Extraction-free direct-expansion rapid PCR (Polymerase Chain Reaction) detection method and kit for staphylococcus aureus and methicillin-resistant staphylococcus aureus

Abstract

The invention discloses a kit and a method for rapid PCR (Polymerase Chain Reaction) detection of Staphylococcus aureus (SA) and methicillin-resistant Staphylococcus aureus (MRSA) without nucleic acid extraction and direct expansion. The kit comprises a primer probe combination which is specifically designed aiming at a specific fragment of SA and an mecA gene of MRSA, wherein an MRSA primer is subjected to LNA base modification so as to improve the specificity. The kit is used for detecting the staphylococcus aureus and the methicillin-resistant staphylococcus aureus through multiple fluorescent PCR amplification. The method is simple, convenient and rapid to operate, the SA and MRSA detection sensitivity can reach 125 CFU / mL, the specificity is good, the method is suitable for various samples such as nasal swabs, sputum and blood culture, and an effective tool is provided for early rapid diagnosis of clinical SA and MRSA infection.

Description

Technical Field

[0001] This invention relates to a rapid PCR detection method and kit for Staphylococcus aureus and methicillin-resistant Staphylococcus aureus without extraction and direct amplification, belonging to the field of microbial detection technology. Background Technology

[0002] Staphylococci are one of the important pathogens causing hospital-acquired infections. Staphylococcus aureus (SA), also known as "Staphylococcus aureus", belongs to the genus Staphylococcus and is a representative of Gram-positive bacteria. It can produce a variety of toxins and invasive enzymes. This bacterium is characterized by its wide distribution, strong pathogenicity, and high drug resistance.

[0003] In recent years, due to the widespread use of hormones and immunosuppressants, as well as the overuse of broad-spectrum antibiotics, hospital-acquired infections caused by methicillin-resistant Staphylococcus aureus (MRSA) have been on the rise. Different MRSA strains arise from multiple Staphylococcus aureus strains independently acquiring a small staphylococcal chromosome (SCCmec), which contains genes encoding proteins that induce resistance in bacteria to most β-lactam antibiotics (such as methicillin). Multiple Staphylococcus aureus strains independently acquire this small chromosome. The development of MRSA is a result of the combination of widespread pathogenicity produced by Staphylococcus aureus, β-lactam resistance, and resistance in most strains to other classes of antibiotics. The methicillin resistance mecA gene plays a decisive role in staphylococcal drug resistance. This gene is carried on a unique mobile genetic element (SCCmec), which encodes penicillin-binding protein 2a (PBP2a) with extremely low affinity for β-lactam antibiotics. This prevents the antibiotics from inhibiting the synthesis of peptidoglycan in the cell wall, thus leading to drug resistance.

[0004] Staphylococcus aureus (SA) is a common bacterium that can cause a variety of infections, but it is usually sensitive to many antibiotics, including methicillin. Methicillin-resistant Staphylococcus aureus (MRSA), however, is a drug-resistant subtype of Staphylococcus aureus that is resistant to all β-lactam antibiotics (including methicillin, oxacillin, all penicillins, cephalosporins, and carbapenems), making treatment very difficult. Therefore, rapid and accurate differential diagnosis between Staphylococcus aureus and methicillin-resistant Staphylococcus aureus is particularly important.

[0005] Currently, the main bacteriological methods for detecting Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA) are culture, immunoassay, and whole-genome sequencing. Culture remains the "gold standard" for diagnosing Staphylococcus and its resistance to methicillin in many countries. However, this method is time-consuming, typically requiring more than 48 hours for results, which is detrimental to early diagnosis and treatment. Immunoassay relies on antigen-antibody reactions, which can lead to false positives and low sensitivity. Whole-genome sequencing, on the other hand, is time-consuming, costly, and requires high-quality samples. This significantly limits the clinical diagnosis of Staphylococcus aureus and MRSA. The lack of simple, rapid, and sensitive detection methods causes a large number of patients infected with Staphylococcus aureus and MRSA to miss the golden period for treatment, leading to more severe bacterial infections and even death. Therefore, there is an urgent need for a more sensitive, specific, and cost-effective method to detect Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA) in order to enable early detection, treatment, and prevention of bacterial infections caused by Staphylococcus aureus and MRSA. Summary of the Invention

[0006] To address the aforementioned issues of long processing time and high cost, this invention provides a rapid PCR detection kit and method for Staphylococcus aureus (SA) and methicillin-resistant Staphylococcus aureus (MRSA) using direct amplification without nucleic acid extraction. This method involves digesting the collected sputum sample, while other samples do not require digestion. After homogenization, nucleic acid is released. LNA base modification of the primers improves the specificity of PCR amplification while maintaining its sensitivity. Ultimately, the identification and detection of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus are achieved through complete synchronization of PCR product formation and fluorescence signal accumulation.

[0007] The first technical solution provided by this invention is a composition for detecting Staphylococcus aureus (SA) and methicillin-resistant Staphylococcus aureus (MRSA). The composition comprises two sets of primers and probes. The first set of primers and probes is used for specific detection of Staphylococcus aureus and includes a first forward primer, a first reverse primer, and a first probe. The sequence of the first forward primer is shown in any one of SEQ ID NO. 1 to 3, the sequence of the first reverse primer is shown in any one of SEQ ID NO. 4 to 6, and the sequence of the first probe is shown in any one of SEQ ID NO. 7 to 9. The second set of primers and probes is used for detection of methicillin-resistant Staphylococcus aureus and includes a second forward primer, a second reverse primer, and a second probe. The sequence of the second forward primer is shown in any one of SEQ ID NO. 10 to 12, the sequence of the second reverse primer is shown in any one of SEQ ID NO. 13 to 15, and the sequence of the second probe is shown in any one of SEQ ID NO. 16 to 18.

[0008] In some embodiments, the 5' ends of both the first and second probes are labeled with fluorescent reporter groups, and the 3' ends are labeled with fluorescent quencher groups.

[0009] In some embodiments, the fluorescent reporter group is selected from any one of FAM, VIC, HEX, CY5, CY3, JOE, and ROX; the fluorescent quencher group is selected from any one of BHQ1, BHQ2, BHQ3, and TAMRA.

[0010] In some embodiments, the second forward primer and the second reverse primer are modified with LNA at a distance of 8-10 bases from the 5' end.

[0011] In some embodiments, the first forward primer sequence is shown in SEQ ID NO.3, the first reverse primer sequence is shown in SEQ ID NO.6, and the first probe sequence is shown in SEQ ID NO.9; the second forward primer sequence is TACTGATTAA*CCCAGTAC, the second reverse primer sequence is TTAATAAGT*GAGGTGCGT, and the second probe sequence is ATAAGTGAGG*TGCGTTAAT, wherein the bases marked with * are all LNA-modified bases.

[0012] The second technical solution provided by the present invention is a kit for simultaneously detecting Staphylococcus aureus and drug resistance genes, wherein the kit includes the composition described in the first technical solution.

[0013] In some embodiments, the kit further includes a third set of primers and probes for amplifying an internal standard, the third set of probes including a third forward primer, a third reverse primer, and a third probe, the third forward primer sequence being shown in SEQ ID NO. 19, the third reverse primer sequence being shown in SEQ ID NO. 20, and the third probe sequence being shown in SEQ ID NO. 21.

[0014] In some embodiments, the 5' end of the third probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group.

[0015] In some embodiments, the kit further includes PCR reaction tubes (containing PCR reaction lyophilized bulbs).

[0016] Furthermore, the PCR reaction lyophilized pellets also include PCR buffer, Taq DNA polymerase, and lyophilization protectant.

[0017] In some embodiments, the PCR buffer is 5-20 mM Tris-HCl, 5-15 mM MgCl2, 20-50 mM NaCl, and 15-25 mM... dNTPs.

[0018] Furthermore, the concentration of the Taq DNA polymerase is 1U to 3U; Furthermore, the freeze-drying protectant includes several of the following: sugars, proteins, and polyols, such as sucrose, trehalose, PEG20000, BSA, mannitol, etc.

[0019] In some embodiments, the kit may also include, but is not limited to, 0.1%-0.5% DMSO, 1%-3% betaine, 1%-10% phosphate buffer, etc.

[0020] In some embodiments, the preparation process of the PCR lyophilized pellets includes: preparing a PCR reaction lyophilization system comprising 5 μL 5×Buffer, 1.4 μL dNTP (25 mM), 1 μL Taq DNA polymerase (1000 U / mL), and 6 μL lyophilization protectant; wherein the 5×Buffer is composed of 20 mM Tris-HCl, 10 mM MgCl2, 12.5 mM NaCl, The PCR reaction lyophilization system consists of 0.8 mM dNTPs, a final primer concentration of 0.24 μmol, and a final probe concentration of 0.12 μmol. The lyophilization protectant includes 4% trehalose, 20% PEG20000, 7% mannitol, and 5% BSA. Prepare the PCR reaction lyophilization system according to the above formula ratio, mix thoroughly, and then add 25 μL dropwise to liquid nitrogen to form spheres. Transfer the spheres to a lyophilizer for lyophilization. The resulting lyophilized PCR reaction spheres are then obtained.

[0021] In some embodiments, the kit also includes a nucleic acid release agent and a diluent.

[0022] Furthermore, the nucleic acid releasing agent comprises 5%-15% Triton X-100, 1%-5% PVP K30, 1%-20% sodium hydroxide, 50mM-200mM guanidine hydrochloride, 50mM-150mM tris-HCl, and 0.1%-1% SDS.

[0023] Furthermore, the diluent comprises one of DNA and RNase-free deionized water and TE buffer.

[0024] In some embodiments, the kit further includes a positive control and a negative control; Positive controls include pUC57 plasmid containing a synthetic Staphylococcus aureus-specific fragment sequence at a concentration of 1×10⁻⁶. 3 ~1×10 6 pUC57 plasmid containing the synthetic methicillin-resistant Staphylococcus aureus mecA gene sequence at a concentration of 1×10⁻¹mL.3 ~1×10 6 The sample contained copies / mL of pUC57 plasmid with a synthetic GAPDH gene sequence at a concentration of 1×10⁻⁶. 3 ~1×10 5 copies / mL; The negative control was a pUC57 plasmid containing a synthetic GAPDH gene sequence at a concentration of 1×10⁻⁶. 3 ~1×10 5 copies / mL.

[0025] The third technical solution provided by the present invention is a method for simultaneously detecting Staphylococcus aureus and drug resistance genes for non-disease diagnosis purposes. The method is to use the kit described in the second technical solution to detect Staphylococcus paraaureus.

[0026] In some embodiments, the method includes the following steps: (1) Extract genomic DNA from the sample to be tested or directly dilute the sample to be tested; (2) Construct a PCR reaction system using the composition described in the first technical solution, perform amplification, and obtain PCR amplification products; (3) The PCR amplification products obtained in step (2) were analyzed using a PCR fluorescence detection system, and the detection results were characterized by fluorescence curves.

[0027] In some implementations, in step (1), when the sample is sputum, it needs to be digested before the nucleic acid is released.

[0028] Furthermore, nucleic acid extraction was performed using magnetic bead method, thermal lysis, chemical lysis, and ultrasonic lysis.

[0029] Optionally, a chemical thermal lysis method can be used for nucleic acid release. The specific method is as follows: If the sample to be tested is a sputum sample, first add 4 times the volume of sputum digestion solution to the sputum sample, shake to mix, and digest at 37°C for 10-15 min; then mix the liquefied sample and nucleic acid release agent at a ratio of 1:4 and lyse at 85°C for 5 min; if the sample to be tested is a nasal swab or blood culture sample, directly mix the sample and nucleic acid release agent at a ratio of 1:4 and lyse at 85°C for 5 min.

[0030] Considering that the collected sputum samples may contain some unknown interfering substances that could affect the subsequent PCR amplification reaction, the lysed nucleic acid was further diluted. The specific treatment method is as follows: 100 μL of the lysed nucleic acid was added to 200 μL of diluent and mixed thoroughly. The mixture was then centrifuged at 6000 rpm for 3 min. Subsequently, 25 μL of the supernatant was taken to dissolve the lyophilized PCR reaction pellets.

[0031] In some embodiments, in step (2), the amount of primers used in the PCR reaction system is 0.24 μM and the amount of probes is 0.12 μM.

[0032] In some implementations, the PCR reaction volume in step (2) is 25 μL.

[0033] In some implementations, in step (2), the PCR amplification program is as follows: pre-denaturation at 95°C for 1 min, then denaturation at 95°C for 1 s, annealing at 60°C for 20 s, for 10 cycles; finally, denaturation at 95°C for 1 s, annealing at 55°C for 20 s, for 35 cycles, and fluorescence is collected.

[0034] The fourth technical solution provided by the present invention is the application of the composition described in the first technical solution or the kit described in the second technical solution in detecting Staphylococcus aureus and / or methicillin-resistant Staphylococcus aureus for non-disease diagnosis purposes.

[0035] Compared with the prior art, the beneficial effects of the present invention are as follows: Using the primer combination of this invention, 1.25 × 10⁻⁶ can be detected simultaneously. 2 It detects Staphylococcus aureus and methicillin-resistant Staphylococcus aureus at CFU / mL; it has high sensitivity and accuracy, providing strong support for clinical detection of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus infections.

[0036] The primer-probe combination of the present invention artificially modifies the MRSA amplification primers with LNA, which significantly improves the detection specificity of the system. At the same time, an enhancer is added to the reaction system to further improve the anti-interference ability of the entire system.

[0037] The Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA) detection kit of this invention is compatible with various sample types. It eliminates the need for complex extraction and purification processes, requiring only a single lysis step to obtain nucleic acid, completely freeing up the hands of testing personnel. Furthermore, the entire detection process is conducted under closed conditions, avoiding false positives and environmental contamination. The PCR reaction is completed within just 30 minutes, making the operation simple and the results easy to read. This facilitates the early diagnosis of Staphylococcus aureus and MRSA, enabling timely control of the disease. Attached Figure Description

[0038] Figure 1 The results of detecting Staphylococcus aureus in this embodiment of the invention are shown.

[0039] Figure 2 The results of detecting methicillin-resistant Staphylococcus aureus in this embodiment of the invention are shown.

[0040] Figure 3 This is the detection result of a methicillin-resistant Staphylococcus aureus-negative sample in an embodiment of the present invention.

[0041] Figure 4 This is a comparison of the amplification efficiency of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus in the experimental examples of this invention.

[0042] Figure 5 This is a comparison of the amplification efficiency of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus in the experimental examples of this invention.

[0043] Figure 6 The results of comparative testing of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus between this invention and commercially available products are presented. Detailed Implementation

[0044] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0045] The strains involved in the following examples: Purchased from the American Type Culture Collection (ATCC) of the United States: Staphylococcus aureus (SA, NO. ATCC 25923), Methicillin-resistant Staphylococcus aureus (MRSA, NO. ATCC 43300), Candida albicans (CA, NO. ATCC 10231DQ), Escherichia coli (Ec, NO. ATCC 43892), Klebsiella pneumoniae (KP, NO. ATCC 51504), Acinetobacter baumannii (Ab, NO. ATCC 19606), Neisseria meningitidis (Nm, NO. ATCC 13102), and Staphylococcus aureus (NO. ATCC 25923). 19606) Streptococcus pneumoniae (SP, NO. ATCC 49619DQ), Haemophilus influenzae (HI, NO. ATCC 51907DQ), Salmonella enterica (Se, NO. ATCC 13314), Mycoplasma pneumoniae (MP, NO. ATCC 15531), Bordetella pertussis (Bp, NO. ATCC 12743), Enterococcus (NO. ATCC 14025), Aspergillus fumigatus (AF, NO. ATCC 1022DQ), Cryptococcus neoformans (CN, NO. ATCC 32045), Neisseria meningitidis. meningitidis (Nm, NO. ATCC 35562) and Proteus species (NO. ATCC 43071) Example 1: Design of primer and probe composition for detecting Staphylococcus aureus and methicillin-resistant Staphylococcus aureus In this embodiment, primers and probes for the Staphylococcus aureus-specific nuc gene and the methicillin-resistant Staphylococcus aureus mecA gene were designed using Premier 5 biological software. All DNA nucleotide sequences were synthesized by Sangon Biotech (Shanghai) Co., Ltd. Sequences with fluorescent group modifications and LNA modifications were purified by HPLC, while other unlabeled DNA nucleic acid chains were purified by ULTRAPAGE.

[0046] The final nucleotide sequences of the primers are shown in Table 1 below.

[0047] Table 1 Primer base sequence listing

[0048] Among them, SEQ ID NO.7-9 has a 5' end labeled with the FAM group and a 3' end labeled with the BHQ1 group; SEQ ID NO.16-18 has a 5' end labeled with the CY5 group and a 3' end labeled with the BHQ2 group; SEQ ID NO.21 has a 5' end labeled with the VIC group and a 3' end labeled with the BHQ1 group.

[0049] All bases marked with * are LNA-modified bases.

[0050] Example 2: Preparation method of PCR reaction system for Staphylococcus aureus and methicillin-resistant Staphylococcus aureus The kit components include: PCR reaction lyophilized bulbs of the primer combination from Example 1 and nucleic acid release agent; The PCR reaction lyophilized pellets contain: 5 μL 5× Buffer, 1.4 μL dNTP (25 mM), 1 μL Taq DNA polymerase (1000 U / mL), and 6 μL lyophilization protectant; the lyophilization protectant includes 4% trehalose, 20% PEG20000, 7% mannitol, and 5% BSA.

[0051] 5×Buffer consists of Tris-HCl, MgCl2, NaCl, dNTPs The composition of the reaction system is as follows: Primer SAF: 0.24 μmol; Primer SAR: 0.24 μmol; Probe SAP: 0.12 μmol; Primer MRSA F: 0.24 μmol; Primer MRSA R: 0.24 μmol; Probe MRSA P: 0.12 μmol; Tris-HCl: 20mM; MgCl2: 10mM; NaCl: 12.5 mM; dNTPs: 0.8 mM; The nucleic acid release agent includes 0.1% SDS, 150mM guanidine hydrochloride, 5% sodium hydroxide, 8% Triton X-100, 3% PVP K30, and 50mM Tris-HCl.

[0052] Prepare the PCR reaction lyophilization system according to the above formula ratio, mix it evenly, and add 25 μL dropwise to liquid nitrogen to form pellets. Then transfer the pellets to a lyophilizer for lyophilization. After lyophilization, the PCR reaction lyophilized pellets are obtained.

[0053] Example 3: Detection method for amplified Staphylococcus aureus and methicillin-resistant Staphylococcus aureus. 1. This embodiment utilizes a kit to detect samples from various sources, including but not limited to nasal swabs, sputum, and blood culture samples. To better illustrate the detection effect of this application, the above three types of samples are used as examples.

[0054] 2. The sputum sample processing procedure is as follows (nasal swabs and blood culture samples do not require pretreatment): Add 2-3 mL of sputum sample to a 50 mL test tube with a screw cap. Depending on the viscosity of the sputum, add 4 times the volume of sputum digestion solution to the sputum tube. Tighten the screw cap, vortex for 1 minute, and digest at 37°C for 10-15 minutes to fully liquefy the sputum.

[0055] 3. Rapid nucleic acid extraction: Take 50 μL of pretreated sputum sample or mixed nasal swab and blood culture sample and add it to a 200 μL lysis buffer tube. Shake and mix well, incubate at 85℃ for 5 min, and mix for 3-5 s before use. Positive control and negative control samples are processed simultaneously with the test sample.

[0056] 4. Sample addition: Transfer 100 μL of the incubation product to a 200 μL dilution tube, mix thoroughly, and then transfer 25 μL to a PCR reaction tube to dissolve the PCR reaction lyophilized bulbs prepared in Example 2. Centrifuge briefly and set aside.

[0057] 5. PCR amplification a) Place the PCR reaction tube into the sample slot of the amplification instrument for testing and set the sample name.

[0058] b) Instrument fluorescence channel selection: Select the FAM channel to detect the Staphylococcus aureus-specific fragment nuc gene; select the VIC / HEX channel to detect the internal control; select the CY5 channel to detect the methicillin-resistant mecA gene.

[0059] Table 2 PCR amplification conditions settings

[0060] C) After setting up, press the "Run" button to start the test.

[0061] 7. Quality Control: Positive control samples: FAM, CY5, and IC (internal standard) channels: Ct≤26; Negative control samples: FAM and CY5 channels have no Ct value, VIC (internal standard) channel Ct < 26; All of the above requirements must be met simultaneously in an experiment. Otherwise, the experiment is considered invalid and needs to be retested.

[0062] 8. Result determination: The FAM channel detection showed a clear amplification curve, and the detection Ct value was ≤30, indicating a positive result for Staphylococcus aureus. The CY5 channel showed a clear amplification curve, and the detection Ct value was ≤30, indicating a positive result for methicillin-resistant antibodies. If no obvious amplification curve or no value is detected in the FAM or CY5 channel, and an obvious amplification curve is detected in the VIC (internal standard) channel with a detection Ct value ≤26, it is determined that Staphylococcus aureus and methicillin-resistant bacteria are positive or the concentration is lower than the detection limit of the kit. If the FAM or CY5 channel shows no obvious amplification curve or no value, or if the VIC (internal standard) channel shows no obvious amplification curve or a Ct value > 26, the test is invalid. Clinical resampling and retesting are recommended. Specific result interpretation criteria are shown in Table 3. Table 3 Criteria for Judging Test Results

[0063] Example 4: Screening of Detection Compositions for Staphylococcus aureus and Methicillin-resistant Staphylococcus aureus The primers and probes shown in Example 1 were serially diluted to 10⁻¹⁰ with the standard strains from the American Type Culture Collection (ATCC) according to the method described in Example 3. 5 cfu / mL, 10 4 cfu / mL, 10 3 cfu / mL, 5×10 2 Screening and detection were performed using cfu / mL.

[0064] The Staphylococcus aureus SA primer-probe combinations 1 (SEQ ID NO. 1, SEQ ID NO. 4, SEQ ID NO. 7), 2 (SEQ ID NO. 2, SEQ ID NO. 5, SEQ ID NO. 8), and 3 (SEQ ID NO. 3, SEQ ID NO. 6, SEQ ID NO. 9) designed according to this invention were used to detect serially diluted Staphylococcus aureus (25923) and Staphylococcus aureus negative samples. Specific detection results are shown in Table 4. Analysis of the detection Ct values ​​shows that primer combination 3 has a smaller Ct value for detecting low concentrations of the target, indicating higher sensitivity; and there was no cross-contamination with other negative samples. Therefore, the designed primer-probe combination 3 (SEQ ID NO. 3, SEQ ID NO. 6, SEQ ID NO. 9) was selected as the optimal primer-probe combination for detecting Staphylococcus aureus.

[0065] The methicillin-resistant Staphylococcus aureus (MRSA) primer and probe combinations 1 (SEQ ID NO. 10, SEQ ID NO. 13, SEQ ID NO. 16), 2 (LNA-modified bases of SEQ ID NO. 10, SEQ ID NO. 13, SEQ ID NO. 16), 3 (SEQ ID NO. 11, SEQ ID NO. 14, SEQ ID NO. 17), 4 (LNA-modified bases of SEQ ID NO. 11, SEQ ID NO. 14, SEQ ID NO. 17), 5 (SEQ ID NO. 12, SEQ ID NO. 15, SEQ ID NO. 18), and 6 (LNA-modified bases of SEQ ID NO. 12, SEQ ID NO. 15, SEQ ID NO. 18) designed according to this invention were used to detect serially diluted methicillin-resistant Staphylococcus aureus (25923) and methicillin-resistant Staphylococcus aureus negative samples. (See illustrations.) Figure 1 The specific detection results are shown in Table 5. Analysis of the Ct values ​​shows that primer combination 4 exhibits a smaller Ct value and higher sensitivity for detecting low-concentration targets; furthermore, there is no cross-contamination with other samples. Therefore, the designed primer-probe combination 4 (SEQ ID NO. 11, SEQ ID NO. 14, SEQ ID NO. 17 with LNA-modified bases) was selected as the optimal primer-probe combination for detecting methicillin-resistant Staphylococcus aureus.

[0066] Table 4. Statistical analysis of primer-probe combination screening results for Staphylococcus aureus

[0067] Table 5. Statistical analysis of primer-probe combination screening results for methicillin-resistant Staphylococcus aureus.

[0068] Using the Staphylococcus aureus primers and probes (SEQ ID NO. 3, SEQ ID NO. 6, SEQ ID NO. 9) and methicillin-resistant Staphylococcus aureus (SEQ ID NO. 11, SEQ ID NO. 14, SEQ ID NO. 17) designed and screened according to this invention, different concentrations of Staphylococcus aureus (25923) or methicillin-resistant Staphylococcus aureus (43300) were detected. According to Table 4, the results show that this kit has high sensitivity for detecting Staphylococcus aureus, and the results are as expected; according to Table 5, Figure 3 The results showed that the primer combinations without LNA modification all exhibited a certain degree of nonspecific amplification in methicillin-resistant negative samples, failing to meet normal detection requirements. The primer combinations with LNA modification specifically designed in this invention showed no Ct values ​​in the detection of methicillin-resistant negative samples, and the sensitivity for detecting methicillin-resistant Staphylococcus aureus was almost identical to that of the primer combinations without LNA modification, indicating that the composition of this invention can accurately distinguish between Staphylococcus aureus and methicillin-resistant Staphylococcus aureus.

[0069] Example 5: Optimization of the reaction system for the detection kit for Staphylococcus aureus and methicillin-resistant Staphylococcus aureus. 1. Optimization of PCR reaction system Considering the rapid lysis of nucleic acid release agents, some impurities in the extraction reagents could affect the test results. To further improve the anti-interference ability of the reaction system, some enhancers were artificially added, such as 0.1%-0.5% DMSO, 1%-3% betaine, and 1%-10% phosphate buffer, in three low-to-medium concentration culture samples. The test results showed that compared with the reaction system without enhancers, the PCR reaction system with 0.2% DMSO had better amplification efficiency and stronger anti-interference ability. The comparative test results are shown below. Figure 4 .

[0070] Considering the high interfering substances in sputum samples, to further enhance the anti-interference ability of the reaction system, the lysed nucleic acid was diluted before reconstitution of the lyophilized bulbs. Three different dilution ratios—1:2 (100uL + 200uL), 1:4 (100uL + 400uL), and 1:8 (100uL + 800uL)—were evaluated, and three low-concentration culture samples were tested. The results showed that the 1:2 (100uL + 200uL) dilution provided better amplification efficiency for low-concentration samples; therefore, the ratio of lysis buffer to diluent was determined to be 1:2. Comparative test results are shown below. Figure 5 .

[0071] 2. PCR reaction program optimization Due to the methicillin-resistant Staphylococcus aureus (MRSA) resistance mechanism and its mutation frequency, repeated testing revealed that using the standard procedure increased the probability of non-specific amplification. Therefore, the standard PCR procedure was modified by artificially setting the first 10 cycles to not collect fluorescence and simultaneously increasing the annealing temperature in this stage. This reduced the probability of primer dimer and other non-specific fragment amplification, thus improving the specificity of the reaction system. Then, the annealing temperature was lowered in the subsequent 35 cycles to further enhance the sensitivity of the reaction system.

[0072] Table 6. Specific PCR amplification procedures before and after optimization.

[0073] By comparing the probabilities of nonspecific amplification in repeated testing of negative samples and cross-samples under different procedures, the results show that the optimized PCR procedure can effectively block nonspecific amplification without affecting the sensitivity of the system. Specific detection results are shown in Table 7.

[0074] Table 7 Statistical analysis of detection results for different amplification procedures

[0075] The optimal reaction system in this embodiment is: The PCR reaction lyophilized pellets contain: 5 μL 5× Buffer, 1.4 μL dNTP (25 mM), 1 μL Taq DNA polymerase (1000 U / mL), and 6 μL lyophilization protectant; the lyophilization protectant includes 4% trehalose, 20% PEG20000, 7% mannitol, and 5% BSA.

[0076] 5×Buffer consists of Tris-HCl, MgCl2, NaCl, dNTPs The composition of the reaction system is as follows: Primer SAF3: 0.24 μmol; Primer SAR3: 0.24 μmol; Probe SA P3: 0.12 μmol; Primer MRSA F2-2: 0.24 μmol; Primer MRSA R2-2: 0.24 μmol; Probe MRSA P2: 0.12 μmol; Tris-HCl: 20mM; MgCl2: 10mM; NaCl: 12.5 mM; dNTPs: 0.8 mM; The nucleic acid release agent includes 0.1% SDS, 150mM guanidine hydrochloride, 5% sodium hydroxide, 8% Triton X-100, 3% PVP K30, 50mM Tris-HCl, and 0.2% DMSO.

[0077] The diluent includes DEPC water.

[0078] Example 6: Detection sensitivity of the Staphylococcus aureus and methicillin-resistant Staphylococcus aureus detection kit Staphylococcus aureus and methicillin-resistant Staphylococcus aureus were serially diluted to 5 × 10⁻⁶ using nasal swabs, sputum, and blood culture-negative samples, respectively. 2 cfu / mL, 2.5×10 2 cfu / mL, 1.25×10 2 CFU / mL and 62.5 CFU / mL were used as test samples. Each gradient was repeated 20 times. Three batches of the composition were used. The best Staphylococcus aureus primers and probes (SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.9) from Example 3 and methicillin-resistant Staphylococcus aureus primers and probes (SEQ ID NO.13, SEQ ID NO.19, SEQ ID NO.23) were used for detection in the best detection system from Example 5. The limit of detection was set at 95%.

[0079] As shown in Table 8, after 20 tests on each of the three test samples (nasal swabs, sputum, and blood cultures) using three batches of combined reagents, the positive sample concentration was 1.25 × 10⁻⁶. 2 The positive detection rate was above 95% at CFU / mL, indicating high sensitivity. This demonstrates that the primer-probe combination reagent of this application exhibits high sensitivity for the detection of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus, and can be used as a kit for nucleic acid detection of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus.

[0080] Table 8. Statistical analysis of the detection results for the three types of samples.

[0081] Example 7: Specificity and interference resistance of the detection kit for Staphylococcus aureus and methicillin-resistant Staphylococcus aureus. 1. Specificity: Eighteen pathogens that are homologous to the Staphylococcus aureus and methicillin-resistant Staphylococcus aureus detection kits and are likely to cause the same or similar clinical symptoms were used as cross-reactive samples. The sample information and test results are shown in Table 9.

[0082] Table 9 Results of Cross-Reactivity Sample Detection

[0083] A Staphylococcus aureus strain ST239-III-t030, which is positive for both the nuc and mecA resistance genes, was selected. Based on the culture concentration, a pretreated Staphylococcus aureus-specific fragment and a methicillin-resistant mecA gene-negative matrix sample were diluted to a 2×LOD concentration to prepare Sample 1. A Staphylococcus aureus strain ST5-Ⅱ-t002, which is positive for both the nuc and mecA resistance genes, was selected. Based on the culture concentration, a pretreated Staphylococcus aureus-specific fragment and a methicillin-resistant mecA gene-negative matrix sample were diluted to a 2×LOD concentration to prepare Sample 2. Both were mixed with the aforementioned 18 cross-reactive substances to prepare Mixed Sample 1 and Mixed Sample 2. Detection was performed using different batches of the kit from this invention, and the results are shown in Tables 10 and 11.

[0084] Table 10. Cross-reactivity test results of mixed sample 1

[0085] Table 11 Results of cross-reactivity test for mixed sample 2

[0086] As can be seen from the test results in Table 9, the cross-reactivity specificity for the 18 pathogens was negative after testing with the three batches of test reagents of this invention, and no false positive results were found.

[0087] As shown in Tables 10 and 11, after mixing 18 cross-reactive pathogens with reference standards of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus at different LOD concentrations for different genotypes, all three batches of the detection reagents of this invention successfully detected the reference standards at the LOD concentrations. Therefore, this invention can eliminate cross-reactivity between different pathogens and accurately detect Staphylococcus aureus and methicillin-resistant Staphylococcus aureus.

[0088] 2. Interfering Substances: For endogenous interfering substances that may be present in the human body and exogenous interfering substances (prescription drugs, over-the-counter drugs, supplements) that may be present in the patient's body, and based on international standards (CLSI EP07, WS / T416-2013), regulatory guidelines, clinical pathology, and pharmacokinetic data, the concentrations of the following substances that may be present in the human body will be detected. Reference samples of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus with different LOD concentrations of the above-mentioned genotypes were added to the following interfering substances as test samples 1 and 2. Three batches of the kit were used to detect test samples 1 and 2, and the detection results are shown in Table 12.

[0089] Table 12 Detection results of different concentrations of interfering substances

[0090] As can be seen from Table 12, the present invention can still accurately detect Staphylococcus aureus and methicillin-resistant Staphylococcus aureus reference samples with different genotypes and LOD concentrations even under different concentrations of exogenous interfering substances, and the Ct value is not significantly different from the control.

[0091] In summary, under different interfering conditions, this invention can accurately detect Staphylococcus aureus and methicillin-resistant Staphylococcus aureus in the test sample.

[0092] Therefore, the present invention has strong anti-interference ability against various interfering substances and good specificity against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus.

[0093] Comparative Example The primer and probe combination of this application was compared and evaluated with commercially available products. First, comparisons were made in terms of sample type, sample processing method, kit composition, PCR reaction time, and detection limit, as shown in Table 13. It is evident that the kit of this invention is adaptable to different sample types. The sample nucleic acid acquisition method eliminates complex nucleic acid extraction and purification steps, requiring only one lysis step, completely freeing up the hands of testing personnel and saving intermediate time for faster test results. Furthermore, the reagent is in a fully premixed lyophilized form, eliminating the need for on-site preparation and cold chain transportation, making it simple and convenient to use. The PCR reaction time is only 30 minutes, significantly reducing patient waiting time and providing a powerful tool for detecting bacterial infections caused by Staphylococcus aureus and methicillin-resistant Staphylococcus aureus. Finally, the accuracy, repeatability, and specificity of this invention and commercially available products were evaluated using low-to-medium concentrations of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus reference samples and cross-samples. The detection results are shown in Table 14. Figure 6 As shown.

[0094] Table 13 Comparison of the present invention combination and commercially available products

[0095] Table 14 Detection Ct values ​​of the combined and comparative kits of the present invention

[0096] From Table 14 and Figure 6It is known that when the template concentration is 2500 CFU / mL, the Ct value of the primer and probe combination of this application is smaller than that of commercially available products, and the coefficient of variation (CV) of the precision calculation of SA and MRSA is less than 5.0%, indicating excellent repeatability. When the template concentration is 500 CFU / mL, the primer and probe combination of this application can effectively detect the sample, but the detection rate of commercially available products is less than 50%. Therefore, the primer and probe combination of this invention can effectively avoid missed detection when detecting low-concentration samples.

[0097] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A composition for detecting Staphylococcus aureus and methicillin-resistant Staphylococcus aureus, characterized in that, The composition comprises two sets of primer probes; The first set of primers and probes is used for the specific detection of Staphylococcus aureus, including a first forward primer, a first reverse primer and a first probe. The sequence of the first forward primer is shown in any one of SEQ ID NO.1~3, the sequence of the first reverse primer is shown in any one of SEQ ID NO.4~6, and the sequence of the first probe is shown in any one of SEQ ID NO.7~9. The second set of primers and probes is used to detect methicillin-resistant Staphylococcus aureus, including a second forward primer, a second reverse primer, and a second probe. The second forward primer sequence is shown in any one of SEQ ID NO. 10-12, the second reverse primer sequence is shown in any one of SEQ ID NO. 13-15, and the second probe sequence is shown in any one of SEQ ID NO. 16-18.

2. The composition according to claim 1, characterized in that, Both the first and second probes have fluorescent reporter groups labeled at their 5' ends and fluorescent quencher groups labeled at their 3' ends; The second forward primer and the second reverse primer are used to modify LNA by 8-10 bases from the 5' end.

3. The composition according to claim 1 or 2, characterized in that, The first forward primer sequence is shown in SEQ ID NO.3, the first reverse primer sequence is shown in SEQ ID NO.6, and the first probe sequence is shown in SEQ ID NO.9; the second forward primer sequence is shown in SEQ ID NO.11, wherein the 10th base at the 5' end is an LNA-modified base; the second reverse primer sequence is shown in SEQ ID NO.14, wherein the 9th base at the 5' end is an LNA-modified base; and the second probe sequence is shown in SEQ ID NO.15, wherein the 10th base at the 5' end is an LNA-modified base.

4. A kit for simultaneously detecting Staphylococcus aureus and drug resistance genes, characterized in that, The kit includes the composition according to any one of claims 1 to 3.

5. The reagent kit according to claim 4, characterized in that, The kit also includes a third set of primers and probes for amplifying the internal standard. The third set of probes includes a third forward primer, a third reverse primer, and a third probe. The sequence of the third forward primer is shown in SEQ ID NO.19, the sequence of the third reverse primer is shown in SEQ ID NO.20, and the sequence of the third probe is shown in SEQ ID NO.

21.

6. The reagent kit according to claim 5, characterized in that, The third probe is labeled with a fluorescent reporter group at its 5' end and a fluorescent quencher group at its 3' end.

7. The reagent kit according to any one of claims 4 to 6, characterized in that, The kit also includes PCR reaction lyophilized pellets, which further include PCR buffer, Taq DNA polymerase, and lyophilization protectant.

8. The reagent kit according to claim 7, characterized in that, The PCR buffer consisted of 5-20 mM Tris-HCl, 5-15 mM MgCl2, 20-50 mM NaCl, and 15-25 mM... dNTPs ; The concentration of the Taq DNA polymerase is 1U~3U; The freeze-drying protectant includes one or more of sugars, proteins, and polyols.

9. The kit according to claim 4 or 5, characterized in that, The kit also includes 0.1%-0.5% DMSO, 1%-3% betaine, and 1%-10% phosphate buffer.

10. The kit according to claim 4 or 5, characterized in that, The kit also includes a nucleic acid release agent and a diluent.

11. The reagent kit according to claim 10, characterized in that, The nucleic acid releasing agent comprises 5%-15% Triton X-100, 1%-5% PVP K30, 1%-20% sodium hydroxide, 50mM-200mM guanidine hydrochloride, 50mM-150mM tris-HCl, and 0.1%-1% SDS. The diluent includes one of DNA and RNase-free deionized water and TE buffer.

12. The reagent kit according to claim 5, characterized in that, The kit also includes a positive control and a negative control; Positive controls include pUC57 plasmid containing a synthetic Staphylococcus aureus-specific fragment sequence at a concentration of 1×10⁻⁶. 3 ~1×10 6 pUC57 plasmid containing the synthetic methicillin-resistant Staphylococcus aureus mecA gene sequence at a concentration of 1×10⁻¹mL. 3 ~1×10 6 copies / mL, and pUC57 plasmid containing the artificially synthesized GAPDH gene sequence, at a concentration of 1×10 3 ~1×10 5 copies / mL; The negative control was a pUC57 plasmid containing a synthetic GAPDH gene sequence at a concentration of 1×10⁻⁶. 3 ~1×10 5 copies / mL.

13. A method for simultaneously detecting Staphylococcus aureus and drug resistance genes for non-disease diagnostic purposes, characterized in that, The method is to detect Staphylococcus paraaureus using the kit described in any one of claims 4 to 12.

14. The method according to claim 13, characterized in that, The method includes the following steps: (1) Extract genomic DNA from the sample to be tested or directly dilute the sample to be tested; (2) Construct a PCR reaction system using the composition according to any one of claims 1 to 3, perform amplification, and obtain PCR amplification products; (3) The PCR amplification products obtained in step (2) were analyzed using a PCR fluorescence detection system, and the detection results were characterized by fluorescence curves.

15. The method according to claim 14, characterized in that, In step (1), when the sample is sputum, it needs to be digested before nucleic acid release; In step (2), the PCR reaction volume is 25 μL, the primer amount in the PCR reaction system is 0.24 μM, the probe amount is 0.12 μM, and the PCR amplification program is: 95℃ pre-denaturation for 1 min, then 95℃ denaturation for 1 s, 60℃ annealing for 20 s, 10 cycles; finally, 95℃ denaturation for 1 s, 55℃ annealing for 20 s, 35 cycles, and fluorescence is collected.

16. The use of the composition according to any one of claims 1 to 3 or the kit according to any one of claims 4 to 12 for the detection of Staphylococcus aureus and / or methicillin-resistant Staphylococcus aureus for non-disease diagnostic purposes.

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