Sextuple PCR detection kit for streptococcus suis drug resistance gene and application of sextuple PCR detection kit

Abstract

The invention relates to the technical field of streptococcus suis drug resistance gene detection, and particularly discloses a streptococcus suis drug resistance gene sextuple PCR detection kit and application thereof. The core of the kit is a specific primer combination, the nucleotide sequences of the specific primer combination are as shown in SEQ ID NO: 1-12, and the specific primer combination respectively aims at six key drug-resistant genes of streptococcus suis: ermB, tet (O), patB, aac (6 '')-Ie-ah (2 ')-Ia, optrA and lsa (E). By optimizing the primer concentration and reaction conditions, an efficient and stable sextuple PCR detection system is established, and amplification and detection of the six genes can be synchronously completed in a single reaction. The method has the advantages of high specificity, high sensitivity, good repeatability, simplicity and convenience in operation and the like. A high-throughput and low-cost molecular detection tool is provided for rapid diagnosis, medication guidance and epidemiological monitoring of streptococcus suis clinical multiple drug resistance, and the application prospect is wide.

Description

Technical Field

[0001] This application relates to the field of Streptococcus suis drug resistance gene detection technology, specifically to a six-fold PCR detection kit for Streptococcus suis drug resistance genes and its application. Background Technology

[0002] Streptococcus suis (SS) is an important zoonotic pathogen. It can cause meningitis, arthritis, pneumonia, and septicemia in pigs, seriously jeopardizing the healthy development of the pig farming industry. It can also infect humans through direct contact, causing serious public health and safety problems. Currently, antibiotic treatment remains the primary clinical method for controlling Streptococcus suis infection.

[0003] However, the effectiveness of existing antibiotic therapies is being weakened by the widespread increase in bacterial resistance. Global surveillance data reveals the severity and prevalence of Streptococcus suis resistance: for example, between 2015 and 2017, the resistance rate to tetracycline in US isolates reached as high as 97%; during the same period, studies in the Netherlands showed resistance rates of 78.4% to tetracycline and 48.1% to clindamycin. In China, studies have shown that the prevalence of the tet(O) gene, which mediates tetracycline resistance, and the ermB gene, which mediates macrolide-lincosamide-streptomycin B (MLSB) resistance, is significantly higher than in other countries. This clearly demonstrates that under environmental antibiotic selection pressure, the widespread dissemination of resistance genes is a key factor leading to the continued formation and spread of bacterial resistance. Therefore, conducting accurate and timely resistance surveillance is of great significance for understanding its prevalence trends, guiding rational clinical drug use, and developing effective prevention and control strategies.

[0004] Currently, the detection of drug resistance in Streptococcus suis mainly relies on the following two traditional methods:

[0005] 1. Phenotypic drug susceptibility test: This method has a high degree of standardization, but it is time-consuming and can only reflect the phenotypic drug resistance results of the strain. It cannot directly reveal the drug resistance genotype it carries, which is not conducive to early warning and transmission tracking of drug resistance mechanisms.

[0006] 2. Single-PCR Genotyping Method: This method can directly detect specific drug resistance genes. However, traditional single-PCR can only detect one target gene per reaction, resulting in low efficiency. For the common clinical problem of multiple drug resistance caused by multiple drug resistance genes, obtaining a complete resistance profile requires multiple independent PCR reactions, leading to a cumbersome, time-consuming, costly, and large sample consumption process.

[0007] Therefore, existing technologies for detecting drug resistance genes in Streptococcus suis suffer from problems such as low efficiency, low throughput, and complex operation, making it difficult to meet the actual needs of rapid clinical diagnosis and large-scale drug resistance monitoring. Summary of the Invention

[0008] In view of this, the purpose of this application is to provide a reagent kit and detection method that can overcome the above-mentioned technical deficiencies. Specifically, it aims to establish a simple, rapid, time-saving, and high-throughput molecular detection scheme to achieve simultaneous one-time detection of six key drug resistance genes (ermB, tet(O), patB, aac(6'')-Ie-aph(2'')-Ia, optrA, and lsa(E)) in Streptococcus suis that are closely related to six commonly used clinical antimicrobial drugs (tetracyclines, macrolides, fluoroquinolones, aminoglycosides, truncated pleurotins, and oxazolidinones). This will significantly improve detection efficiency and provide strong technical support for rapid diagnosis of clinical drug resistance in Streptococcus suis, epidemiological monitoring, and precision medicine.

[0009] To achieve the above objectives, this application provides at least the following technical solutions:

[0010] In a first aspect, this application provides a primer set for detecting drug resistance genes in Streptococcus suis: specific primers including the following nucleotide sequences such as SEQ ID NO. 1~12.

[0011] The 12 primers in the primer set are divided into 6 pairs. Each pair of primers has been carefully designed and screened to specifically target and bind to 6 different drug resistance gene loci of significant clinical importance in the Streptococcus suis genome:

[0012] Primer pair targeting the fluoroquinolone resistance gene patB: SEQ ID NO.1 (upstream primer), SEQ ID NO.2 (downstream primer);

[0013] Primer pair targeting the oxazolidinone resistance gene optrA: SEQ ID NO.3 (upstream primer) and SEQ ID NO.4 (downstream primer);

[0014] Primer pair targeting the tetracycline resistance gene tet(O): SEQ ID NO.5 (upstream primer) and SEQ ID NO.6 (downstream primer);

[0015] Primer pair targeting the macrolide resistance gene ermB: SEQ ID NO.7 (upstream primer) and SEQ ID NO.8 (downstream primer);

[0016] Primer pair targeting the aminoglycoside resistance gene aac(6″)-Ie-aph(2′)-Ia: SEQ ID NO.9 (upstream primer), SEQ ID NO.10 (downstream primer);

[0017] Primer pair targeting the truncated pleurodontia drug resistance gene lsa(E): SEQ ID NO.11 (upstream primer) and SEQ ID NO.12 (downstream primer).

[0018] In the same polymerase chain reaction (PCR) system, these 12 (six pairs) primers can simultaneously, independently and efficiently guide DNA polymerase to specifically amplify their respective drug resistance gene target fragments, thereby achieving the goal of "one reaction, simultaneous detection of 6 targets".

[0019] Secondly, this application provides the use of the primer set described in the first aspect in the preparation of products for detecting streptococcal resistance genes.

[0020] In some preferred embodiments, the drug resistance gene includes at least one of ermB, tet(O), patB, aac(6'')-Ie-aph(2'')-Ia, optrA, and lsa(E).

[0021] Thirdly, this application provides a reagent for detecting drug resistance genes in Streptococcus suis, comprising the primer set described in the first aspect.

[0022] Fourthly, this application provides a kit for detecting drug resistance genes in Streptococcus suis, which includes the reagents described in the third aspect.

[0023] In some embodiments, the kit further includes Mg 2+ 10×PCR buffer, dNTPs, and ExTaq enzyme.

[0024] In some embodiments, the kit further comprises a positive control and / or a negative control, wherein the positive control is a mixed DNA template containing gene fragments from ermB, tet(O), patB, aac(6'')-Ie-aph(2'')-Ia, optrA, and lsa(E).

[0025] Fifthly, this application provides a method for detecting drug resistance genes in Streptococcus suis for non-diagnostic purposes, which includes the following steps:

[0026] Using Streptococcus suis DNA as a template, and employing the primer set described in the first aspect, the reagents described in the third aspect, or the kit described in the fourth aspect, ermB, tet(O), patB, aac(6'')-Ie-aph(2'')-Ia, optrA, and lsa(E) can be simultaneously amplified via multiplex PCR.

[0027] The PCR amplification products are analyzed, and the presence or absence of specific amplification bands determines whether the sample contains the corresponding drug resistance gene.

[0028] In some preferred embodiments, the reaction system (50 μL) for the multiplex PCR is as follows:

[0029] 25 μL of 2×Rapid Taq Master Mix, 2 μL of DNA template, and primers for each gene are as follows: optrA 0.5 μL, ermB 0.75 μL, aac(6″)-Ie-aph(2′)-Ia 1 μL, patB and tet(O) 1.25 μL each, lsa(E) 1.5 μL, with a concentration of 10 μmol·L⁻¹; add ddH₂O to 50 μL.

[0030] In some preferred embodiments, the reaction procedure for the multiplex PCR is as follows:

[0031] Pre-denaturation at 95℃ for 5 min; followed by 35 cycles (denaturation at 95℃ for 15 s, annealing at 54℃ for 30 s, extension at 72℃ for 30 s); and finally extension at 72℃ for 10 min.

[0032] In a sixth aspect, this application provides a method for screening or preparing anti-streptococcal drugs, including the step of detecting the drug resistance gene profile of the test strain using the primer set described in the first aspect or the kit described in the third aspect.

[0033] Compared with the prior art, the technical solution of this application has at least the following advantages and beneficial effects:

[0034] 1. The six primer pairs provided in this application do not interfere with each other in a single reaction system, and can simultaneously amplify six different Streptococcus suis drug resistance gene targets, integrating the traditionally multiple-stage detection into a single reaction, thus improving efficiency several times over. Each primer is designed to target the conserved region of each drug resistance gene, ensuring the accuracy of amplification and effectively avoiding non-specific binding and cross-reaction, thereby ensuring the accuracy of the detection results from the source. The primer set has been systematically optimized and has similar and high amplification efficiency under shared reaction conditions, solving the common problems of amplification competition and inhibition in multiplex PCR, and ensuring the stability and reliability of the multiplex detection system.

[0035] 2. The method for detecting Streptococcus suis resistance genes constructed based on the above primer set in this application has a simplified procedure, significantly reducing technical complexity and operation time. It is easy to carry out in routine laboratories and can be clearly and intuitively interpreted simultaneously by conventional agarose gel electrophoresis, without the need for complex instruments. Methodologically, negative and positive quality controls can be easily introduced to ensure the reliability and credibility of each batch of tests.

[0036] 3. The application of this application in drug resistance diagnosis and monitoring has significant practical value. It can rapidly generate resistance gene profiles of bacterial strains against six major classes of antimicrobial drugs, providing direct molecular evidence for the development of precise and effective anti-infective treatment plans in clinical practice, and helping to reduce the blind use of empirical medications. The high throughput and simplicity of this technology make it ideal for large-scale bacterial strain screening, enabling efficient tracking of the prevalence and spread of drug resistance genes, and providing crucial data support for public health decision-making. Attached Figure Description

[0037] Figure 1 The electrophoresis diagram for the six-fold PCR detection provided in Example 2 of this application is shown below, where M: DL2000 Marker; 1: six-fold PCR positive control; 2: patB; 3: optrA; 4: tet(O); 5: ermB; 6: aac(6″)-Ie-aph(2′)-Ia; 7: lsa(E); 8: negative control.

[0038] Figure 2 The results are specificity test results for the six-fold PCR detection method provided in Example 3 of this application, wherein M: DL2000 Marker; 1: Porcine Escherichia coli; 2: Porcine Pasteurella multocida; 3: Porcine Actinobacillus pleuropneumoniae; 4: Plasmodium parasuis; 5: Porcine Streptococcus positive strain; 7: patB; 8: optrA; 9: tet(O); 10: ermB; 11: aac(6″)-Ie-aph(2′)-Ia; 12: lsa(E); 13: Negative control.

[0039] Figure 3 The results of the sensitivity test for the six-fold PCR method provided in Example 4 of this application are shown; where M: DL2000 Marker: 1: 100 ng / μL; 2: 10 ng / μL; 3: 1 ng / μL; 4: 100 pg / μL; 5: 10 pg / μL; 6: 1 pg / μL; 7: 10 -1 pg / μL; 8:10 -2 pg / μL; 9:10 -3 pg / μL; 10:10 -4 pg / μL; 11: negative control.

[0040] Figure 4The results of the repeatability test for the six-fold PCR method provided in Example 5 of this application are shown, where M: DL2000 Marker; 1-3: three different positive strain templates; 4: negative control; AC: intra-batch repeat; DF: inter-batch repeat.

[0041] Figure 5 The results show a comparison between the six-fold PCR method provided in Example 6 of this application and the single-gene PCR detection method.

[0042] Figure 6 The results show a comparison between the six-fold PCR detection method provided in Example 6 of this application and the drug sensitivity test results of the micro-broth dilution method.

[0043] Figure 7 The statistical results of drug resistance genes carried by the Streptococcus suis isolate provided in Example 7 of this application. Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0045] The materials used in the following embodiments are not limited to those listed below, and other similar materials may be used instead. Unless otherwise specified, the instruments shall be used under conventional conditions or as recommended by the manufacturer. Those skilled in the art should have relevant knowledge of the use of conventional materials and instruments.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of this application pertains. Before a detailed description of this application, the following definitions are provided to better understand it.

[0047] In this application, unless the context clearly indicates otherwise, the terms “including,” “comprising,” “containing,” “having,” etc., shall be understood as open-ended and mean “including but not limited to.”

[0048] To better understand this teaching and without limiting its scope, all figures and other numerical values ​​used in the specification and claims to express quantities, percentages, or proportions should, in all cases, be understood to be modified by the term "about." Therefore, unless otherwise stated, the numerical parameters set forth in the following specification and appended claims are approximate values ​​that may vary depending on the desired properties sought. At a minimum, each numerical parameter should be interpreted based at least on the reported significant figures and by applying common rounding techniques.

[0049] The primer set provided in this application for detecting drug resistance genes of Streptococcus suis includes primer pairs for drug resistance genes patB, optrA, tet(O), ermB, aac(6″)-Ie-aph(2′)-Ia, and lsa(E), specifically:

[0050] The nucleotides of the upstream and downstream primers for amplifying the fluoroquinolone resistance gene patB are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively;

[0051] The upstream and downstream primers for amplifying the oxazolidinone resistance gene optrA are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0052] The upstream and downstream primers for amplifying the tetracycline resistance gene tet(O) are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively;

[0053] The upstream and downstream primers for amplifying the macrolide resistance gene ermB are shown in SEQ ID NO.7 and SEQ ID NO.8, respectively.

[0054] The upstream and downstream primers for amplifying the aminoglycoside resistance gene aac(6″)-Ie-aph(2′)-Ia are shown in SEQ ID NO.9 and SEQ ID NO.10, respectively.

[0055] The upstream and downstream primers for amplifying the truncated pleurodontia drug resistance gene lsa(E) are shown in SEQ ID NO.11 and SEQ ID NO.12, respectively.

[0056] In this application, specific primers were designed using Primer Primer 5 software based on the gene sequences of patB, optrA, tet(O), ermB, aac(6″)-Ie-aph(2′)-Ia, and lsa(E) in Streptococcus suis from the Comprehensive Antibiotic Resistance Database (CARD, https: / / card.mcmaster.ca / home). The six primer pairs were subjected to PCR amplification in the same system at the same annealing temperature, without interference between them, ensuring accurate amplification of the six drug resistance genes. The source of the primer sets is not particularly limited; primer synthesis methods well-known in the art can be used. In the embodiments of this application, the primers were synthesized by Wuhan Qingke Biotechnology Co., Ltd., Sangon Biotech (Shanghai) Co., Ltd., and Genscript Biotech Co., Ltd.

[0057] Based on this, embodiments of this application provide reagents or kits for detecting drug resistance genes in Streptococcus suis, wherein the reagents or kits include the aforementioned primer set. In addition, the kit preferably further includes the following reagents: Mg... 2+ The reagents include 10×PCR buffer, dNTPs, and Ex Taq enzyme. This application does not have specific restrictions on the source of the above reagents; any source well-known in the art may be used. In the embodiments of this application, Mg... 2+ The 10×PCR buffer, dNTPs, and Ex Taq enzyme were obtained from Nanjing Novizan Biotechnology Co., Ltd.

[0058] In some preferred embodiments, the kit further includes positive and / or negative controls, wherein the positive control is a mixed DNA template containing gene fragments from ermB, tet(O), patB, aac(6'')-Ie-aph(2'')-Ia, optrA, and lsa(E). The primary function of the positive control is to demonstrate that the entire PCR reaction system, from primers, enzymes, and buffers to the thermal cycling procedure, functions correctly in the current experiment. The negative control is a sample known to be free of any target nucleic acids (in this application, referring to the six drug resistance genes, including ermB and tet(O)) and is used to monitor whether the entire detection process is subject to nonspecific contamination or interference.

[0059] Based on this, embodiments of this application provide the application of the aforementioned primer set in the preparation of products for detecting Streptococcus suis drug resistance genes. The Streptococcus suis drug resistance genes include at least one of ermB, tet(O), patB, aac(6'')-Ie-aph(2'')-Ia, optrA, and lsa(E).

[0060] Based on this, this application provides a method for detecting drug resistance genes in Streptococcus suis for non-diagnostic purposes, which includes the following steps: using the genomic DNA of Streptococcus suis as a template, and using the aforementioned primer set, simultaneously amplifying ermB, tet(O), patB, aac(6'')-Ie-aph(2'')-Ia, optrA, and lsa(E) through multiplex PCR; analyzing the PCR amplification products, and determining whether the sample to be tested contains the corresponding drug resistance gene based on the presence or absence of specific amplification bands.

[0061] In this application, there are no special limitations on the method for extracting the genomic DNA of Streptococcus suis; any bacterial DNA extraction method well known in the art can be used. In the embodiments of this application, the EasyPure® Bacteria Genomic DNA Kit (containing RNase A) from Beijing TransGen Biotechnology Co., Ltd. was used for extraction.

[0062] In some preferred embodiments, the reaction system (50 μL) for the multiplex PCR is as follows:

[0063] 25 μL of 2×Rapid Taq Master Mix, 2 μL of DNA template, and primers for each gene are as follows: optrA 0.5 μL, ermB 0.75 μL, aac(6″)-Ie-aph(2′)-Ia 1 μL, patB and tet(O) 1.25 μL each, lsa(E) 1.5 μL, with a concentration of 10 μmol·L⁻¹; add ddH₂O to 50 μL.

[0064] In some preferred embodiments, the reaction procedure for the multiplex PCR is as follows:

[0065] Pre-denaturation at 95℃ for 5 min; followed by 35 cycles (denaturation at 95℃ for 15 s, annealing at 54℃ for 30 s, extension at 72℃ for 30 s); and finally extension at 72℃ for 10 min.

[0066] Based on this, embodiments of this application also provide a method for screening or preparing anti-streptococcal drugs, including the step of using the aforementioned primer set or the aforementioned kit to detect the drug resistance gene spectrum of the test strain.

[0067] The technical solution of this application and the technical effects achieved will be described in detail below with reference to more specific embodiments.

[0068] Example 1: Design and synthesis of primers for six-fold PCR detection of Streptococcus suis drug resistance genes

[0069] 1. Target gene selection: The core drug resistance genes corresponding to the six key antimicrobial drugs used in the clinical treatment of Streptococcus suis infection (fluoroquinolones, oxazolidinones, tetracyclines, macrolides, aminoglycosides, and truncated pleurotins) were selected as detection targets, namely: patB, optrA, tet(O), ermB, *aac(6″)-Ie-aph(2′)-Ia*, and lsa(E).

[0070] 2. Primer Design: Standard nucleotide sequences of the six genes in *Streptococcus suis* were obtained from the Comprehensive Antibiotic Resistance Database (CARD). Specific primers were designed within the conserved regions of each gene using Primer Premier 5.0 software. The design principle was to ensure no significant secondary structures or dimer formation between primer pairs, and that the final amplified products showed clear differences in length for easy electrophoresis differentiation. The designed primer sequences and expected product sizes are shown in Table 1.

[0071] Table 1. PCR primers for detecting drug resistance genes.

[0072]

[0073] 3. Primer synthesis: The 12 primers shown in Table 1 were synthesized by Wuhan Qingke Biotechnology Co., Ltd., Sangon Biotech (Shanghai) Co., Ltd., and Genscript Biotech Co., Ltd.

[0074] Example 2: Establishment and optimization of a six-fold PCR reaction system

[0075] 1. Template DNA preparation

[0076] Clinical isolates of Streptococcus suis (isolated and preserved by the Institute of Animal Husbandry and Veterinary Medicine, Hubei Academy of Agricultural Sciences) were inoculated into liquid culture medium and cultured overnight. Genomic DNA was extracted using a standard bacterial genomic DNA extraction kit (Beijing TransGen Biotech Co., Ltd.), and its concentration and purity were determined using an ultra-micro spectrophotometer (A260 / A280 ratio should be between 1.8 and 2.0). The DNA was then diluted to approximately 50 ng / μL as a working template.

[0077] 2. Single-gene PCR verification

[0078] First, use each primer pair in Table 1 above to perform singleton PCR amplification to verify the specificity and effectiveness of each primer pair.

[0079] Using DNA from multidrug-resistant strains carrying six drug resistance genes—patB, optrA, tet (O), ermB, aac (6″)-Ie-aph (2′)-Ia, and lsa (E)—as PCR templates, single-gene PCR amplification experiments were conducted using specifically designed primers for each drug resistance gene. The single-gene PCR reaction system (total system 20 μL) was prepared as follows: 10 μL of 2×RapidTaq Master Mix, 7 μL of ultrapure water, and 10 μmol·L⁻¹. -1 1 μL each of forward and reverse primers and 1 μL of DNA template were used. The reaction program was set as follows: 95 ℃ pre-denaturation for 5 min, followed by 30 cycles (95 ℃ denaturation for 15 s, annealing at the primer Tm value for 15 s, and extension at 72 ℃ for 30 s), with a final extension at 72 ℃ for 10 min after each cycle. After PCR amplification, the amplified product was loaded onto a 2% agarose gel and electrophoresed at 140 V for 25 min. After electrophoresis, the gel was photographed using a gel imaging system to observe and confirm that the target band was single and the fragment size was consistent with expectations. PCR products with acceptable bands were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were further uploaded to the NCBI database for sequence alignment analysis to verify the accuracy of the amplified fragments.

[0080] Electrophoresis confirmed that each single gene could amplify a specific band.

[0081] 3. Orthogonal optimization of the six-fold PCR system

[0082] To balance the amplification efficiency of the six primer pairs in the same reaction system and avoid competitive inhibition, a six-factor, five-level L25 (6^5) orthogonal experimental design was adopted to systematically optimize the final concentration, annealing temperature and PCR cycle number of each of the six primer pairs.

[0083] After optimization, the optimal 50 μL six-fold PCR reaction system was determined as follows:

[0084] 25 μL of 2×Rapid Taq Master Mix, 2 μL of DNA template, and primers for each gene as follows: optrA 0.5 μL, ermB 0.75 μL, aac(6″)-Ie-aph(2′)-Ia 1 μL, patB and tet(O) 1.25 μL each, lsa(E) 1.5 μL, and add ddH2O to 50 μL.

[0085] Optimal amplification program: pre-denaturation at 95 °C for 5 min; followed by 35 cycles of amplification (denaturation at 95 °C for 15 s, annealing at 54 °C for 30 s, extension at 72 °C for 30 s); and finally, final extension at 72 °C for 10 min.

[0086] Results Interpretation: Take 5 μL of PCR product, mix with loading buffer, and perform electrophoresis on a 2% agarose gel (containing GoldView nucleic acid dye) (120 V, 30 min). Observe and photograph the gel under a gel imaging system. Based on the expected product size in Table 1, compare with the DNA molecular weight standard (DL2000) to determine whether there are corresponding specific amplification bands in each sample.

[0087] Figure 1 The results of amplification using the optimized system on a positive control DNA template containing all six drug resistance genes are shown. The bands of all six genes are clearly visible, of the correct size, and there is no nonspecific amplification.

[0088] Example 3: Specificity validation of the six-fold PCR detection method

[0089] To verify the specificity of the method in this application, common porcine pathogens were selected as controls for amplification.

[0090] Test strains: Streptococcus suis positive strain (known to carry all six target genes), Escherichia coli, Pasteurella multocida, Actinobacillus pleuropneumoniae, and Haemophilus parasuis (formerly Haemophilus parasuis) were all clinical isolates, preserved by the Institute of Animal Husbandry and Veterinary Medicine, Hubei Academy of Agricultural Sciences.

[0091] 1. Method: Genomic DNA of each strain was extracted as a template according to the method described in Example 2, and amplification was performed using the optimized six-fold PCR system and program of Example 2.

[0092] 2. Results: such as Figure 2 As shown, only the template from the positive Streptococcus suis strain amplified all six clear, specific bands. The templates from the other four non-Streptococcus suis pathogens did not amplify any bands matching the target size, indicating that the six-fold PCR primer set and method of this application have high specificity for the six target genes of Streptococcus suis and show no cross-reactivity with other common swine bacteria.

[0093] Example 4: Sensitivity Validation of the Six-fold PCR Detection Method

[0094] To determine the limit of detection of the method of this application, the genomic DNA of Streptococcus suis positive strains was serially diluted 10-fold before detection.

[0095] 1. Method: Streptococcus suis DNA with an initial concentration of 100 ng / μL was serially diluted 10-fold with ultrapure water, successively to 10⁻¹, 10⁻², 10⁻³, and 10⁻⁻⁴. 4 10⁻ 5 10⁻ 6 Take 2 μL of DNA at each dilution as a template and perform hexapplication PCR amplification according to the method in Example 2.

[0096] result: Figure 3 The results are shown as a sensitivity test of the six-fold PCR method, where M: DL2000 Marker: 1: 100 ng / μL; 2: 10 ng / μL; 3: 1 ng / μL; 4: 100 pg / μL; 5: 10 pg / μL; 6: 1 pg / μL; 7: 10 -1 pg / μL; 8:10 -2 pg / μL; 9:10 -3 pg / μL; 10:10 -4 pg / μL; 11: negative control. (e.g., pg / μL) Figure 3 As shown, the amplified bands of all six drug resistance genes were obtained after diluting the DNA template to 10⁻⁻⁶. 5 It was still clearly visible at 1 pg / μL or approximately 2 pg of total template, and when diluted to 10⁻ 6 The bands disappeared upon application. Therefore, the limit of detection (LOD) of the six-fold PCR method established in this application is 10 pg / μL DNA for each target gene, which is highly sensitive and can meet the detection requirements of samples with low bacterial count or low DNA concentration.

[0097] Example 5: Repeatability Validation of a Six-fold PCR Detection Method

[0098] The repeatability of the method was evaluated at both the intra-batch and inter-batch levels.

[0099] 1. Intra-batch reproducibility: Using all PCR reagents prepared in the same batch, different operators independently tested the DNA templates of three known positive strains three times on the same day on different PCR instruments.

[0100] 2. Inter-batch repeatability: Three independent batches of PCR reaction reagents prepared at three different times (5 days apart) were used to detect the DNA templates of the above three positive strains.

[0101] 3. Results: such as Figure 4 As shown, in both intra-batch and inter-batch repeatability tests, all three positive strains stably amplified the expected six target gene bands, with consistent band brightness and a clean background. The negative controls showed no amplification. These results demonstrate that the six-fold PCR detection method established in this application possesses excellent repeatability and stability.

[0102] Example 6: Comparison and Validation of the Six-fold PCR Method and the Conventional Method

[0103] 1. Comparison with single-gene PCR

[0104] Twelve clinical isolates of Streptococcus suis were randomly selected and tested using the six-fold PCR method established in this application and the traditional single-gene PCR method (each of the six genes was detected separately).

[0105] The results are as follows Figure 5 As shown, the two methods yielded completely consistent results for the detection of all six genes in all strains, with a concordance rate of 100%. This demonstrates that the accuracy of the six-fold PCR method is equivalent to that of the "gold standard" single-gene PCR, but with significantly improved efficiency.

[0106] 2. Comparison with micro-broth dilution method for drug sensitivity testing

[0107] The minimum inhibitory concentrations (MICs) of the above 12 strains against the corresponding six classes of antibiotics were determined by micro-broth dilution method and compared with the genotype detection results of hexapolyPCR.

[0108] The results are as follows Figure 6As shown, for macrolides, tetracyclines, and oxazolidinones, the concordance rate between genotype (positive / negative) and phenotype (resistance / susceptibility) was 100% (12 / 12). For truncated pleurotins and fluoroquinolones, the concordance rate was 91.67% (11 / 12). For aminoglycosides, the concordance rate was 83.33% (10 / 12). Overall, the genotype detection method established in this application showed a high degree of consistency with the phenotypic drug susceptibility test results, indicating that detecting these resistance genes can effectively predict bacterial resistance phenotypes and provide a rapid and accurate reference for clinical drug use.

[0109] Example 7: Application of drug resistance gene detection in clinically isolated Streptococcus suis strains

[0110] The six-fold PCR detection method established in this application was used to screen for drug resistance genes in 62 clinical isolates of Streptococcus suis from pig farms in different regions of Hubei Province, including Suizhou and Xiangyang.

[0111] 1. Method: Genomic DNA was extracted from 62 bacterial strains according to the method in Example 2, and then amplified by hexaplex PCR.

[0112] 2. Results: Figure 7 The statistical results of drug resistance genes carried by Streptococcus suis isolates are shown in the figure. Among the 62 strains, the carrying rates of the six drug resistance genes were as follows: ermB (macrolides): 96.77% (60 / 62); tet(O) (tetracyclines): 80.65% (50 / 62); patB (fluoroquinolones): 79.03% (49 / 62); optrA (oxazolidinones): 40.32% (25 / 62); aac(6″)-Ie-aph(2′)-Ia (aminoglycosides): 19.35% (12 / 62); lsa(E) (truncated pleurotins): 16.13% (10 / 62).

[0113] The above results reveal the prevalence of multidrug resistance genes in Streptococcus suis in the investigated swine population. This application example demonstrates that the method described in this application can be used efficiently and conveniently for large-scale epidemiological surveys of drug resistance genes in clinical samples.

[0114] Example 8: Six-fold PCR Detection Kit

[0115] Based on the above embodiments, this application also provides a six-fold PCR detection kit for Streptococcus suis drug resistance genes, which comprises the following components:

[0116] PCR reaction solution A (lyophilized or liquid): contains the six pairs of specific primers shown in SEQ ID NO. 1-12 of Example 1, premixed at the optimal concentration ratio.

[0117] PCR reaction solution B: contains 2×Rapid Taq Master Mix (containing DNA polymerase, dNTPs, Mg²⁺ and optimized buffer).

[0118] Positive control: Mixed plasmid DNA or genomic DNA containing six gene-specific fragments: patB, optrA, tet(O), ermB, aac(6″)-Ie-aph(2′)-Ia, and lsa(E).

[0119] Negative control: sterile ultrapure water or DNA from a sensitive strain of Streptococcus suis that does not contain the target gene.

[0120] Instructions: Describe in detail the operating methods, reaction procedures, and result interpretation criteria in Example 2.

[0121] In summary, this application provides a six-fold PCR detection kit for Streptococcus suis drug resistance genes and its applications. Through carefully designed specific primer combinations and optimized reaction systems, it successfully achieves efficient and simultaneous detection of six key drug resistance genes. This method has significant advantages such as high specificity, high sensitivity, good repeatability, simple operation, and short detection cycle. Compared with existing technologies, its high-throughput characteristic is particularly outstanding, which can greatly improve detection efficiency and reduce costs. Validation experiments show that the detection results of this method are accurate and reliable, highly consistent with the results of single-gene PCR and drug susceptibility testing. It can be effectively applied to the rapid diagnosis of drug resistance in clinical isolates of Streptococcus suis, medication guidance, and epidemiological monitoring, and has important application value.

[0122] The present application has been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present application. The descriptions of the embodiments above are only for the purpose of helping to understand the present application and its core ideas. It should be noted that those skilled in the art can make several improvements and modifications to the present application without departing from the principles of the present application, and these improvements and modifications also fall within the protection scope of the claims of the present application.

Claims

1. A primer set for detecting Streptococcus suis drug resistance genes: characterized in that, The specific primers include the following nucleotide sequences as SEQ ID NO. 1~12.

2. The primer set according to claim 1, characterized in that, The primer set includes: The primer pair for the fluoroquinolone resistance gene patB has the nucleotide sequences as shown in SEQ ID NO. 1 and SEQ ID NO. 2; The primer pair for the oxazolidinone resistance gene optrA has the nucleotide sequences as shown in SEQ ID NO. 3 and SEQ ID NO. 4; The primer pair for the tetracycline resistance gene tet(O) has the nucleotide sequences as shown in SEQ ID NO. 5 and SEQ ID NO. 6; The primer pair for the macrolide resistance gene ermB has the nucleotide sequences as shown in SEQ ID NO. 7 and SEQ ID NO. 8; The primer pair for the aminoglycoside resistance gene aac(6'')-Ie-aph(2'')-Ia has the nucleotide sequences as shown in SEQ ID NO. 9 and SEQ ID NO. 10; The primer pair for the lentinan resistance gene lsa(E) has the nucleotide sequences as shown in SEQ ID NO. 11 and SEQ ID NO.

12.

3. Use of the primer set of claim 1 or 2 in the preparation of a product for detecting Streptococcus suis resistance genes, which include at least one of ermB, tet(O), patB, aac(6'')-Ie-aph(2'')-Ia, optrA and lsa(E).

4. A reagent for detecting a Streptococcus suis drug resistance gene, characterized by, The primer set of claim 1 or 2 is included.

5. A kit for detecting a Streptococcus suis drug resistance gene, characterized by, The reagent of claim 4 is included.

6. The kit of claim 5, wherein The kit also includes a 10x PCR buffer containing Mg 2+ , dNTPs, and ExTaq enzyme.

7. The kit according to claim 5 or 6, characterized in that, The kit further contains a positive control and / or a negative control, wherein the positive control is a mixed DNA template containing gene fragments of ermB, tet(O), patB, aac(6'')-Ie-aph(2'')-Ia, optrA and lsa(E).

8. A method for detecting a drug-resistant gene of Streptococcus suis for non-diagnostic purposes, characterized by, The following steps are included: Using the primer set of the first aspect or the reagent of the third aspect or the kit of the fourth aspect, the ermB, tet(O), patB, aac(6'')-Ie-aph(2'')-Ia, optrA and lsa(E) are simultaneously amplified by multiplex PCR reaction with Streptococcus suis DNA as template; The PCR amplification products are analyzed, and whether the corresponding resistance genes are contained in the sample to be tested is determined according to whether specific amplification bands exist.

9. The detection method according to claim 8, characterized in that, The reaction system of the multiplex PCR is as follows: 2x Rapid Taq Master Mix 25 μL, DNA template 2 μL, each gene primer as follows: optrA 0.5 μL, ermB 0.75 μL, aac(6'')-Ie-aph(2'')-Ia 1 μL, patB and tet(O) each 1.25 μL, lsa(E) 1.5 μL, and ddH2O to 50 μL; The reaction program of the multiplex PCR is as follows: 95 °C pre-denaturation for 5 min; followed by 35 cycles, each cycle including 95 °C denaturation for 15 s, 54 °C annealing for 30 s, 72 °C elongation for 30 s; final 72 °C terminal elongation for 10 min.

10. A method for screening or preparing anti-Streptococcus suis drugs, comprising the step of detecting the drug resistance gene profile of a test strain using the primer set of claim 1 or 2 or the kit of any one of claims 5-7.

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