Primer and probe for qPCR detection of drug-resistant mutation of dermatophytes SQLE, implementation method and detection system
By designing specific primers and TaqMan-MGB probes, combined with multiplex qPCR technology, and optimizing the reaction system, the problems of complexity and high false positive rate in the detection of SQLE gene mutations in dermatophytes in existing technologies have been solved, achieving rapid and accurate drug resistance detection applicable to a variety of clinical samples.
Patent Information
- Application Number
- CN202511166263.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-20
- Publication Date
- 2025-11-28
AI Technical Summary
Existing technologies struggle to efficiently and accurately identify gene mutations in dermatophytes that resist terbinafine, especially key site mutations in the SQLE gene. This results in long testing cycles, complex procedures, and high false positive rates, making it impossible to achieve rapid and accurate resistance assessments in clinical practice.
By designing specific primers and TaqMan-MGB probes, combining multiplex qPCR technology, and optimizing the reaction system, sensitive detection of mutation sites such as F397L, A448T, and L393S in the SQLE gene was achieved. Wild-type blocking probes were used to improve the specificity and stability of the detection.
It enables rapid and accurate detection of SQLE resistance mutations in dermatophytes, with a sensitivity of up to 0.25‰ and high specificity. It can simultaneously detect multiple high-frequency mutation sites in a single tube, with a coverage of over 75%, significantly improving detection efficiency and accuracy.
Smart Images
Figure CN121023074A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to primers and probes, implementation methods, and detection systems for qPCR detection of SQLE drug resistance mutations in dermatophytes. Background Technology
[0002] Dermatophyte infections are the most common superficial fungal infections, affecting approximately 20% to 25% of the global population. In recent years, with the widespread use of antifungal drugs, the resistance rate of dermatophytes has been steadily increasing, particularly with the rapid global spread of *Trichophyton indotineae*, which is resistant to terbinafine (TRB). The emergence of local cases in my country further highlights the severity of this problem.
[0003] Although both EUCAST and CLSI standards currently recommend using the microbroth dilution method (such as EUCAST E.Def11.0) for drug susceptibility testing, its limitations have been repeatedly pointed out in multiple studies, including: long testing cycle; strict requirements on strain activity, inoculum concentration, and culture conditions; inability to accurately identify small mutant subpopulations in mixed infection samples; inability to directly determine the relationship between gene-level mutation types and threshold values; high operator skill requirements; and high misjudgment rate. 1 .
[0004] In molecular detection, single-site qPCR or HRM technologies are currently widely used. While these technologies have some promise, most studies remain at the laboratory validation stage and lack multi-site combined detection or clinical simulation validation. Furthermore, although commercial qPCR kits (such as DermaGenius PCR) are available on the market, their functionality is still limited to detecting specific sites (F397L, L393F), covering low mutation frequencies, unable to distinguish mutation sites, and struggling to identify drug-resistant subtypes in wild-type interference backgrounds. 2 .
[0005] Numerous clinical studies have clearly demonstrated a causal relationship between squalene epoxidase (SQLE) gene mutations and TRB resistance. 34 Mutations at key sites of the SQLE gene (such as F397L, L393F, L393S, A448T, etc.) significantly reduce the binding affinity of TRB to the target site by altering the conformation of the active site of squalene cyclooxygenase, directly leading to the clinical drug resistance phenotype. Summary of the Invention
[0006] The purpose of this invention is to provide primers and probes, implementation methods, and detection systems for qPCR detection of SQLE drug resistance mutations in dermatophytes.
[0007] According to a first aspect of the present invention, primers and probes for qPCR detection of SQLE resistance mutations in dermatophytes are characterized by comprising at least one of the following specific primers and probes for detecting gene mutations in dermatophytes:
[0008] Specific primers and probes for detecting the F397L mutation in the SQLE gene of dermatophytes, wherein the F397L mutation corresponds to the nucleotide mutation g.1189T>C;
[0009] Specific primers and probes for detecting the A448T mutation in the SQLE gene of dermatophytes, wherein the SQLE gene A448T mutation corresponds to the nucleotide mutation g.1404G>A;
[0010] Specific primers and probes for detecting the L393S mutation in the SQLE gene of dermatophytes, wherein the L393S mutation corresponds to the nucleotide mutation g.1178T>C.
[0011] Preferably, the nucleotide sequences of the specific primers and probes used to detect the F397L mutation in the SQLE gene of dermatophytes are as follows:
[0012] Forward primer: 5'-CAGTCCTTATACTCTATACTCGC-3';
[0013] Reverse primer: 5'-GGGCCATCTATAAGTCCAAGTT-3';
[0014] TaqMan-MGB fluorescent probe: FAM-CTGGTGGTAAGCATATGT-MGB;
[0015] MGB probe modified wild-type blocking probe: 5'-GTCCTTATACTCTATATTCG-MGB.
[0016] Preferably, the nucleotide sequences of the specific primers and probes used for detecting the A448T mutation in the SQLE gene of dermatophytes are as follows:
[0017] Forward primer: 5'-TTACTCCGTAGCCTTCCTCA-3';
[0018] Reverse primer: 5'-TGCACTCGAAAAGGGTCAAG-3';
[0019] TaqMan-MGB fluorescent probe: TAMRA-TATCTGGCTACACCTTACT-MGB;
[0020] Wild-type blocking probe modified with MGB probe: 5'-CGTAGCCTTCCTCGCT-MGB.
[0021] Preferably, the nucleotide sequences of the specific primers and probes used to detect the L393S mutation in the SQLE gene of dermatophytes are as follows:
[0022] Forward primer: 5'-AATATTCTTGCCCAGTCCTC-3';
[0023] Reverse primer: 5'-GGGCCATCTATAAGTCCAAGTT-3';
[0024] TaqMan-MGB fluorescent probe: CY5-TACTCTATATTCGCCGCTGGT-MGB;
[0025] Wild-type blocking probe modified with MGB probe: 5'-TCTTGCCCAGTCCTTAT-MGB.
[0026] According to a second aspect of the present invention, a method for implementing primers and probes for qPCR detection of SQLE drug resistance mutations in dermatophytes is characterized by comprising the following steps:
[0027] Drug susceptibility testing was performed on the obtained dermatophytes. By comparing the SQLE gene sequences of drug-resistant dermatophytes and wild-type dermatophytes, multiple drug resistance mutation sites of the SQLE gene in dermatophytes were obtained.
[0028] Primers, TaqMan-MGB probes, and wild-type blocking probes were designed based on the drug resistance mutation sites of the SQLE gene to specifically detect SQLE drug resistance mutations in each dermatophyte.
[0029] The primers and probes designed for detecting SQLE resistance mutations in dermatophytes were subjected to specificity testing, and primers and probes that meet the specificity requirements for detecting SQLE resistance mutations in dermatophytes were screened out.
[0030] Specific primers and probes for the SQLE resistance mutation of dermatophytes that meet the specificity requirements are validated at the strain level to obtain primers and probes as described in any one of claims 1-4 with validated sensitivity and specificity.
[0031] Preferably, the strain-level validation includes: using primers and probes that have been validated for specificity, using pure cultured strain DNA as a template, preparing qPCR amplification templates through a serial dilution method (40ng / μL-40fg / μL), establishing a standard curve, and evaluating the sensitivity and specificity of specific primers and probes for dermatophyte SQLE drug-resistant mutations that meet the specificity requirements.
[0032] According to a third aspect of the present invention, a method for optimizing the reaction system for qPCR detection of SQLE drug resistance mutations in dermatophytes includes the following steps:
[0033] Construct a mixed DNA sample model containing DNA from drug-resistant strains, wild-type strains, and human DNA;
[0034] Single qPCR and multiplex qPCR were used to detect the F397L, L393S and A448T mutation sites in a mixed DNA sample model, respectively, and the stability and anti-interference ability of the reaction system were evaluated.
[0035] By comparing the detection performance under different reaction ratios, the optimal reaction system was determined.
[0036] The DNA sample to be tested is mixed with the primers and probes described in any one of claims 1-4 in an optimized ratio and then subjected to qPCR amplification.
[0037] Analyze the amplification data to evaluate the detection efficacy.
[0038] Preferably, the reaction system supports both single and multiple detection, and the reaction system volume is 20 μL, as detailed below:
[0039] Reaction system for single qPCR detection:
[0040] reagents F397L A448T L393S 10 μmol / L forward primer 0.4μL 0.4μL 0.4μL 10 μmol / L reverse primer 0.4μL 0.4μL 0.4μL 10 μmol / L fluorescent probe 0.1μL 0.6μL 0.4μL 10 μmol / L wild-type blocking probe 1μL 0.4μL 0.4μL probe-based qPCR premix 10μL 10μL 10μL ROX reference dye 0.4μL 0.4μL 0.4μL ddH2O Quantitatively up to 20 μL Quantitatively up to 20 μL Quantitatively up to 20 μL DNA sample 2μL 2μL 2μL
[0041] Reaction system for multiplex qPCR detection:
[0042]
[0043] Preferably, the qPCR amplification reaction program is as follows: pre-denaturation, 37℃ for 2 minutes, 95℃ for 5 minutes; amplification and signal acquisition, the first 5 cycles are 95℃ for 10 seconds, 55℃ for 15 seconds, 72℃ for 30 seconds; the next 40 cycles are 95℃ for 10 seconds, 55℃ for 15 seconds, 72℃ for 30 seconds, and fluorescence signal acquisition is performed.
[0044] According to a fourth aspect of the present invention, a detection system for SQLE drug resistance mutations in dermatophytes includes:
[0045] A mixing device is used to mix the DNA of the sample to be tested with the aforementioned synthetic primers and probes for detecting SQLE resistance mutations in dermatophytes according to a single or multiplex qPCR reaction system to obtain a mixture of the DNA of the sample to be tested and the aforementioned universal primers and probes.
[0046] The detection device is used to detect the DNA of the sample to be tested in a mixture of the aforementioned primers and probes to determine whether there is a SQLE resistance mutation site in the sample to be tested.
[0047] This invention addresses the triple challenges of "wild-type interference + low-abundance mutation + operational efficiency" in clinical samples by designing and optimizing an MGB probe with wild-type blocking function for the first time, and achieving reliable results under complex clinical simulation conditions.
[0048] The TaqMan-MGB qPCR assay of this invention has significant advantages in detecting terbinafine resistance in dermatophytes. Regarding detection sensitivity, this technology can stably detect low-abundance mutations as low as 0.25‰. Furthermore, this technology can not only distinguish between "mutant" and "wild-type" mutations but also precisely locate specific mutation sites. In terms of throughput, this invention employs a multiplex detection design, adding the two high-frequency sites A448T and L393S, and can simultaneously detect multiple high-frequency mutation sites in a single tube, achieving a coverage rate of over 75%. Attached Figure Description
[0049] Figure 1 This is a schematic flowchart illustrating the primer and probe implementation method for qPCR detection of SQLE drug resistance mutations in dermatophytes provided in this embodiment of the invention;
[0050] Figure 2 This is a schematic flowchart illustrating the design and validation of primers and probes for qPCR detection of SQLE drug resistance mutations in dermatophytes, as well as the optimization and validation of the qPCR detection reaction system, provided in this embodiment of the invention.
[0051] Figure 3a This is the result of validation at the strain level using primers and probes with the F397L mutation site.
[0052] Figure 3b This is the result of validation at the strain level using primers and probes with the mutation site A448T.
[0053] Figure 3c This is the result of validation at the strain level using primers and probes with the mutation site L393S.
[0054] Figure 4a This is a graph showing the results of a single qPCR detection using primers and probes with the F397L mutation site in a mixed DNA sample model.
[0055] Figure 4b This is a graph showing the results of a single qPCR detection using primers and probes with the A448T mutation site in a mixed DNA sample model.
[0056] Figure 4c This is a graph showing the results of a single qPCR detection using primers and probes with the L393S mutation site in a mixed DNA sample model.
[0057] Figure 5a This is a graph showing the results of multiplex qPCR detection using primers and probes with the F397L mutation site in a mixed DNA sample model.
[0058] Figure 5b This is a graph showing the results of multiplex qPCR detection using primers and probes with the A448T mutation site in a mixed DNA sample model.
[0059] Figure 5c This is a graph showing the results of multiplex qPCR detection using primers and probes with the L393S mutation site in a mixed DNA sample model. Detailed Implementation
[0060] The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the preferred embodiments described below are only for illustration and explanation of the present invention and are not intended to limit the present invention.
[0061] Figure 1 This is a schematic flowchart illustrating the primer and probe implementation method for qPCR detection of dermatophyte SQLE resistance mutations provided in this invention. The implementation method of the primers and probes for qPCR detection of dermatophyte SQLE resistance mutations is described in detail below.
[0062] The first step involved subculturing common dermatophyte strains preserved at the Center for Fungal and Fungal Disease Research, Peking University, and determining their minimum inhibitory concentration (MIC) against antifungal drugs such as terbinafine using the microdilution method to assess the drug resistance of dermatophytes.
[0063] The second step involved extracting genomic DNA from terbinafine-resistant strains confirmed by drug susceptibility testing and determining their concentration using a Nanodrop 2000 ultra-micro spectrophotometer. PCR amplification was performed using universal primers for the SQLE gene, and the full-length SQLE gene sequence was obtained via Sanger sequencing.
[0064] The third step is to compare the sequencing results with the SQLE genome reference sequences reported in the NCBI database and manually verify and screen for base mutation sites that affect the drug resistance phenotype.
[0065] Step 4: Based on the mutation sites obtained through screening, primers and probes for qPCR detection of SQLE resistance mutations in dermatophytes are designed using Primer Express 3.0 software.
[0066] By repeating the above four steps, this embodiment identified three drug resistance mutations: F397L (corresponding to the nucleotide mutation g.1189T>C), A448T (corresponding to the nucleotide mutation g.1404G>A), and L393S (corresponding to the nucleotide mutation g.1178T>C), and designed specific primer and probe combinations for each mutation.
[0067] Step 5: Compare the amplified DNA sequence based on the designed primers in the NCBI database to ensure primer specificity.
[0068] The results showed that the primer and probe combination designed in this invention had 100% specificity, meaning it only targeted the target mutation site and no non-specific amplification was detected.
[0069] Step 6: Select sequencing-validated bacterial strains for methodological validation, including the F397L mutant (6 strains), A448T mutant (2 strains), L393S mutant (1 strain), and wild-type strains (10 strains). Inoculate them into Sabouraud dextrose agar and culture for 3-7 days. Prepare bacterial suspensions of a certain concentration, extract DNA, and quantify it using a Nanodrop 2000 micro-spectrophotometer. Serially dilute the DNA solution to verify the limits of detection (LOD) and cross-reactivity of the designed primers and probes. After repeated trials, e.g., 2-3 times, the final primers and probes are shown in Table 1.
[0070] Table 1. Sequence information of primers and probes.
[0071]
[0072]
[0073] like Figure 2 As shown, after designing primers and probes, the designed primers and probes were validated at the strain level, specifically as follows: Dermatophytes preserved at the Center for Fungal and Fungal Diseases Research, Peking University, were selected for validation. The extracted bacterial DNA solution was quantified using a Nanodrop 2000 micro-volume spectrophotometer, and serially diluted (40 ng / μL-40 fg / μL) to detect the lowest detection limit and R0 of the primers and probes, as well as the standard curve. 2 The results, along with the Ct difference between wild-type and resistant strains at the same concentration, are shown in Table 2. Specific results are as follows: Figures 3a to 3c As shown.
[0074] Table 2. Results of strain-level validation.
[0075]
[0076] like Figure 2 As shown, after performing strain-level validation, clinical sample testing was conducted, as detailed below:
[0077] 1. To verify the clinical applicability of qPCR detection, this invention constructs a standardized mixed DNA sample model. This model comprises three components: drug-resistant strains, wild-type strains, and human DNA, with a fixed total DNA concentration of 40 ng / μL. A dual gradient is established: the first gradient simulates the proportion of pathogen DNA in clinical samples, with three concentration levels of 0.1%, 1%, and 2% of total DNA from drug-resistant and wild-type strains; the second gradient simulates the proportion of drug-resistant strains in the pathogen, with five gradients: 100%, 75%, 50%, 25%, and 0%. This comprehensively simulates various drug resistance scenarios that may be encountered clinically. This design can systematically evaluate the ability of qPCR detection to identify different drug resistance ratios.
[0078] Based on the DNA sample model described above, a single qPCR reaction was used to detect the three mutation sites: F397L, A448T, and L393S.
[0079] The reaction system for a single qPCR assay (20 μL):
[0080]
[0081]
[0082] qPCR amplification program: pre-denaturation, 37℃ for 2 minutes, 95℃ for 5 minutes; amplification and signal acquisition, first 5 cycles 95℃ for 10 seconds, 55℃ for 15 seconds, 72℃ for 30 seconds; the next 40 cycles 95℃ for 10 seconds, 55℃ for 15 seconds, 72℃ for 30 seconds, and fluorescence signal acquisition.
[0083] Through the above experiments, we systematically evaluated the detection performance, focusing on three key indicators: detection sensitivity, specificity, and decision threshold. Detailed data are shown in Table 3 and... Figures 4a-4c .
[0084] Table 3. Power of single qPCR detection
[0085]
[0086] Note:
[0087] Detection sensitivity: The lowest concentration of mutant DNA that can be reliably detected in a mixed sample with a total DNA concentration of 40 ng / μL (the corresponding percentage is the proportion of mutant DNA in the mixed sample DNA);
[0088] Upper limit of specificity: The maximum concentration of wild-type DNA that will not produce false positives (the value in parentheses represents the proportion of wild-type DNA to total DNA);
[0089] Criterion threshold (Ct): The critical Ct value used to determine whether a sample is positive. That is, when the Ct value of a sample is less than the threshold, it is determined to be positive, and when it is greater than the threshold, it is determined to be negative.
[0090] 2. To achieve simultaneous single-tube detection of three key drug resistance mutation sites—F397L, L393S, and A448T—this invention establishes an innovative multiplex qPCR detection method. Based on a previously constructed mixed DNA sample model, the simultaneous detection of the three mutation sites—F397L, A448T, and L393S—was achieved by optimizing the reaction system for multiplex qPCR detection.
[0091] Reaction system for multiplex qPCR detection (20 μL):
[0092]
[0093] The results showed that, even with 99.975% human and wild-type DNA, it could still accurately identify 0.025% of extremely low-abundance drug resistance mutations and establish a reliable cutoff value standard. Detailed data are shown in Table 4 and... Figures 5a-5c .
[0094] Table 4. Power of multiplex qPCR detection
[0095]
[0096]
[0097] Note:
[0098] Detection sensitivity: The lowest concentration of mutant DNA that can be reliably detected in a mixed sample with a total DNA concentration of 40 ng / μL (the corresponding percentage is the proportion of mutant DNA in the mixed sample DNA);
[0099] Upper limit of specificity: The maximum concentration of wild-type DNA that will not produce false positives (the value in parentheses indicates the proportion of wild-type DNA to total DNA);
[0100] Criterion threshold (Ct): The critical Ct value used to determine whether a sample is positive. That is, when the Ct value of a sample is less than the threshold, it is determined to be positive, and when it is greater than the threshold, it is determined to be negative.
[0101] Twenty-one clinical samples were tested using drug susceptibility testing and SQLE gene sequencing. Seven samples were identified as drug-resistant strains, and 14 as wild-type strains. qPCR detection was performed using primers and probes designed according to this invention. Results showed that all drug-resistant samples (7 / 7) were accurately detected, achieving a 100% mutation detection rate; no false positives were observed in the 14 wild-type samples; and compared to traditional drug susceptibility testing, this method can complete the detection within 3 hours. This method enables early and rapid identification of drug resistance mutations, providing important evidence for precision medicine in clinical practice and contributing to improved patient prognosis.
[0102] The following section compares the SQLE drug resistance mutation-specific primers and probes for dermatophytes of this invention with existing literature. 5 The laboratory methods for detecting terbinafine-resistant dermatophytes in the study were compared, as detailed in Table 5.
[0103] Table 5 Comparison of Methods for Detecting Drug Resistance in Dermatophytes
[0104]
[0105] As can be seen from the table above, the TaqMan-MGB qPCR detection developed in this invention has significant advantages in detecting terbinafine resistance in dermatophytes. Regarding detection sensitivity, this technology can stably detect low-abundance mutations as low as 0.25‰. Furthermore, this technology can not only distinguish between "mutant" and "wild-type" mutations but also accurately locate specific mutation sites. In terms of throughput, this invention employs a multiplex detection design, adding the two high-frequency sites A448T and L393S, enabling simultaneous detection of multiple high-frequency mutation sites in a single tube, achieving a coverage rate of over 75%. Its modular primer and probe design allows for flexible expansion of new mutation sites according to clinical needs, ensuring that the detection method always keeps pace with pathogen mutation trends.
[0106] In clinical applications, this technology has significant advantages: First, the detection time is only 2-3 hours, much faster than the 1-3 weeks of traditional drug sensitivity testing; second, it can directly detect clinical samples such as skin flakes and nail flakes without the need for time-consuming pure culture processes; and finally, the cost of a single test is controlled within a reasonable range, making it of significant value for promotion and application at the grassroots level.
[0107] This invention designs specific primers and TaqMan-MGB probes based on SQLE gene mutation hotspots, enabling precise molecular diagnosis of terbinafine resistance in dermatophytes, and providing direct evidence for the selection of clinical antifungal drugs.
[0108] After rigorous validation at the strain level, this detection system exhibits excellent stability. The sensitivity for strain DNA samples reaches 40 fg / μL, while for clinical samples it can identify low-abundance drug-resistant mutations as low as 10 pg / μL (0.25‰). Employing multiplex qPCR technology, it can simultaneously detect multiple key mutation sites (including F397L, L393S, A448T, etc.), and achieve dual validation through wild-type blocked probe design, significantly improving diagnostic accuracy, with a clinical pathogen detection rate of up to 100%. This technology is applicable to various clinical sample types, such as skin flakes, nail dandruff, and hair, and has high detection sensitivity, providing clinicians with rapid and reliable etiological diagnostic results, facilitating early and precise treatment of dermatophyte infections.
[0109] This invention also provides a detection system for SQLE drug resistance mutations in dermatophytes, the system comprising:
[0110] A mixing device for mixing the DNA of the sample to be tested with the aforementioned synthetic primers and probes for detecting SQLE resistance mutations in dermatophytes, to obtain a mixture of the DNA of the sample to be tested with the aforementioned primers and probes;
[0111] The detection device is used to detect the DNA of the sample to be tested with the aforementioned primers and probes to determine whether the sample to be tested contains SQLE drug-resistant mutations of dermatophytes.
[0112] A fluorescent probe is labeled with a specific fluorescence that indicates the presence of mutations; then the DNA of the sample to be tested is mixed with the fluorescently labeled primers and probes, and the resulting mixture is provided to the detection device to detect whether there is a SQLE resistance mutation in the sample.
[0113] The aforementioned detection device is a qPCR detection device. This qPCR detection device detects the mixture of the sample DNA to be tested and the primers and probes of this invention. It can quickly and accurately detect whether there are SQLE drug-resistant mutations in different clinical samples, guide clinical treatment, facilitate early and precise treatment of dermatophytes, and improve prognosis.
[0114] The specific implementation process is as follows:
[0115] First, the primers and probes designed in this invention are artificially synthesized, and the probes are labeled with specific fluorescence. The fluorescent labels of the probes corresponding to different mutations are different. After synthesis, the concentrations of the primers and probes are quantified using a NanoDrop2000 micro-volume spectrophotometer for later use.
[0116] Secondly, after the clinical samples were collected, DNA was extracted, and the nucleic acid concentration of the samples was quantified using a NanoDrop2000 ultra-micro spectrophotometer for later use.
[0117] Finally, the synthesized primers and probes were mixed with clinical sample DNA in a specific ratio and then tested using a qPCR instrument. This instrument can detect the sample reaction in real time through visual signal changes.
[0118] Result interpretation: If a clinical sample contains a dermatophyte SQLE resistance mutation, it will bind to the designed primers and probes, producing fluorescent signals of different colors. These signals are displayed on the qPCR instrument as exponential amplification curves of different colors and corresponding Ct values. The accuracy of each test is controlled by comparing the curves and Ct values of negative and positive controls. The type of resistance mutation in the test sample is determined by comparing the curve color and Ct value of the test sample.
[0119] This invention provides a qPCR detection method for SQLE drug resistance mutations in dermatophytes suitable for clinical samples. It can improve the sensitivity and specificity of dermatophyte drug resistance detection and can be directly used for rapid detection of drug resistance in dermatophyte patient samples, which is conducive to early and accurate treatment and improves prognosis. It is applicable to different clinical samples such as bronchoalveolar lavage fluid, tissue samples, and exudates.
[0120] Although the present invention has been described in detail above, it is not limited thereto, and those skilled in the art can make various modifications based on the principles of the present invention. Therefore, all modifications made in accordance with the principles of the present invention should be understood to fall within the protection scope of the present invention.
Claims
1. Primers and probes for qPCR detection of SQLE drug resistance mutations in dermatophytes, characterized in that... Includes at least one of the following specific primers and probes for detecting dermatophyte gene mutations: Specific primers and probes for detecting the F397L mutation in the SQLE gene of dermatophytes, wherein the F397L mutation corresponds to the nucleotide mutation g.1189T>C; Specific primers and probes for detecting the A448T mutation in the SQLE gene of dermatophytes, wherein the SQLE gene A448T mutation corresponds to the nucleotide mutation g.1404G>A; Specific primers and probes for detecting the L393S mutation in the SQLE gene of dermatophytes, wherein the L393S mutation corresponds to the nucleotide mutation g.1178T>C.
2. The primers and probes according to claim 1, characterized in that, The nucleotide sequences of the specific primers and probes used to detect the F397L mutation in the SQLE gene of dermatophytes are as follows: Forward primer: 5'-CAGTCCTTATACTCTATACTCGC-3'; Reverse primer: 5'-GGGCCATCTATAAGTCCAAGTT-3'; TaqMan-MGB fluorescent probe: FAM-CTGGTGGTAAGCATATGT-MGB; MGB probe modified wild-type blocking probe: 5'-GTCCTTATACTCTATATTCG-MGB.
3. The primers and probes according to claim 1 or 2, characterized in that, The nucleotide sequences of the specific primers and probes used to detect the A448T mutation in the SQLE gene of dermatophytes are as follows: Forward primer: 5'-TTACTCCGTAGCCTTCCTCA-3'; Reverse primer: 5'-TGCACTCGAAAAGGGTCAAG-3'; TaqMan-MGB fluorescent probe: TAMRA-TATCTGGCTACACCTTACT-MGB; Wild-type blocking probe modified with MGB probe: 5'-CGTAGCCTTCCTCGCT-MGB.
4. The primers and probes according to claim 1, 2, or 3, characterized in that, The nucleotide sequences of the specific primers and probes used to detect the L393S mutation in the SQLE gene of dermatophytes are as follows: Forward primer: 5'-AATATTCTTGCCCAGTCCTC-3'; Reverse primer: 5'-GGGCCATCTATAAGTCCAAGTT-3'; TaqMan-MGB fluorescent probe: CY5-TACTCTATATTCGCCGCTGGT-MGB; Wild-type blocking probe modified with MGB probe: 5'-TCTTGCCCAGTCCTTAT-MGB.
5. A method for implementing primers and probes for qPCR detection of SQLE drug resistance mutations in dermatophytes, wherein the primers and probes are those described in any one of claims 1-4, characterized in that, Includes the following steps: Drug susceptibility testing was performed on the obtained dermatophytes. By comparing the SQLE gene sequences of drug-resistant dermatophytes and wild-type dermatophytes, multiple drug resistance mutation sites of the SQLE gene in dermatophytes were obtained. Primers, TaqMan-MGB probes, and wild-type blocking probes were designed based on the drug resistance mutation sites of the SQLE gene to specifically detect SQLE drug resistance mutations in each dermatophyte. The primers and probes designed for detecting SQLE resistance mutations in dermatophytes were subjected to specificity testing, and primers and probes that meet the specificity requirements for detecting SQLE resistance mutations in dermatophytes were screened out. Specific primers and probes for the SQLE resistance mutation of dermatophytes that meet the specificity requirements were validated at the strain level to verify the sensitivity and specificity of the primers and probes.
6. The method for implementing primers and probes for qPCR detection of SQLE drug resistance mutations in dermatophytes according to claim 5, characterized in that... ； The strain-level validation includes: using primers and probes that have been validated for specificity, using pure cultured strain DNA as a template, preparing qPCR amplification templates through serial dilution, establishing a standard curve, and evaluating the sensitivity and specificity of specific primers and probes for SQLE drug-resistant mutations of dermatophytes that meet the specificity requirements.
7. A method for optimizing the reaction system for qPCR detection of SQLE drug resistance mutations in dermatophytes, characterized in that, Includes the following steps: Construct a mixed DNA sample model containing DNA from drug-resistant strains, wild-type strains, and human DNA; Single qPCR and multiplex qPCR were used to detect the F397L, L393S and A448T mutation sites in a mixed DNA sample model, respectively, and the stability and anti-interference ability of the reaction system were evaluated. By comparing the detection performance under different reaction ratios, the optimal reaction system was determined. The DNA sample to be tested is mixed with the primers and probes described in any one of claims 1-4 in an optimized ratio and then subjected to qPCR amplification. Analyze the amplification data to evaluate the detection efficacy.
8. The method for optimizing the reaction system for qPCR detection according to claim 7, characterized in that, The reaction system supports both single and multiple detection, and the reaction volume is 20 μL, as detailed below: Reaction system for single qPCR detection: Reaction system for multiplex qPCR detection:
9. The method for optimizing the reaction system for qPCR detection according to claim 7, characterized in that, The qPCR amplification reaction program is as follows: pre-denaturation, 37℃ for 2 minutes, 95℃ for 5 minutes; amplification and signal acquisition, the first 5 cycles are 95℃ for 10 seconds, 55℃ for 15 seconds, 72℃ for 30 seconds; the next 40 cycles are 95℃ for 10 seconds, 55℃ for 15 seconds, 72℃ for 30 seconds, and fluorescence signal is acquired.
10. A detection system for SQLE drug resistance mutations in dermatophytes, characterized in that, include: A mixing device is used to mix the DNA of the sample to be tested with the aforementioned synthetic primers and probes for detecting SQLE resistance mutations in dermatophytes according to a single or multiplex qPCR reaction system to obtain a mixture of the DNA of the sample to be tested and the aforementioned universal primers and probes. The detection device is used to detect the DNA of the sample to be tested in a mixture of the aforementioned primers and probes to determine whether there is a SQLE resistance mutation site in the sample to be tested.