein (ISP) is highly conserved, and the protein it encodes has strong immunogenicity and is the main antigen of Streptococcus dysgalactiae. Studies have shown that PCR with specific primers designed based on the ISP gene of Streptococcus dysgalactiae can amplify the target band. The sequencing results of its PCR amplification product were compared with the corresponding sequence in GenBank, and the sequence homology was analyzed to be 94.2% to 100%, proving that the ISP gene is a highly conserved sequence of Streptococcus dysgalactiae, and primers can be designed based on the ISP gene to detect Streptococcus dysgalactiae.

Recombinase-aided amplification (RAA) is an amplification technology that does not require thermostable enzymes and complex thermal cyclers. This technology uses single strand DNA binding protein (SSB), recombinase and DNA polymerase to complete the reaction. At present, isothermal amplification technologies are gradually increasing. Different isothermal amplification technologies are designed according to different reaction principles. Commonly used isothermal amplification technologies include: nuclear acid sequence-based amplification detection technology (NASBA), loop-mediated isothermal amplification method (LAMP) and recombinase-mediated isothermal amplification technology (RAA). However, NASBA requires preheating; LAMP requires the design of 4 to 6 pairs of primers; and NASBA and LAMP require a higher reaction temperature. RAA has been widely used in pathogenic microorganism detection, disease diagnosis and other fields in recent years due to its advantages such as high efficiency, good sensitivity and short time. The application of RAA-LFD technology in bacterial detection is slightly later than that in virus detection. However, the bacterial genome is DNA nucleic acid, which does not require reverse transcription compared to some RNA viruses. It is more conducive to being out of the laboratory environment and applied to on-site testing.

At present, there is a lack of a simple, effective, sensitive and low technical requirement method for detecting Streptococcus dysgalactiae. Therefore, a specific primer is provided for the detection of Streptococcus dysgalactiae, which has the advantages of easy reaction temperature, short reaction time, strong specificity, high sensitivity and high consistency of test results. The method for detecting Streptococcus dysgalactiae is of great significance in the field of pathogenic bacteria detection.

Summary of the invention

In view of the above-mentioned deficiencies in the prior art, the present invention provides a primer pair and a probe combination for detecting Streptococcus dysgalactiae and an application thereof. The primer pair and the probe of the present invention are used for RAA-LFD detection, which has the advantages of easy reaction temperature, short reaction time, strong specificity, high sensitivity and high detection result compliance rate. In addition, the present invention has low requirements on the operating technology of equipment and test personnel, and is suitable for detecting Streptococcus dysgalactiae in the field or in scenarios where there is a lack of laboratory testing equipment.

The present invention adopts the following technical solutions:

A primer pair and a probe for detecting Streptococcus dysgalactiae by RAA-LFD, wherein the primer pair is: ISP-F1/ISP-R1, ISP-F1/ISP-R2, ISP-F2/ISP-R1, ISP-F2/ISP-R2, ISP-F3/ISP-R1 or ISP-F3/ISP-R2; wherein the nucleotide sequences of ISP-F1, ISP-F2, ISP-F3, ISP-R1 and ISP-R2 are as follows:

ISP-F1: 5’-AATTTAAATCAGAGGCAAAAGGTGACCACGGAA-3’ (SEQ ID NO. 1);

ISP-F2: 5’-AATGCTCCACATAGGCCGCATAAGTATTAACAGA-3’ (SEQ ID NO. 2);

ISP-F3: 5’-TCCCAAAGAACTTTCTGACATGGCAATAGCAAC-3’ (SEQ ID NO. 3);

ISP-R1: 5’-ATCTGATGTTCTTAATCCTGCAGAGCCTACTCG-3’ (SEQ ID NO. 4);

ISP-R2: 5’-GATGTTCTTAATCCTGCAGAGCCTACTCGTCCA-3’ (SEQ ID NO. 5);

The nucleotide sequence of the probe is:

5'-TATCTCCAAATTTAAATCAGAGGCAAAAGGT GACCACGGAAGAGGTGT-3' (SEQ ID NO. 6).

Furthermore, the 5' end of the downstream primer is labeled with biotin, the 5' end of the probe is labeled with fluorescein, the 3' end is labeled with C ₃ Spacer, and at 30 bp of the probe, the base T is replaced with dSpacer;

The probe is:

5’-FAM-TATCTCCAAATTTAAATCAGAGGCAAAAGG/dSpace

r/GACCACGGAAGAGGTGT(C ₃ Spacer)-3'.

Application of the primer pair and probe combination in detecting Streptococcus dysgalactiae.

A kit for detecting Streptococcus dysgalactiae comprises the above primer pair and probe.

A method for detecting Streptococcus dysgalactiae comprises the following steps: extracting DNA of a sample to be tested, performing RAA amplification using the primer pair and probe combination, and detecting the RAA amplification product using LFD.

Furthermore, the RAA amplification system is: 23-25 μL of tris buffer, 1.8-2.2 μL of 10 μM forward primer, 1.8-2.2 μL of 10 μM reverse primer, 0.5-0.7 μL of 10 μM probe, and 17-17.3 μL of the sample DNA to be tested and ddH ₂ O; preferably, 25 μL of buffer, 2.1 μL of 10 μM forward primer, 2.1 μL of 10 μM reverse primer, 0.6 μL of 10 μM probe, and 17.2 μL of the sample DNA to be tested and ddH ₂ O.

Furthermore, the above RAA amplification procedure is 39°C to 44°C for 15 to 30 min; preferably 39°C for 15 min.

By adopting the above technical solution, the beneficial effects of the present invention are as follows:

The invention provides a primer and probe combination for detecting Streptococcus dysgalactiae by RAA-LFD and its application. The method of the invention designs primers and probes based on the ISP gene sequence of Streptococcus dysgalactiae at the same time to reduce the probability of false positive, thereby improving the feasibility of the successful RAA-LFD detection method; by repeatedly optimizing the reaction system and reaction conditions, it is determined that the optimal reaction temperature is 39°C and the minimum reaction time is 15min. The invention has good specificity and the minimum detection sensitivity can reach 1.002×10 ² pg/μL. The RAA-LFD method established by the invention and the traditional PCR method are used to detect 50 zebrafish samples infected with poison. The results show that the positive coincidence rate of the two methods is 97.6%, the negative coincidence rate is 90.0%, and the total coincidence rate is 98.0%. Compared with the traditional PCR method, the RAA-LFD method has an area under the ROC curve (AUC) of 0.950, and the sensitivity and specificity are 100% and 90% respectively. Therefore, the present invention adopts the primers and probes designed simultaneously based on the ISP gene sequence of Streptococcus dysgalactiae in combination with the RAA-LFD detection method, which has the advantages of easy reaction temperature, short reaction time, strong specificity, high sensitivity and high compliance rate of detection results, and has low requirements on the operating skills of equipment and test personnel, and is suitable for the detection of Streptococcus dysgalactiae in scenarios where there is a lack of laboratory testing equipment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a screening diagram of the RAA-LFD primer pair for Streptococcus dysgalactiae.

FIG. 2 is a screening diagram of RAA-LFD primer pairs of Streptococcus dysgalactiae at different temperatures.

FIG. 3 is a sensitivity analysis diagram of the RAA-LFD primer pair of Streptococcus dysgalactiae.

FIG. 4 is a schematic diagram of RAA-LFD detection of Streptococcus dysgalactiae.

FIG. 5 is a diagram showing the optimization of reaction conditions for detecting Streptococcus dysgalactiae using the RAA-LFD method.

FIG. 6 is a graph showing the sensitivity and specificity of the RAA-LFD method for detecting Streptococcus dysgalactiae.

FIG. 7 is a test strip image of RAA-LFD testing clinical samples.

FIG8 is a gel electrophoresis diagram of PCR detection of clinical samples.

FIG. 9 is a ROC analysis diagram of RAA-LFD detecting Streptococcus dysgalactiae.

DETAILED DESCRIPTION

The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

Example 1 Primer design and screening

1. Primer design

According to the ISP gene detected by Streptococcus dysgalactiae, the homology of the ISP gene sequence was analyzed by DNAMAN and the conserved region was determined. Oligo 7 software was used to design a total of 6 pairs of primers and a probe based on the conserved region. The primer pairs were ISP-F1/ISP-R1, ISP-F1/ISP-R2, ISP-F2/ISP-R1, ISP-F2/ISP-R2, ISP-F3/ISP-R1 and ISP-F3/ISP-R2; among them, the nucleotide sequences of ISP-F1, ISP-F2, ISP-F3, ISP-R1 and ISP-R2 are as follows:

ISP-F1: 5’-AATTTAAATCAGAGGCAAAAGGTGACCACGGAA-3’ (SEQ ID NO. 1);

ISP-F2: 5’-AATGCTCCACATAGGCCGCATAAGTATTAACAGA-3’ (SEQ ID NO. 2);

ISP-F3: 5’-TCCCAAAGAACTTTCTGACATGGCAATAGCAAC-3’ (SEQ ID NO. 3);

ISP-R1: 5’-ATCTGATGTTCTTAATCCTGCAGAGCCTACTCG-3’ (SEQ ID NO. 4);

ISP-R2: 5’-GATGTTCTTAATCCTGCAGAGCCTACTCGTCCA-3’ (SEQ ID NO. 5);

The nucleotide sequence of the probe is

5'-TATCTCCAAATTTAAATCAGAGGCAAAAGGT GACCACGGAAGAGGTGT-3' (SEQ ID NO: 6).

2. Primer screening

The PCR method was used for primer screening. The PCR reaction system and PCR reaction operating parameters are shown in Table 1 and Table 2. The following PCR reactions all used the PCR reaction system and PCR reaction operating parameters in Table 1 and Table 2.

Table 1 PCR reaction system

Table 2 PCR reaction operating parameters

(1) Preliminary screening: Using 2 μL of Streptococcus dysgalactiae DNA as a template, PCR reactions were performed using the 6 pairs of specific primers designed according to the ISP gene of Streptococcus dysgalactiae in step 1, and the results were observed by 1% agarose gel electrophoresis. The results are shown in FIG1 , wherein M is a DNA molecular mass standard; NC is a negative control; 1 is Streptococcus dysgalactiae; 2 is Streptococcus dolphinus; and 3 is Streptococcus agalactiae. The target bands were all amplified by 1% agarose gel electrophoresis.

(2) Screening the optimal temperature: Using 2 μL of Streptococcus dysgalactiae DNA as a template, PCR was performed using the six pairs of specific primers designed according to the ISP gene of Streptococcus dysgalactiae in step 1 at 38.2°C, 40.9°C, 44.7°C, 49.5°C and 53.5°C, respectively. The results were observed by 1% agarose gel electrophoresis. The results are shown in FIG2 , wherein M is a DNA molecular mass standard; NC is a negative control; 1 is ISP-F1/ISP-R1; 2 is ISP-F1/ISP-R2; 3 is ISP-F2/ISP-R1; 4 is ISP-F2/ISP-R2; 5 is ISP-F3/ISP-R1; and 6 is ISP-F3/ISP-R2. The target bands were all amplified by 1% agarose gel electrophoresis, and the brightness of the bands was almost the same;

(3) Primer concentration screening: The DNA of Streptococcus dysgalactiae was diluted 10 times in succession to concentrations of 1.613×10 ³ ng/μL, 1.613×10 ² ng/μL, 1.613×10 ¹ ng/μL, 1.613×10 ⁰ ng/μL, 1.613×10 ² pg/μL, 8.065×10 ¹ pg/μL and 4.033×10 ¹ pg/μL, respectively. The diluted DNA of Streptococcus dysgalactiae was used as a template and the primers obtained in step 1 were used for PCR reaction. The results were observed by 1% agarose gel electrophoresis. The results are shown in FIG3 . The target bands were all amplified by 1% agarose gel electrophoresis. When the DNA concentration was lower than 1.613×10 ² pg/μL, the brightness of the six specific target bands was weakened.

It can be seen that the six pairs of specific primers designed based on the ISP gene of Streptococcus dysgalactiae all meet the requirements.

Example 2 Establishment of RAA-LFD detection method

1. Screening primers:

ISP-F2/ISP-R2 designed in Example 1 were randomly selected as primers, and DNA of Streptococcus dysgalactiae was used as a template. PCR amplification was performed according to the reaction system and reaction procedure in Table 1 and Table 2, and the amplified product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and the sequencing results were compared with the corresponding sequences in GenBank to analyze the sequence homology. The results are shown in Table 3;

Table 3 Sequence homology comparison using MEGA 7.0 and BioEdit

As shown in Table 3, the sequence homology is 94.2% to 100%, so ISP-F2/ISP-R2 is selected to detect the ISP gene of Streptococcus dysgalactiae, and biotin is labeled at the 5′ end of its downstream primer, named ISP-RAA-LR; carboxyfluorescein (FAM) is labeled at the 5′ end of the probe, and C ₃ Spacer is labeled at the 3′ end. At 30 bp of the probe, the base T is replaced by dSpacer, named ISP-Probe, and used for LFD band detection of subsequent amplification products, as shown below:

ISP-RAA-LF:

5’-AATGCTCCACATAGGCCGCATAAGTATTAACAGA-3’;

ISP-RAA-LR:

5’-BiotinGATGTTCTTAATCCTGCAGAGCCTACTCGTCCA-3’;

ISP-Probe:

5’-FAMTATCTCCAAATTTAAATCAGAGGCAAAAGG/dSpace r/GACCACGGAAGAGGTGT(C3Spacer)-3’.

2. Establishment of RAA-LFD detection system and method for Streptococcus dysgalactiae

The establishment of the RAA-LFD detection system and method for Streptococcus dysgalactiae is shown in Figure 4, and the specific process is as follows:

(1) Take out the reagents required for the test stored at -20°C and melt them to room temperature, mix them on a vortex oscillator, and centrifuge them instantly using a handheld centrifuge; open the metal water bath in advance and adjust the temperature to 39°C. The required reagents are shown in Table 4;

(2) Prepare a reaction premix with the required reagents prepared in step (1), mix thoroughly and centrifuge briefly. The system of the reaction premix is shown in Table 4;

(3) Transfer 47 μL of the reaction premix prepared in step (2) to a reaction tube containing 2.5 mg of RAA lyophilized powder, dissolve the lyophilized powder fully, and centrifuge briefly;

(4) Add 3 μL of the initiator (magnesium acetate solution) to the reaction tube obtained in step (3), cap the tube tightly and centrifuge briefly to allow the initiator to enter the premixed solution, mix well, and centrifuge briefly;

(5) Perform RAA amplification by placing the reaction tube obtained in step (4) into the water bath prepared in step (1) and reacting for 20 min;

(6) After the reaction is completed, the amplified product obtained in step (5) is diluted 10 times with PBS buffer;

(7) Take out the required number of LFD test strips and mark the absorbent pads of the LFD test strips;

(8) Take out a corresponding number of PCR centrifuge tubes, mark them, add 100 μL of the diluted reactant in step (6) into the centrifuge tubes, and mix by pipetting;

(9) placing the test strip obtained in step (7) into a centrifuge tube with the sample point facing downward and leaving it to stand for 5 to 10 minutes;

(10) The test results were interpreted in a place with sufficient natural light. No strips or only the test line (T line) appeared, which was considered an invalid result; if only the quality control line (C line) appeared, it was considered a negative result; if both the quality control line (C line) and the test line (T line) appeared, it was considered a positive result. The following RAA-LFD tests were performed according to the above established method.

Table 4 RAA reaction system

3. Optimization of RAA-LFD reaction conditions

(1) Optimization of the optimal reaction temperature: Using Streptococcus dysgalactiae DNA as a template, using ISP-RAA-LF: F and ISP-RAA-LR: R obtained in 1 as primers, and using ISP-Probe as a probe, the RAA-LFD detection established in 2 was performed at different temperatures. Five groups of temperature gradients were set, namely 29°C, 34°C, 39°C, 44°C and 49°C. An equal amount of ddH ₂ O was added to replace the negative reaction as a negative control. The results are shown in Figure 5, where NC is a negative control.

As shown in Figure 5A, within the temperature range of 39°C to 44°C, the RAA amplification products were all detected by the test strips, and the brightness of the band on the test strip detection line T line showed a trend of first increasing and then decreasing with the increase of temperature. When the temperature was lower than 39°C or higher than 44°C, the brightness of the band on the test strip T line was not obvious; as shown in Figure 5B, there was no significant difference in the gray value of the T line analyzed by ImageJ software (P>0.05). In order to make the detection temperature closer to the ambient temperature, 39°C was selected as the optimal reaction temperature;

(2) Optimizing the best reaction time: Using Streptococcus dysgalactiae DNA as a template, using ISP-RAA-LF: F and ISP-RAA-LR: R obtained in 1 as primers, and using ISP-Probe as a probe, the reaction time was optimized at 39°C. RAA-LFD detection was performed at 5 min, 10 min, 15 min, 20 min, 25 min and 30 min, respectively. An equal amount of ddH ₂ O was added to replace the negative reaction as a negative control. The experimental results were observed after the reaction was carried out for 5 min. The experimental results are shown in Figure 5C;

As shown in Figure 5C, the test strip detection line T line initially showed a weak bright band, and as the reaction time increased, the detection line gradually brightened; as shown in Figure 5D, when the reaction time was 15min, 20min, 25min and 30min, respectively, there was no significant difference in the gray value of the T line (P>0.05). In order to ensure the rapidity of the experiment, 15min was selected as the optimal reaction time for RAA.

4. Analysis of sensitivity and specificity of RAA-LFD method for detecting Streptococcus dysgalactiae

(1) Analysis of the sensitivity of RAA-LFD method for detecting Streptococcus dysgalactiae: Using different concentrations of Streptococcus dysgalactiae DNA as template, ISP-F2/ISP-R2 primers were selected for PCR amplification, and the reaction procedures and reaction systems are shown in Tables 1 and 2; ISP-RAA-LF and ISP-RAA-LR obtained in 1 were used as primers for RAA-LFD detection, and the results are shown in Figure 6, where M is the DNA molecular mass standard; NC is the negative control; 1 is 1.002×10 ³ ng/μL; 2 is 1.002×10 ² ng/μL; 3 is 1.002×10 ¹ ng/μL; 4 is 1.002×10 ⁰ ng/μL; 5 is 1.002×10 ² pg/μL; 6 is 1.002×10 ¹ pg/μL; 7 is 1.002×10 ⁰ pg/μL; a is Streptococcus dysgalactiae; b is Streptococcus agalactiae; c is Streptococcus iniae; d is Staphylococcus aureus; e is Vibrio mimicus; f is Aeromonas hydrophila; g is Edwardsiella;

As shown in Figure 6A, when the DNA concentration of Streptococcus dysgalactiae is 1.002×10 ³ ng/μL～1.002×10 ⁰ ng/μL, the target band is bright, indicating that the minimum detection limit of PCR amplification is 1.002×10 ⁰ ng/μL; as shown in Figure 6B, when the DNA concentration of Streptococcus dysgalactiae decreases, the brightness of the T line of the test strip gradually weakens. When the DNA concentration of Streptococcus dysgalactiae is lower than 1.002×10 ² pg/μL, the brightness of the band on the T line is not obvious; as shown in Figure 6C, there is no significant difference in the gray value of the T line analyzed by ImageJ software (P>0.05). Therefore, the minimum detection limit of RAA-LFD method for Streptococcus dysgalactiae is 1.002×10 ² pg/μL, and the genome sensitivity detected by RAA-LFD method is higher than that of traditional PCR method.

(2) Analysis of the specificity of RAA-LFD method for detecting Streptococcus dysgalactiae: DNA of Streptococcus dysgalactiae, Streptococcus agalactiae, Streptococcus iniae, Staphylococcus aureus, Vibrio mimicus, Aeromonas hydrophila and Edwardsiella pneumoniae were used as templates, and ISP-RAA-LF:F and ISP-RAA-LR:R: obtained in 1 were used as primers to perform RAA-LFD detection, respectively, to verify the specificity of this method for detecting Streptococcus dysgalactiae. The results are shown in FIG6D .

As shown in Figure 6D, Streptococcus dysgalactiae has a bright positive band on the T line of the test strip, while the other six pathogens have no positive bands on the T line of the test strip. Therefore, the RAA-LFD method has a strong specificity for Streptococcus dysgalactiae and is not affected by other pathogenic bacteria in aquatic products.

Example 3

50 zebrafish were tested by PCR and RAA-LFD respectively to evaluate the performance of RAA-LFD detection method for Streptococcus dysgalactiae. The two methods were compared and the results are shown in Table 5 and Figures 7-9.

Table 5 Comparison of RAA-LFD and traditional PCR in experimental samples

As shown in Table 5, the positive coincidence rate of the two methods is 97.6%, the negative coincidence rate is 90.0%, and the total coincidence rate is 98.0%.

As shown in Figure 7, the RAA-LFD method detected 41 positive samples and 9 negative samples, with a positive rate of 82.0% and a negative rate of 18.0%; as shown in Figure 8, the traditional PCR detected 40 positive samples and 10 negative samples, with a positive rate of 80.0% and a negative rate of 20.0%; as shown in Figure 9, the two lines belong to the RAA group and the low line belongs to the PCR group. Compared with the traditional PCR, the AUC of RAA-LFD is 0.950, the sensitivity is 100%, and the specificity is 90%. The actual sample detection results show that the RAA-LFD method has a higher detection accuracy and a better practical application effect.

Claims (3)

1. A primer pair and probe combination for detecting streptococcus agalactiae by using RAA-LFD, which is characterized in that the primer pair is as follows: ISP-F1/ISP-R1, ISP-F1/ISP-R2, ISP-F2/ISP-R1, ISP-F2/ISP-R2, ISP-F3/ISP-R1 or ISP-F3/ISP-R2; wherein the nucleotide sequences of ISP-F1, ISP-F2, ISP-F3, ISP-R1 and ISP-R2 are as follows:

ISP-F1：5’-AATTTAAATCAGAGGCAAAAGGTGACCACGGAA-3’；

ISP-F2：5’-AATGCTCCACATAGGCCGCATAAGTATTAACAGA-3’；

ISP-F3：5’-TCCCAAAGAACTTTCTGACATGGCAATAGCAAC-3’；

ISP-R1：5’-ATCTGATGTTCTTAATCCTGCAGAGCCTACTCG-3’；

ISP-R2：5’-GATGTTCTTAATCCTGCAGAGCCTACTCGTCCA-3’；

The 5' end of the downstream primer is marked with biotin; fluorescein is marked at the 5 'end of the probe, C3 Spacer is marked at the 3' end of the probe, and dSpacer is marked at the position of the probe 31 bp;

the probe is as follows:

5’-FAM-TATCTCCAAATTTAAATCAGAGGCAAAAGG/dSpacer/GACCACGGAAGAGGTGT(C3 Spacer)-3’。

2. Use of a primer pair and probe combination according to claim 1 for the preparation of a kit for detecting streptococcus dysgalactiae.

3. A kit for detecting streptococcus dysgalactiae comprising the primer pair and probe combination of claim 1.