CN117821627A - LAMP primer group, kit and detection method for streptococcus agalactiae - Google Patents
LAMP primer group, kit and detection method for streptococcus agalactiae Download PDFInfo
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Abstract
The invention relates to the field of detection of microbial technology, in particular to an LAMP primer group, a kit and a detection method of streptococcus dysgalactiae. The primer group provided by the invention designs a loop-mediated isothermal amplification primer by taking the streptococcus dysgalactiae gapC gene as a target gene, and realizes rapid identification and detection of streptococcus dysgalactiae by using the LAMP technology. Meanwhile, the invention provides a method for detecting streptococcus agalactiae in the sample to be detected by adopting the LAMP technology, specifically amplifying target genes of the streptococcus agalactiae by using the LAMP primer group, and observing amplification results by using a color reaction or gel electrophoresis image to realize detection of the streptococcus agalactiae in the sample to be detected. The primer group and the detection method provided by the invention are novel detection technologies for clinical rapid visualization of streptococcus dysgalactiae, have high sensitivity and high specificity, can efficiently detect streptococcus dysgalactiae in a sample, and can be used for epidemic prevention and food safety improvement.
Description
Technical Field
The invention relates to the field of detection of microbial technology, in particular to an LAMP primer group, a kit and a detection method of streptococcus dysgalactiae.
Background
Streptococcus dysgalactiae of bovine origin is one of the main pathogenic bacteria causing cow mastitis, and seriously endangers cow health. The harm of streptococcus dysgalactiae to human is not ignored, and with the gradual increase of old people suffering from chronic diseases, the bacteremia report rate caused by streptococcus dysgalactiae is between 2% and 18%, and the harm to human is expanding continuously.
The loop-mediated isothermal amplification technology is used as a novel nucleic acid detection technology, 4 specific primers are designed aiming at 6 regions of a target gene, the strand displacement activity of Bst DNA polymerase is utilized to amplify at a constant temperature and high efficiency in the same reaction system, and white magnesium pyrophosphate precipitate is generated while a large amount of target DNA is synthesized. Since the amplification reaction relies on 6 separate regions of target DNA, the specificity is very high. Generally, the requirement of experimental temperature can be met by means of a constant-temperature water bath, and the method is suitable for rapid detection of a base layer. However, the sensitivity of the LAMP detection technology for streptococcus agalactiae in the prior art is not high, and only the detection result of the streptococcus agalactiae after extraction and purification is shown, so that the infection condition of the streptococcus agalactiae in the sampled sample is not directly indicated, and the detection limit and the sensitivity of the sampled sample are not considered.
Disclosure of Invention
Aiming at the technical problems, the invention provides an LAMP primer group, a kit and a detection method for streptococcus dysgalactiae. The streptococcus agalactiae gapC gene is used as a target gene to design a loop-mediated isothermal amplification primer, and the LAMP technology is used for realizing the rapid detection of the streptococcus agalactiae, so that an effective and rapid screening method is provided for the detection of the streptococcus agalactiae.
In order to solve the technical problems, the invention adopts the following technical scheme:
in a first aspect, the invention provides a streptococcus agalactiae detection primer group based on the LAMP technology, wherein the nucleotide sequence of the primer group is as follows:
F3：ACTGGTGACCAAATGATCCTTG；
B3：GTTGATTTCGTCAACAGAAACGT；
FIP：TAGCTTTAGCAGCACCAGTTGAGTT-TGGTGGTGACCTTCGTCG；
BIP：AATGGTAAACTTGATGGTGCTGCAC-TCAAGAGTTACAACCAACTCAGT；
LF：GTTTGCAGCACCAGCACGAG；
LB：AACGTGTTCCTGTTCCAACTGGAT。
wherein, the nucleotide sequence of the forward outer primer F3 is shown as SEQ ID NO. 1; the nucleotide sequence of the reverse outer primer B3 is shown as SEQ ID NO. 2; the nucleotide sequence of the forward inner primer FIP is shown as SEQ ID NO. 3; the nucleotide sequence of the reverse inner primer BIP is shown as SEQ ID NO.4, the nucleotide sequence of the forward loop primer LF is shown as SEQ ID NO.5, and the nucleotide sequence of the reverse loop primer LB is shown as SEQ ID NO.6. The primer group is designed by taking streptococcus agatus gene as a target gene according to a primer design principle, and is finally determined after screening analysis. The primer group can specifically amplify DNA of streptococcus dysgalactiae, and can be combined with chromogenic reaction or gel electrophoresis image to rapidly identify and detect streptococcus dysgalactiae.
In a second aspect, the invention provides the use of the primer set described above in the preparation of a kit for identifying streptococcus dysgalactiae or for detecting whether a sample to be tested contains streptococcus dysgalactiae.
In a third aspect, a kit for detecting streptococcus dysgalactiae comprises the primer set and a reaction system required for the LAMP reaction.
In a fourth aspect, the present invention provides a method for detecting whether a sample to be tested contains streptococcus agalactiae, comprising the primer set as described above, comprising the steps of:
s1: extracting total DNA of a sample to be detected;
s2: performing LAMP amplification by using the primer set according to claim 1 or the kit according to claim 3 with the total DNA extracted in S1 as a template;
s3: identification result: if the primer group or the kit in the step S2 can be used for realizing the specific amplification by taking the total DNA as a template, the sample to be detected contains or is suspected to contain streptococcus agalactiae;
the methods are useful for non-disease diagnosis and treatment.
And adding the total bacterial DNA of the sample to be detected into the LAMP reaction system containing the specific primer group provided by the invention for isothermal amplification reaction, and judging whether the fresh milk contains streptococcus dysgalactiae or not according to the result. In actual detection, low concentration bacteria in the sample to be detectedAnd the presence of interfering substances can hinder the extraction of DNA or can result in too low a concentration of DNA, and the detection method used cannot correctly reflect whether the sample to be detected contains streptococcus dysgalactiae or not and the concentration thereof. Compared with the prior art, the method has the advantages of simplicity, convenience, rapidness, high specificity, strong sensitivity and visual sensitivity up to 10 2 The sensitivity of agarose gel electrophoresis can reach 10 1 copies/μL。
Further, the method of the identification result in S3 is to add a chromogenic material for judging whether the DNA of the sample to be identified is successfully amplified into the LAMP reaction system, and the chromogenic state of the reaction system is used as the identification result; or taking LAMP amplification products, spotting the LAMP amplification products on agarose gel, and observing the imaging result in a gel imaging system to be used as an identification result.
Adding a color developing material into the reaction system after amplification is completed, uniformly mixing, and visually observing the color developing state, so as to judge whether the raw fresh milk contains streptococcus dysgalactiae or not by contrasting the color developing states of the positive control substance and the negative control substance; sucking a certain amount of LAMP amplification products, adding the LAMP amplification products into agarose gel for electrophoresis detection, and observing electrophoresis results by naked eyes, if step-type strips appear, judging positive, namely detecting that fresh milk contains streptococcus dysgalactiae; if no stepped strip appears, the method is judged as negative, namely, the fresh milk is detected to not contain streptococcus agalactiae.
Further, LAMP amplification was performed in 25. Mu.L or 50. Mu.L LAMP amplification system; the LAMP amplification system comprises an inner primer, an outer primer, a loop primer, dNTPs, a DNA template, bst DNA polymerase, buffer and enzyme-free water; the concentration ratio of the inner primer to the outer primer in the system is 4-10:1, and the final concentration of the inner primer in the system is 8.0-20.0 mu M/L.
The single LAMP reaction system of the streptococcus agalactiae gapC gene is established, and has no cross reaction with common streptococcus agalactiae, streptococcus uberis, escherichia coli, staphylococcus aureus, listeria and the like, and has high specificity verification and visualization sensitivity of 9.53 multiplied by 10 2 The sensitivity of agarose gel electrophoresis detection was 9.53X10 at the copies/. Mu.L 1 copies/μL。
Illustratively, the present invention provides a visualized LAMP amplification system of 25. Mu.L comprising:
WarmStart Colorimetric LAMP 2×Master Mix 12.5μL
10×Primers 2.5μL(8.0-20.0μM FIP/BIP、2μM F3/B3、4μM LF/LB)
1.0-5.0 mu L of DNA template
Enzyme-free water was added to 25. Mu.L.
The LAMP color-changing premix is used, the color reaction can be observed by naked eyes after the reaction is finished, and the sample to be detected is judged to be positive in yellow on the premise that negative control is red or orange red and positive control is yellow, namely the raw fresh milk contains streptococcus agalactiae; and judging that the sample to be detected is red or orange red, namely detecting that the fresh milk does not contain streptococcus dysgalactiae.
Further, the concentration ratio of the inner primer to the outer primer in the system is 8:1, and the final concentration of the inner primer in the system is 16.0 mu M/L.
The influence of the primer proportion on the whole visual LAMP detection method can be reduced by optimizing the internal and external primer proportion.
Further, the LAMP amplification was carried out at a temperature of 61-64 ℃.
Further, the reaction time is 30-60min.
Further, the LAMP amplification was carried out at a temperature of 62-63 ℃.
Further, the reaction time was 30min.
Further, the sample to be tested is fresh milk; the method for extracting the total DNA of the sample to be detected is an EDTA-Tris method.
The low concentration of bacteria in raw milk and the presence of calcium ions, fat, proteins and the like can prevent effective DNA extraction, and the problems of incomplete removal of milk proteins, poor cell disruption efficiency, high residual impurities and the like lead to low concentration and low purity of extracted milk microorganism DNA, so that false negative easily occurs in the result. The EDTA-Tris method is applied to the extraction of bacterial DNA in fresh milk, so that the DNA recovery rate of streptococcus dysgalactiae detection is improved, and interference factors affecting subsequent LAMP amplification are shielded. The method is verified by clinical detection and simulation, and the method has the following steps of detecting and verifying the artificial polluted fresh milkHas good specificity, and the visualization sensitivity and the sensitivity of the liposaccharide gel electrophoresis detection are about 2.94 multiplied by 10 2 CFU/mL。
Drawings
FIG. 1 is a non-specific verification of the LAMP primer of the gapC gene of Streptococcus dysgalactiae in example 2 of the present invention;
m, marker;1, positive control; 2, negative control; 3, blank control;
FIG. 2 shows the results of optimizing the reaction temperature of the LAMP detection method for Streptococcus dysgalactiae in experimental example 1 of the present invention;
m, marker;1-8, 58-65 ℃;9, negative control;
FIG. 3 shows the results of optimization of the reaction time of the LAMP detection method for Streptococcus dysgalactiae in Experimental example 1 of the present invention;
m, marker;1-6, 10-60min;7, negative control;
FIG. 4 shows the results of optimizing the ratio of the inner primer to the outer primer in the LAMP detection method of Streptococcus dysgalactiae in experimental example 1 of the present invention;
m,100bp DNA Marker;1-6, wherein the ratio of the inner primer to the outer primer is sequentially 1:1, 2:1, 4:1, 6:1, 8:1 and 10:1;7 represents a negative control;
FIG. 5 shows the results of specific verification of the LAMP detection method for Streptococcus dysgalactiae in Experimental example 2 of the present invention;
m,100bp DNA Marker;1, streptococcus agalactiae; 2-12, which are staphylococcus chromogenes, staphylococcus slowly, streptococcus uberis, staphylococcus hominis, staphylococcus aureus, streptococcus agalactiae, escherichia coli, enterococcus haii, streptococcus dysgalactiae, listeria monocytogenes LM90 and listeria monocytogenes LM-EDGe in sequence; 13, negative control;
FIG. 6 is a sensitivity evaluation result of the LAMP detection method of Streptococcus dysgalactiae in Experimental example 3 of the present invention;
m,100bp DNA Marker;1-10 in order of 9.53×10 9 -9.53×10 0 cobies/. Mu.L; 11, negative control;
FIG. 7 shows the LAMP detection effect of Streptococcus dysgalactiae of the invention in example 4 and comparative examples 1-7 by different DNA extraction methods from artificially contaminated raw milk;
m,100bp DNA Marker;1, EDTA-Tris method, 2, naOH-Tris-HCl method, 3, naOH cracking method, 4, PBS cracking method; 5, sample boiling method; 6, chelex100 process; 7, etNa method; 8, a kit method; 9 represents a negative control; 10 represents a blank;
FIG. 8 is a result of verification of specificity of a method for detecting Streptococcus dysgalactiae in raw milk in example 4 of the present invention;
m,100bp DNA Marker;1, artificially polluting milk by streptococcus dysgalactiae; 2-11 are in turn staphylococcus chromogenes, staphylococcus slowly, streptococcus uberis, staphylococcus hominis, staphylococcus aureus, streptococcus agalactiae, escherichia coli, enterococcus haii, streptococcus dysgalactiae and listeria monocytogenes LM90 artificially contaminated milk; 12, negative control; 13, blank control;
FIG. 9 is a sensitivity evaluation result of a method for detecting Streptococcus dysgalactiae in raw milk in example 4 of the present invention;
m,100bp DNA Marker;1-8 are 2.94×10 in sequence 8 -2.94×10 1 CFU/mL;9, negative control.
Detailed Description
The present invention will be described in further detail with reference to specific embodiments in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
The following microorganisms were all stored in a zoonotic laboratory at the university of Shihe. The experimental methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents, and the like used are all commercially available. The streptococcus-stop positive plasmid standard substance is self-constructed by the primer provided by the invention.
Example 1 primer design
The streptococcus agalactiae gapC gene sequence downloaded from NCBI query is imported into an on-line design LAMP Primer software Primer ExplorerV5, a plurality of groups of primers are generated on line, and the primers are screened by referring to a Primer design instruction manual provided by a website. And inputting the selected Primer into NCBI for Primer Blast comparison, and further selecting the Primer with strong specificity for subsequent experiments. And finally determining the primer of streptococcus dysgalactiae through screening analysis, wherein primer synthesis is completed by the responsibility of Qingdao Rui Boxing family.
TABLE 1 primer design
The nucleotide sequence of the outer primer F3 is shown as SEQ ID NO.1, the nucleotide sequence of the outer primer B3 is shown as SEQ ID NO.2, the nucleotide sequence of the inner primer FIP is shown as SEQ ID NO.3, the nucleotide sequence of the inner primer BIP is shown as SEQ ID NO.4, the nucleotide sequence of the loop primer LF is shown as SEQ ID NO.5, and the nucleotide sequence of the reverse loop primer LB is shown as SEQ ID NO.6.
EXAMPLE 2 preliminary exploration of the LAMP reaction System of Streptococcus dysgalactiae
The LAMP reaction system (Table 2) was constructed with reference to WarmStart Colorimetric LAMP X Master Mix instructions from NEB, tube 1 for the DNA template for Streptococcus dysgalactiae gene DNA (DNA extraction kit extraction), tube 2 for the negative control (no enzyme water), and tube 3 for the blank control (template-removed solution). After the LAMP reaction was carried out at 65℃for 30min, the color change of the LAMP solution in the tube was observed. mu.L of the reaction product was taken out and subjected to agarose gel electrophoresis, and the electrophoresis was observed, and the result was shown in FIG. 1.
TABLE 2 Streptococcus dysgalactiae LAMP reaction System
Note that: the LAMP primer group mixture consisted of 16. Mu.L of 100. Mu.M FIP, 16. Mu.L of 100. Mu.M BIP, 2. Mu.L of 100. Mu.M F3, 2. Mu.L of 100. Mu.M B3, and 64. Mu.L of enzyme-free water.
The color of the reaction solution in the LAMP reaction of the gapC gene of Streptococcus dysgalactiae in tube 1 was changed, while neither the negative control (tube 2) nor the blank (tube 3) was changed (FIG. 1A), which also corresponds to the agarose gel electrophoresis result (FIG. 1B). Therefore, the kit prepared by the primer group provided by the invention can be used for identifying streptococcus dysgalactiae or detecting whether the sample to be detected contains streptococcus dysgalactiae.
Example 3
The embodiment provides a method for detecting whether streptococcus agalactiae is contained in a sample to be detected.
The sample to be detected is streptococcus agalactiae, and the steps are as follows:
s1: extracting total DNA of streptococcus dysgalactiae by using a bacterial genome DNA extraction kit;
s2: LAMP amplification was performed using the total DNA extracted in S1 as a template and the primer set prepared in example 1; the reaction system: warmStart Colorimetric LAMP 2 XMaster Mix 12.5. Mu.L, 10 XPrimers2.5. Mu.L (8. Mu.M FIP/BIP, 2. Mu. M F3/B3, 4. Mu.M LF/B), DNA template 2.0. Mu.L, enzyme-free water was added to 25. Mu.L.
Reaction conditions: constant temperature at 62 ℃ for 30min.
S3: the color change of the reaction system solution was observed. mu.L of the reaction product was taken out, subjected to agarose gel electrophoresis, and the result of the electrophoresis was observed. The results are shown in FIG. 2.
Experimental example 1 optimization of the reaction System
1. Reaction temperature optimization
The reaction temperature was adjusted in a gradient manner using Streptococcus dysgalactiae positive plasmid standard as a template and the reaction system of example 3 at 58, 59, 60, 61, 62, 63, 64, 65℃respectively, and enzyme-free water as a negative control, the negative control reaction temperature being 65 ℃. After 30min of reaction, the presence or absence of the color change and the electrophoresis band were observed to determine the results.
The LAMP reaction solution started to undergo macroscopic change at 61-64℃and the color change was more pronounced at 62-63 ℃. In combination with the electrophoresis result (FIG. 2B), the LAMP reaction optimum temperature of the gapC gene of Streptococcus dysgalactiae was 62-63 ℃. Therefore, the optimum temperature of the reaction system was 62 ℃.
2. Reaction time optimization
The LAMP reaction system in example 3 was used, and the LAMP reaction was carried out by setting the reaction time to a gradient (10, 20, 30, 40, 50, 60 min) at 62℃using Streptococcus dysgalactiae positive plasmid standard as a template. The presence or absence of the color change and the presence or absence of the electrophoresis band were observed with enzyme-free water as a negative control, and the reaction time was 30min.
When the amplification time was 30-60min, the LAMP reaction solution of Streptococcus dysgalactiae was changed to orange-yellow in color (FIG. 3A), and for further determination of the optimal reaction time, it was found that the bands started to appear when the reaction time was 20min, but were more clear and bright when 30-60min, and the visual perception was not significantly different (FIG. 3B). In order to realize the rapidity of the LAMP detection technology, 30min is selected as the optimal reaction time of the reaction system.
3. Optimization of inner and outer primer proportion in streptococcus agalactiae LAMP detection method
To reduce the effect of primer ratios on the overall visual LAMP detection method. The standard plasmid is used as a template, and the reaction conditions are as follows: keeping the temperature at 62 ℃ for 30min, and respectively adjusting the ratio of the inner primer to the outer primer to be 1:1, 2:1, 4:1, 6:1, 8:1 and 10:1. The LAMP system was used for the test, and after the completion of the reaction, the test results were determined.
As is apparent from FIG. 4A, when the ratio of the primers was 2:1, the reaction solution in the tube became orange-yellow, and the color change was gradually increased as the ratio was increased. As a result of agarose gel electrophoresis (FIG. 4B), the gradient bands were observed at a primer ratio of 4-10:1, and the bands were significantly clearer when the inner and outer primer ratios were 8:1. Therefore, the optimal ratio of the inner primer to the outer primer of the reaction system is 8:1.
Experimental example 2 evaluation of specificity
Based on the optimized streptococcus agalactiae LAMP detection method, the genome DNA of strains such as streptococcus agalactiae, staphylococcus chromogenes, staphylococcus slow, streptococcus uberis, human staphylococcus, staphylococcus aureus, streptococcus agalactiae, escherichia coli, enterococcus hainanensis, salmonella, listeria monocytogenes LM90, listeria monocytogenes LM-EDGe and the like are used as templates for detection. The enzyme-free water was used as a negative control.
The visualized LAMP reaction found that only the LAMP reaction solution of the positive sample changed from red to orange, and the rest of the samples were unchanged (FIG. 5A). Agarose gel results were consistent with the visualization results (fig. 5B). The LAMP detection method of streptococcus agalactiae established by the test is proved to have high specificity and do not generate cross reaction with other strains.
Experimental example 3 sensitivity evaluation
The concentration of the constructed streptococcus dysgalactiae positive plasmid was measured using Nano Drop 2000 and specific copy numbers were calculated. Using ddH 2 O performed gradient dilution to plasmid concentration of 9.53×10 9 -9.53×10 0 COPies/. Mu.L. And (3) taking the LAMP reaction as a template, and carrying out LAMP reaction based on the optimized detection method, and measuring and calculating the sensitivity of the established LAMP detection method.
The visual sensitivity of the LAMP detection method of streptococcus agalactiae established by the invention is 9.53 multiplied by 10 2 The sensitivity of agarose gel electrophoresis detection was 9.53X10, with copies/. Mu.L (FIG. 6A) 1 COPIES/. Mu.L (FIG. 6B).
Example 4
The embodiment provides a method for detecting streptococcus agalactiae in fresh milk.
The detection sample is artificial pollution fresh milk: mixing the cultured streptococcus agalactiae with fresh milk, and carrying out pollution of an artificial sample.
S1: extracting total DNA in artificially contaminated fresh milk by adopting an EDTA-Tris method;
s2: LAMP amplification was performed using the total DNA extracted in S1 as a template and the primer set prepared in example 1; the reaction system: warmStart Colorimetric LAMP 2 XMaster Mix 12.5. Mu.L, 10 XPrimers 2.5. Mu.L (16. Mu.M FIP/BIP, 2. Mu. M F3/B3, 4. Mu.M LF/B), DNA template 2.0. Mu.L, enzyme-free water was added to 25. Mu.L. Reaction conditions: constant temperature at 62 ℃ for 30min.
S3: the color change of the reaction system solution was observed. mu.L of the reaction product was taken out, subjected to agarose gel electrophoresis, and the result of the electrophoresis was observed.
Comparative examples 1 to 6
Comparative examples 1 to 6 bacterial DNA was extracted from fresh milk samples by NaOH-Tris-HCl method, naOH lysis method, PBS lysis method, sample boiling method, chelex100 method, etNa method, and kit method, and other steps and parameters were the same as in example 4.
The results of example 4 and comparative examples 1-6 are shown in FIG. 7. From the results, the LAMP detection effect is optimal by extracting DNA from fresh milk by using an EDTA-Tris method. From the visual result in fig. 7A, only test tube 1 has a color change, and test tubes 2-8 are all false negative, and cannot correctly display the result.
Experimental example 4 method for detecting Streptococcus dysgalactiae in raw fresh milk specificity evaluation
Artificially contaminated raw fresh milk was respectively prepared from staphylococcus chromogenes, staphylococcus lentus, streptococcus uberis, staphylococcus hominis, staphylococcus aureus, streptococcus agalactiae, escherichia coli, enterococcus haii, salmonella, listeria monocytogenes LM90 and the like, and the specificity was verified by the method for detecting streptococcus dysgalactiae in raw fresh milk in example 4, and the result is shown in fig. 8 by using raw fresh milk which is not artificially contaminated as a negative control and sterile water as a blank control.
The result shows that the fresh milk containing streptococcus dysgalactiae shows positive results, the rest are negative, and no false positive appears with other bacteria, so that the method for detecting streptococcus dysgalactiae in fresh milk is high in specificity.
Experimental example 5 sensitivity evaluation of method for detecting Streptococcus dysgalactiae in raw fresh milk
Mixing activated streptococcus agalactiae with fresh milk, wherein the concentration of streptococcus agalactiae in the emulsion is 2.94×10 8 CFU/mL. Diluting fresh milk infected with Streptococcus dysgalactiae by multiple ratio to obtain Streptococcus dysgalactiae with concentration of 2.94×10 8 、2.94×10 7 、2.94×10 6 、2.94×10 5 、2.94×10 4 、2.94×10 3 、2.94×10 2 、2.94×10 1 Artificial contaminated raw milk of CFU/mL. The method for detecting streptococcus agalactiae in raw fresh milk established in example 4 was adopted to detect the streptococcus agalactiae respectively.
As can be seen from FIG. 9, the LAMP detection method for streptococcus agalactiae in raw milk established by the invention has a visual sensitivity of 2.94×10 in a simulated clinical test 2 CFU/mL, sensitivity of agarose gel electrophoresis detection was 2.94X10 2 CFU/mL。
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (10)
1. The streptococcus agalactiae detection primer group based on the LAMP technology is characterized by comprising the following nucleotide sequences:
F3：ACTGGTGACCAAATGATCCTTG；
B3：GTTGATTTCGTCAACAGAAACGT；
FIP：TAGCTTTAGCAGCACCAGTTGAGTT-TGGTGGTGACCTTCGTCG；
BIP：AATGGTAAACTTGATGGTGCTGCAC-TCAAGAGTTACAACCAACTCAGT；
LF：GTTTGCAGCACCAGCACGAG；
LB：AACGTGTTCCTGTTCCAACTGGAT。
2. the use of the primer set according to claim 1 in the preparation of a kit: the application of the kit is to identify streptococcus agalactiae or detect whether the sample to be detected contains streptococcus agalactiae.
3. A kit for detecting Streptococcus dysgalactiae, comprising the primer set according to claim 1 and a reaction system required for LAMP reaction.
4. A method for detecting streptococcus agalactiae in a sample to be tested, comprising the primer set of claim 1, comprising the steps of:
s1: extracting total DNA of a sample to be detected;
s2: performing LAMP amplification by using the primer set according to claim 1 or the kit according to claim 3 with the total DNA extracted in S1 as a template;
s3: identification result: if the primer group or the kit in the step S2 can be used for realizing the specific amplification by taking the total DNA as a template, the sample to be detected contains or is suspected to contain streptococcus agalactiae;
the methods are useful for non-disease diagnosis and treatment.
5. The method according to claim 4, wherein the step S3 is characterized in that a chromogenic material for judging whether the DNA of the sample to be identified is successfully amplified is added into the LAMP reaction system, and the chromogenic state of the reaction system is used as the identification result; or taking LAMP amplification products, spotting the LAMP amplification products on agarose gel, and observing the imaging result in a gel imaging system to be used as an identification result.
6. The method according to claim 4, wherein the LAMP amplification is carried out in a LAMP amplification system of 25. Mu.L or 50. Mu.L; the LAMP amplification system comprises an inner primer, an outer primer, a loop primer, dNTPs, a DNA template, bst DNA polymerase and enzyme-free water; the concentration ratio of the inner primer to the outer primer in the system is 4-10:1, and the final concentration of the inner primer in the system is 8.0-20.0 mu M/L.
7. The method according to claim 6, wherein the concentration ratio of the inner primer to the outer primer in the system is 8:1, and the final concentration of the inner primer in the system is 16.0. Mu.M/L.
8. The method for detecting streptococcus agalactiae in a sample according to any one of claims 4-7, wherein: the temperature for LAMP amplification is 61-64 ℃; and/or
The reaction time is 30-60min.
9. The method for detecting streptococcus agalactiae in a sample according to claim 8, wherein the temperature for performing LAMP amplification is 62-63 ℃; and/or
The reaction time was 30min.
10. The method for detecting streptococcus agalactiae in a sample according to claim 4, wherein said sample is fresh milk; the method for extracting the total DNA of the sample to be detected is an EDTA-Tris method.
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