Kit for detecting penicillin and amoxicillin resistance of streptococcus suis and application thereof

Abstract

The application provides a kit for detecting penicillin and amoxicillin resistance of streptococcus suis and application thereof, and belongs to the technical field of molecular biology detection. The kit comprises a primer pair and AlwNI restriction endonuclease; the primer pair comprises primers PBP1a-2083F and PBP1a-2083R, and the sequences are shown in SEQ ID NO:1 and SEQ ID NO:2 respectively. The kit is used for detecting penicillin and amoxicillin resistance of streptococcus suis, has the advantages of high specificity, high sensitivity, low detection cost and low technical requirement, and provides reliable technology for monitoring penicillin and amoxicillin resistance of streptococcus suis in clinic.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology detection technology, specifically relating to a kit for detecting penicillin and amoxicillin resistance in Streptococcus suis and its application. Background Technology

[0002] Streptococcus suis ( Streptococcus suis, SS Streptococcus suis is a major pathogenic bacterium in pigs, primarily colonizing the upper respiratory tract and tonsils. It can cause meningitis, arthritis, pneumonia, septicemia, and even death, resulting in significant economic losses for the pig farming industry. β-lactam antibiotics, including penicillin and amoxicillin, are among the main antibiotics used to treat streptococcal infections in pigs. However, in recent years, due to the overuse of antibiotics, Streptococcus suis has developed resistance to β-lactam antibiotics. If the strain has already developed resistance to penicillin and amoxicillin, continued use may lead to treatment failure, delay in treatment, and increased mortality and farming costs in sick pigs.

[0003] Currently, the resistance mechanism of Streptococcus suis to β-lactam antibiotics is not clear. Clinical detection of Streptococcus suis resistance is mainly determined by measuring the minimum inhibitory concentration (MIC) of antibiotics against bacteria, which takes 3-4 days and requires high technical skills, making it unsuitable for rapid detection at the grassroots level. Therefore, there is a lack of reliable rapid detection methods for Streptococcus suis resistance to penicillin and amoxicillin in existing technologies. Summary of the Invention

[0004] The purpose of this invention is to provide a kit for detecting penicillin and amoxicillin resistance in Streptococcus suis, which has the advantages of high specificity, high sensitivity, low detection cost, and low technical requirements, providing a reliable technology for clinical monitoring of penicillin and amoxicillin resistance in Streptococcus suis.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] A kit for detecting penicillin and amoxicillin resistance in Streptococcus suis includes primer pairs and AlwNI restriction endonuclease; the primer pairs include primers PBP1a-2083F and PBP1a-2083R, with sequences shown in SEQ ID NO:1 and SEQ ID NO:2, respectively.

[0007] A method for detecting penicillin and amoxicillin resistance in Streptococcus suis using the aforementioned kit for non-diagnostic purposes, characterized by comprising the following steps:

[0008] (1) Using the Streptococcus suis bacterial suspension as a template, PCR amplification reaction was carried out using primers PBP1a-2083F and PBP1a-2083R. The obtained PCR product was subjected to agarose gel electrophoresis. If a fragment of 570 bp appeared, the PCR product was purified.

[0009] (2) The purified PCR product was digested with AlwNI restriction endonuclease. The digested product was subjected to agarose gel electrophoresis. When two bands of 479bp and 91bp appeared in the lane, the streptococcus suis to be tested was determined to be a penicillin and amoxicillin resistant strain.

[0010] In this invention, the PCR reaction system is as follows: 12.5 μL of 2×Taq Master Mix, 8.5 μL of ddH2O, 1 μL of PBP1a-2083F, 1 μL of PBP1a-2083R, and 2 μL of bacterial culture.

[0011] In this invention, the PCR reaction procedure is as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, primer annealing at 55°C for 30 s, extension at 72°C for 30 s, 30 cycles; extension at 72°C for 10 min.

[0012] In this invention, the enzyme digestion reaction system is as follows: 1 μg of purified PCR product, 1 μL of AlwNI restriction endonuclease, 5 μL of 10× rCutSmart buffer, and ddH2O to make up to 50 μL.

[0013] In this invention, the enzyme digestion reaction procedure is as follows: after incubating in a water bath at 35-39℃ for 5-15 minutes, incubate in a water bath at 75-85℃ for 15-25 minutes to thermally inactivate the endonuclease.

[0014] The beneficial effects of this invention: Conventional 96-well polystyrene plate micro-broth dilution method for detecting penicillin and amoxicillin resistance in Streptococcus suis takes 3-4 days to complete. This invention's kit can detect penicillin and amoxicillin resistance in Streptococcus suis within 4-5 hours. Therefore, clinically, this kit can be used for preliminary testing. Although it may miss penicillin and amoxicillin-resistant strains, if a Streptococcus suis strain is quickly found to be resistant to penicillin and amoxicillin using this kit, other antibiotic susceptibility tests can be conducted as early as possible to identify highly effective antibiotics and reduce losses in pig farms. This invention's kit offers a simple and rapid detection method, with results interpreted by enzyme digestion. It is easy to operate, low-cost, and significantly reduces the workload of traditional drug susceptibility testing methods, greatly improving efficiency. Attached Figure Description

[0015] Figure 1Electrophoresis images of PCR amplification products of various Streptococcus suis strains using the kit of this invention are shown. Lanes 1-10 are, in order: 2018WUSS145, 2022WUSS042, 2022WUSS043, 2023WUSS072, 2023WUSS078, YS626, YS628, YS682, YS683, and YS801; Lane 11 is the negative control; Lane M is the molecular weight marker. Lanes 12-21 are, in order: 2018WUSS005, 2018WUSS007, 2018WUSS008, 2018WUSS033, 2018WUSS107, 2022WUSS001, 2022WUSS007, 2022WUSS031, 2022WUSS046, 2022WUSS097.

[0016] Figure 2 Electrophoresis images of the PCR amplification products of each Streptococcus suis strain, obtained by digestion with AlwNI restriction endonuclease, are shown. Lanes 1-10 are, in order: 2018WUSS145, 2022WUSS042, 2022WUSS043, 2023WUSS072, 2023WUSS078, YS626, YS628, YS682, YS683, and YS801; Lane 11 is the negative control; Lane M is the molecular weight marker. Lanes 12-21 are, in order: 2018WUSS005, 2018WUSS007, 2018WUSS008, 2018WUSS033, 2018WUSS107, 2022WUSS001, 2022WUSS007, 2022WUSS031, 2022WUSS046, 2022WUSS097. Detailed Implementation

[0017] The following detailed embodiments further illustrate the above-described content of the present invention, but should not be construed as limiting the scope of the subject matter of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Unless otherwise specified, the following embodiments are all implemented using conventional prior art.

[0018] Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art.

[0019] Example 1: Kit for detecting penicillin and amoxicillin resistance in Streptococcus suis

[0020] SNP sites for detecting penicillin and amoxicillin resistance in Streptococcus suis

[0021] To find a rapid method for detecting penicillin and amoxicillin resistance in Streptococcus suis, nearly 700 strains of Streptococcus suis were tested. Streptococcus suis The MIC values ​​of the strain for penicillin and amoxicillin were measured, and its genomic DNA was sequenced and compared. Numerous mutations were found in the *Streptococcus suis* genome, for example, in… pbp2a, pbp2b, pbp2x pbp1a, pbp1b In these genes, not only single-point mutations exist, but also multiple-point mutations. Some results of the amino acid mutations in the corresponding protein sequences they encode are listed in Table 1.

[0022] Table 1. Streptococcus suis ( Streptococcus suis List of amino acid mutations corresponding to genomic mutations in strains

[0023]

[0024] During the study, it was found that numerous mutations in the genome of Streptococcus suis strains were not related to their penicillin and amoxicillin resistance. For example, the mutation at position 455 of PBP1a from asparagine to aspartic acid did not alter its penicillin and amoxicillin resistance. Some of the results are listed in Table 2.

[0025] Table 2. Genomic mutations of Streptococcus suis strains and their resistance to penicillin and amoxicillin.

[0026]

[0027] Note: a and b are antibiotic resistance breakpoints. "Penicillin MIC value" a MIC ≥ 1 This column indicates that the data represents the penicillin MIC value. When the MIC ≥ 1, the bacterium is resistant to penicillin. "Amoxicillin MIC value" b MIC>2 "This indicates that the data in this column is the MIC value of amoxicillin. When the MIC>2, the bacteria is resistant to amoxicillin."

[0028] Through extensive and creative work, strains of Streptococcus suis that exhibited high levels of resistance to penicillin (MIC value ≥ 8 μg / mL) and resistance to amoxicillin (MIC value ≥ 4 μg / mL) were discovered among nearly 700 strains. PBP1a The 2083rd base of the gene is G; among the remaining Streptococcus suis strains (i.e., strains that cannot simultaneously achieve a MIC value ≥8 μg / mL for penicillin and a MIC value ≥4 μg / mL for amoxicillin), PBP1a The 2083rd base of the gene is T, and some results are listed in Table 3. Therefore... PBP1a The 2083rd position of the gene is an SNP site. PBP1aThe T-to-G mutation at position 2083 of the gene leads to an amino acid mutation, changing from serine to alanine. This results in Streptococcus suis exhibiting high levels of resistance to penicillin (MIC value ≥ 8 μg / mL) and resistance to amoxicillin (MIC value ≥ 4 μg / mL).

[0029] Table 3. Streptococcus suis strains PBP1a The 2083rd base of the gene and its resistance to penicillin and amoxicillin

[0030]

[0031] Note: a and b are antibiotic resistance breakpoints. "Penicillin MIC value" a MIC ≥ 1 This column indicates that the data represents the penicillin MIC value. When the MIC ≥ 1, the bacterium is resistant to penicillin. "Amoxicillin MIC value" b MIC>2 "This indicates that the data in this column is the MIC value of amoxicillin. When the MIC>2, the bacteria is resistant to amoxicillin."

[0032] 2. Kit for detecting penicillin and amoxicillin resistance in Streptococcus suis

[0033] Targeting Streptococcus suis PBP1a Primer pairs (PBP1a-2083F and PBP1a-2083R) were designed to amplify the 570bp SNP at position 2083 of the gene. PBP1a Gene fragments were analyzed, and the presence or absence of AlwNI restriction endonuclease was used to detect whether Streptococcus suis exhibited high-level resistance to penicillin (MIC value ≥ 8 μg / mL) and resistance to amoxicillin (MIC value ≥ 4 μg / mL).

[0034] A kit for detecting penicillin and amoxicillin resistance in Streptococcus suis includes primer pairs and AlwNI restriction endonucleases; the primer pairs include a forward primer PBP1a-2083F and a reverse primer PBP1a-2083R. The sequence of the forward primer PBP1a-2083F (SEQ ID NO:1) is as follows: 5'-GAAGGCTGTTATGTCTTACG-3'; the sequence of the reverse primer PBP1a-2083R (SEQ ID NO:2) is as follows: 5'-TATTGACCATCTGTCTGTCC-3'.

[0035] The forward primer PBP1a-2083F and the reverse primer PBP1a-2083R were synthesized by Beijing Qingke Biotechnology Co., Ltd., with a concentration of 10 nmol / L.

[0036] AlwNI restriction endonuclease, at a concentration of 10,000 units / ml, was purchased from New England Biotechnology (Beijing) Co., Ltd.

[0037] 2. Method for detecting penicillin and amoxicillin resistance using the kit of this invention.

[0038] The method for detecting penicillin and amoxicillin resistance in Streptococcus suis using the kit of the present invention includes the following steps:

[0039] (1) Using the Streptococcus suis bacterial suspension as a template, PCR amplification was performed using primers PBP1a-2083F and PBP1a-2083R. The reaction system was 25 μL, and the composition was as follows: 12.5 μL of 2×Taq MasterMix (purchased from Nanjing Novizan Biotechnology Co., Ltd.), 8.5 μL of ddH2O, 1 μL of 10 μM forward primer PBP1a-2083F, 1 μL of 10 μM reverse primer PBP1a-2083R, and 2 μL of bacterial suspension. The PCR amplification reaction program was as follows: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 55°C primer annealing for 30 s, 72°C extension for 30 s, 30 cycles; 72°C extension for 10 min. Simultaneously, enzyme-free ultrapure water (Labshark 130114010) was used as a negative control instead of the tested Streptococcus suis bacterial solution. The obtained PCR products were subjected to agarose gel electrophoresis. If a 570 bp fragment appeared, the PCR product was purified using a standard DNA product purification kit (DP204) (purchased from Tiangen Biotech (Beijing) Co., Ltd.). The negative control should show no PCR amplification bands.

[0040] (2) The purified PCR product was digested with AlwNI restriction endonuclease in a 50 μL volume. The specific steps were as follows: Add the following reaction mixture to ice (AlwNI restriction endonuclease was added last): 1 μg of purified PCR product, 1 μL of AlwNI restriction endonuclease, 5 μL of 10× rCutSmart buffer (purchased from New England Biotechnology (Beijing) Co., Ltd.), and finally, add ddH2O to bring the volume to 50 μL. The digestion procedure was as follows: Incubate at 37°C for 10 min, then incubate at 80°C for 20 min to inactivate the endonuclease. Simultaneously, enzyme-free ultrapure water (labshark130114010) was used as a negative control instead of PCR product for digestion.

[0041] (3) Perform agarose gel electrophoresis on the enzyme digestion products obtained in step (2). When two bands of 479bp and 91bp appear in the lane, the streptococcus suis to be tested is determined to be highly resistant to penicillin (MIC value ≥ 8 μg / mL) and resistant to amoxicillin (MIC value ≥ 4 μg / mL); the negative control has no band.

[0042] Example 2: Reliability of the reagent kit of the present invention

[0043] One hundred other Streptococcus suis strains isolated in the laboratory were tested for resistance to penicillin and amoxicillin using the kit of this invention. Simultaneously, the minimum inhibitory concentrations (MICs) of penicillin and amoxicillin against these Streptococcus suis strains were determined using the conventional microbroth dilution method. Comparison of the test results demonstrates the reliability of the kit of this invention.

[0044] The determination of the minimum inhibitory concentration (MIC) of penicillin and amoxicillin against Streptococcus suis using the traditional micro-broth dilution method includes the following steps:

[0045] (1) The drug susceptibility test of Streptococcus suis was performed using the broth dilution method recommended by the American Clinical and Laboratory Standards Institute (M100-Ed31). Staphylococcus aureus ATCC29213 was used as the quality control strain. The MICs of penicillin and amoxicillin against each Streptococcus suis strain were determined.

[0046] (2) Streptococcus suis to be tested was streaked onto THB blood agar plates (prepared by adding 1.5% agar powder and 5% sterile defibrinated sheep blood to THB liquid medium; THB medium was purchased from Qingdao Haibo Biotechnology Co., Ltd., and agar powder and sterile defibrinated sheep blood were purchased from Nanjing Dingguo Biotechnology Co., Ltd.). The bacteria were revived by culturing in a 37℃ CO2 incubator for 12 hours. Single colonies were picked and placed in THB medium, and cultured in a shaker at 37℃ and 180 rpm until the logarithmic phase. Then, the bacteria were transferred to THB medium at a volume ratio of 1:100 and cultured in a shaker at 37℃ and 180 rpm until the OD phase. 600 Reaching 0.1-0.15;

[0047] (3) OD 600 The Streptococcus suis bacterial suspension with a pH of 0.1-0.15 was transferred to MH broth medium (purchased from Qingdao Haibo Biotechnology Co., Ltd.) at a volume ratio of 1:1000 to obtain a diluted bacterial suspension. In a 96-well polystyrene plate, 180 μL of the diluted bacterial suspension was added to well 1, 100 μL of the diluted bacterial suspension was added to wells 2-11 respectively, and 100 μL of pure MH broth medium was added to well 12.

[0048] (4) Add 20 μL of antibiotic stock solution (penicillin or amoxicillin) with a concentration of 2560 μg / mL to well 1, mix by pipetting, and then add 100 μL of the liquid to well 2 for 1:1 dilution. Then, in the same way, add 100 μL of the liquid from the previous well to the next well for 1:1 dilution until 100 μL of the liquid is added to well 10. Then, take 100 μL of the liquid from well 10 and discard it.

[0049] (5) Place the 96-well polystyrene plate in a 37°C incubator and incubate for 16-24 hours. Observe the results with the naked eye. The highest concentration of antibiotic in the wells without bacterial precipitation is taken as the MIC value.

[0050] Results: Both methods detected 10 Streptococcus suis strains exhibiting high-level resistance to penicillin (MIC ≥ 8 μg / mL) and resistance to amoxicillin (MIC ≥ 4 μg / mL). Other strains did not simultaneously exhibit high-level resistance to both penicillin (MIC ≥ 8 μg / mL) and amoxicillin (MIC ≥ 4 μg / mL). The test results for the strains exhibiting high-level resistance to penicillin (MIC ≥ 8 μg / mL) and resistance to amoxicillin (MIC ≥ 4 μg / mL), along with some other strains, are presented in Table 4. Figure 1 and Figure 2 middle.

[0051] Depend on Figure 1 It is known that *Streptococcus suis* strains exhibiting high levels of resistance to penicillin (MIC value ≥ 8 μg / mL) and resistance to amoxicillin (MIC value ≥ 4 μg / mL), along with other *Streptococcus suis* strains... PBP1a All genes were amplified normally, and the negative control showed no amplification band. The AlwNI restriction endonuclease was used to amplify the 570bp gene. PBP1a Gene fragments were digested with enzymes, and the detection results were as follows: Figure 2 .Depend on Figure 2 It can be seen that the negative control enzyme digestion product had no specific band, while the PCR amplification products of Streptococcus suis 2018WUSS145, 2022WUSS042, 2022WUSS043, 2023WUSS072, 2023WUSS078, YS626, YS628, YS682, YS683, and YS801 were 570bp. PBP1aThe gene fragments were all digested into two bands, 479 bp and 91 bp, indicating that these strains showed high-level resistance to penicillin (MIC value ≥ 8 μg / mL) and resistance to amoxicillin (MIC value ≥ 4 μg / mL). Streptococcus suis strains 2018WUSS005, 2018WUSS007, 2018WUSS008, 2018WUSS033, 2018WUSS107, 2022WUSS001, 2022WUSS007, 2022WUSS031, 2022WUSS046, and 2022WUSS097 did not show two bands in their digestion products, indicating that these strains could not simultaneously achieve high-level resistance to penicillin (MIC value ≥ 8 μg / mL) and resistance to amoxicillin (MIC value ≥ 4 μg / mL).

[0052] As shown in Table 4, using the kit of this invention, a 570bp fragment appeared in the PCR products of all strains. Among them, 10 strains showed two bands, 479bp and 91bp, in their enzyme digestion products. The results of the traditional microbroth dilution method also showed MIC values ​​≥8 μg / mL for penicillin and ≥4 μg / mL for amoxicillin. Therefore, the concordance rate between the kit of this invention and the microbroth dilution method is 100%, indicating that the kit of this invention is accurate and reliable.

[0053] Table 4. Comparison of the detection results of the kit of the present invention with the MIC values ​​of various antibiotics against Streptococcus suis detected by the microbroth dilution method.

[0054]

[0055] Note: a and b are antibiotic resistance breakpoints. "Penicillin MIC value" a MIC ≥ 1 This column indicates that the data represents the penicillin MIC value. When the MIC ≥ 1, the bacterium is resistant to penicillin. "Amoxicillin MIC value" b MIC>2 "This indicates that the data in this column is the MIC value of amoxicillin. When the MIC>2, the bacteria is resistant to amoxicillin."

Claims

1. The application of primer pairs and AlwNI restriction endonuclease in the preparation of a kit for detecting penicillin and amoxicillin resistance in Streptococcus suis, characterized in that... The primer pair includes primers PBP1a-2083F and PBP1a-2083R, with sequences shown in SEQ ID NO:1 and SEQ ID NO:2, respectively. PCR amplification is performed using the primer pair. If a fragment of 570 bp appears, the PCR product is purified. The purified PCR product is digested with AlwNI restriction endonuclease. The digested product is then subjected to agarose gel electrophoresis. When only two bands of 479 bp and 91 bp appear in the lanes, the tested Streptococcus suis is determined to be a penicillin and amoxicillin-resistant strain.

2. A method for detecting penicillin and amoxicillin resistance in Streptococcus suis for non-disease diagnostic purposes, characterized in that... Includes the following steps: (1) Using the Streptococcus suis bacterial suspension as a template, PCR amplification reaction was performed using the primers PBP1a-2083F and PBP1a-2083R described in claim 1. The obtained PCR product was subjected to agarose gel electrophoresis. If a fragment of 570 bp appeared, the PCR product was purified. (2) The purified PCR product was digested with AlwNI restriction endonuclease. The digested product was subjected to agarose gel electrophoresis. When only two bands of 479bp and 91bp appeared in the lane, the streptococcus suis to be tested was determined to be a penicillin and amoxicillin resistant strain.

3. The method according to claim 2, characterized in that... The PCR reaction system is as follows: 12.5 μL of 2×TaqMasterMix, 8.5 μL of ddH2O, 1 μL of PBP1a-2083F, 1 μL of PBP1a-2083R, and 2 μL of bacterial culture.

4. The method according to claim 3, characterized in that... The PCR reaction procedure is as follows: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 55°C primer annealing for 30 s, 72°C extension for 30 s, 30 cycles; 72°C extension for 10 min.

5. The method according to claim 4, characterized in that... The enzyme digestion reaction system is as follows: 1 μg of purified PCR product, 1 μL of AlwNI enzyme, 5 μL of 10× rCutSmart buffer, and ddH2O to make up to 50 μL.

6. The method according to claim 5, characterized in that... The enzyme digestion reaction procedure is as follows: after incubating in a water bath at 35-39℃ for 5-15 minutes, incubate in a water bath at 75-85℃ for 15-25 minutes to thermally inactivate the endonuclease.

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