A candida auris and its drug-resistant mutation site detection primer probe combination, kit and method

Abstract

This invention discloses a primer-probe combination for detecting Candida auris and its drug-resistant mutation sites. The Candida auris drug-resistant mutation sites include azole resistance mutation sites: mutation sites A395T and A428G in the ERG11 gene, and an anti-echinocandin resistance mutation site: mutation site C1916T in the FKS1 gene. The primer-probe combination includes primer and probe sequences as shown in SEQ ID NO. 1-13, and corresponding kits and detection methods are also provided. In this invention, the primer-probe combination is prepared as a lyophilized reagent, eliminating the need for detection system preparation and directly detecting the nucleic acid extracted from the sample. It features simple operation, good stability, and wide applicability. Furthermore, by detecting the sample, it is possible to quickly determine whether the sample is positive for Candida auris and whether Candida auris drug-resistant mutations have occurred, while also identifying the drug-resistant mutation targets, meeting clinical needs and achieving precision medicine.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a primer-probe combination, kit, and method for detecting Candida auris and its drug-resistant mutation sites. Background Technology

[0002] Candida auris is a novel, multidrug-resistant fungus isolated from the external auditory canal of a patient in Japan. Candida auris easily infects immunocompromised patients and can cause bloodstream infections, urinary tract infections, and skin infections, primarily through hospital-acquired infections, with bloodstream infection being the most common type. The mortality rate of fungemia caused by Candida auris is 49%–68%. Due to its rapid spread, diverse invasiveness, wide range of infected individuals, difficulty in identification, and high mortality rate, Candida auris is known as a "superbug." Furthermore, it readily produces drug-resistant mutant strains, thus posing a serious threat to global health security.

[0003] Data shows that the resistance rate of *Candida auris* to azoles is approximately 90%, with multidrug resistance rates of 25% and polyazole resistance rates of 13%. The resistance mechanisms of *Candida auris* to azoles include: 1. Mutation and overexpression of the ERG11 gene: The main drug target, lanosterol 14-α-demethylase, is encoded by the ERG11 gene. Mutations in ERG11 prevent the drug from properly recognizing and binding to the target. ERG11 mutations may also affect the catalytic efficiency of proheme; 2. Overexpression of the efflux pump: Increased expression of the CDR1 gene in efflux proteins; 3. Biofilm formation, etc. The resistance mechanism of *Candida auris* to echinocandins: Mutations in the hotspot region of the FKS1 gene can lead to decreased sensitivity of β-(1,3)-D-glucan synthase to echinocandins, thus resulting in resistance.

[0004] Traditional clinical detection methods include microscopic examination, histopathological examination, culture, and serology. However, *Candida auris* is phenotypically similar to other *Candida* species in various genetic traits, making identification difficult, traditional culture time lengthy, and prone to misdiagnosis using these methods. In recent years, rDNA sequencing based on the internal transcribed spacer region or D1 / D2 region and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry have been increasingly applied to the identification of *Candida auris*. However, these detection methods are costly, require expensive equipment, and necessitate specialized personnel, limiting their applicability and making them unsuitable for rapid early screening.

[0005] Fluorescent PCR technology is a widely used identification method for Candida auris detection, characterized by its speed, simplicity, low cost, high sensitivity, and good specificity. It can efficiently detect the presence of related drug resistance genes and mutations. While there are existing research reports on the application of quantitative fluorescent PCR in the detection of Candida auris, research on the detection of drug resistance mutation sites in Candida auris strains is limited. Furthermore, there is a lack of commercially available detection reagents that offer good stability, ease of operation, and room-temperature storage. Therefore, there is a need to develop a kit and detection method for detecting drug resistance mutation sites in Candida auris strains to meet clinical needs. Summary of the Invention

[0006] To address at least one of the above problems, the present invention provides a primer-probe combination, kit, and method for detecting Candida auris and its drug-resistant mutation sites.

[0007] To achieve the above objectives, the present invention employs the following technical means:

[0008] The first aspect of the present invention provides a primer-probe combination for detecting Candida auris and its drug-resistant mutation sites, wherein the Candida auris drug-resistant mutation sites include azole resistance mutation sites: mutation sites A395T and A428G of the ERG11 gene and echinocandin resistance mutation site: mutation site C1916T of the FKS1 gene.

[0009] The primer-probe combination includes:

[0010] (1) Primer pairs for targeted amplification of Candida auris: the forward primer is shown in SEQ ID NO.1, the reverse primer is shown in SEQ ID NO.3, and the probe for detecting Candida auris is shown in SEQ ID NO.2;

[0011] (2) Targeted amplification of anti-azole drug resistance mutation sites: Primer pairs for the mutation sites A395T and A428G of the ERG11 gene: forward primers are shown in SEQ ID NO. 4, reverse primers are shown in SEQ ID NO. 6 and 7, and probes for detecting the A395T and A428G mutation sites are shown in SEQ ID NO. 5;

[0012] (3) Targeted amplification of the anti-echinocandin resistance mutation site: Primer pair for the C1916T mutation site of the FKS1 gene: forward primer as shown in SEQ ID NO. 8, reverse primer as shown in SEQ ID NO. 10, and probe for detecting the C1916T mutation site as shown in SEQ ID NO. 9.

[0013] Furthermore, the primer-probe composition further includes: (4) primer pairs for targeting and amplifying human internal standards: the forward primer is shown in SEQ ID NO. 11, the reverse primer is shown in SEQ ID NO. 13, and the probe for detecting human internal standards is shown in SEQ ID NO. 12.

[0014] In some embodiments of the present invention, the primer-probe combination has a fluorescent group at the 5' end and a quenching group at the 3' end. The fluorescent groups on probes detecting the same target are the same, and the fluorescent groups on probes detecting different targets are different. In some embodiments of the present invention, the fluorescent group is one of FAM, HEX, VIC, ROX and CY5, and the quenching group is one of BHQ1, MGB and BHQ2.

[0015] In a specific embodiment of the present invention, the fluorescent group modified at the 5' end of the Candida auris probe is VIC, and the quenching group modified at the 3' end is BHQ1; the fluorescent group modified at the 5' end of the probes for mutation sites A395T and A428G is FAM, and the quenching group modified at the 3' end is BHQ1; the fluorescent group modified at the 5' end of the probe for mutation site C1916T is ROX, and the quenching group modified at the 3' end is BHQ2; the fluorescent group modified at the 5' end of the probe for the human internal standard is CY5, and the quenching group modified at the 3' end is BHQ2.

[0016] A second aspect of the present invention provides a detection kit for Candida auris and its drug-resistant mutation sites, the kit comprising the primer-probe combination described in the first aspect.

[0017] In some embodiments of the present invention, the primer-probe combination is configured as a primer-probe working solution. After adding the PCR reaction reagent and the lyophilization protectant in a ratio of PCR reaction reagent: primer-probe working solution: lyophilization protectant = 10:3:2, the solution is then lyophilized to prepare a lyophilized reagent. The lyophilization procedure is as follows:

[0018] (1) Pre-freezing: Plate temperature -40℃, cooling time 30min, hold for 90min;

[0019] (2) Sublimation: Plate temperature -30℃, heating time 30min, holding time 360min, pressure 10Pa;

[0020] (3) Analysis: Plate temperature 30℃, heating time 60min, holding time 240min, pressure 5Pa.

[0021] In some embodiments of the present invention, the final concentrations of primers for Candida auris, mutant site A395T, mutant site A428G, and mutant site C1916T in the primer-probe working solution are 1.85-2.15 μM; the final concentrations of probes for Candida auris, mutant site A395T, and mutant site A428G are 1.07-1.08 μM; the final concentration of probe for mutant site C1916T is 0.85-0.86 μM; the final concentration of primers for human internal standard is 0.7-0.72 μM; and the final concentration of probe for human internal standard is 0.35-0.36 μM.

[0022] In some embodiments of the present invention, the freeze-drying protectant comprises the following raw materials: 4%~5% sucrose by weight and volume, 2%~3% trehalose by weight and volume, 4%~5% mannitol by weight and volume, 0.02~0.05% BSA by weight and volume, 0.05%~0.2% Tween-80 by volume, and the balance being DEPC water.

[0023] A third aspect of the present invention provides a method for detecting Candida auris and its drug-resistant mutation sites for non-diagnostic and non-therapeutic purposes using the kit described in the second aspect, comprising the following steps:

[0024] S1. Extract nucleic acid from the sample to be tested;

[0025] S2. Add the nucleic acid of the sample to be tested to the lyophilization reagent, vortex to mix, centrifuge, and then put it into the PCR instrument for detection.

[0026] S3. Analysis of test results.

[0027] In some embodiments of the present invention, the method for analyzing the detection results is as follows:

[0028] (1) If the human internal standard has no Ct value in the fluorescence channel, and the Ct value of the Candida auris fluorescence channel is ≤36, then Candida auris is positive; at the same time, if

[0029] a. If the Ct value of the fluorescence channel corresponding to the three mutations A395T, A428G, and C1916T is ≤36, and the difference between the Ct value of the fluorescence channel corresponding to the mutation and the fluorescence channel of Candida auris is ≤10, then the corresponding drug resistance site mutation will occur simultaneously.

[0030] b. If any one of the following does not meet the requirements in a, then the resistance site corresponding to the three mutations A395T, A428G, and C1916T, or the difference between the fluorescence channel corresponding to the mutation and the fluorescence channel of Candida auris, then the resistance site corresponding to the mutation that does not meet the requirements in a has not undergone mutation.

[0031] (2) If the Ct value of the human internal standard fluorescence channel is ≤31, and the Ct value of the Candida auris fluorescence channel is >36, and other fluorescence channels have no Ct value, then Candida auris is negative;

[0032] (3) If the Ct value of the human internal standard fluorescence channel is >31 and the Ct value of the Candida auris fluorescence channel is >36, and other fluorescence channels have no Ct value, then the sample is considered invalid and needs to be extracted or sampled again.

[0033] In some embodiments of the present invention, the PCR detection reagent is the 2×HieffUnicon® Multiplex ARMS qPCR Mix reagent manufactured by Yisheng Biotechnology Co., Ltd.

[0034] In some embodiments of the present invention, the PCR detection process includes the following reaction procedure: 45-50℃ for 5 min, 1 cycle; 90-95℃ for 2 min, 1 cycle; 90-95℃ for 3 s, 50-54℃ for 5 s, 70-72℃ for 5 s, 5 cycles; 90-95℃ for 5 s, 55-58℃ for 10 s, 40 cycles.

[0035] In a specific embodiment of the present invention, the PCR reaction procedure is as follows: 50℃ for 5 min, 1 cycle; 95℃ for 2 min, 1 cycle; 95℃ for 3 s, 54℃ for 5 s, 72℃ for 5 s, 5 cycles; 95℃ for 5 s, 58℃ for 10 s, 40 cycles.

[0036] In some embodiments of the present invention, 20 μL of nucleic acid from the sample to be tested is added to the lyophilized reagent prepared from a 15 μL system. In some embodiments of the present invention, the data acquisition temperature is 58 °C, and the reaction volume per well is 20 μL.

[0037] In some embodiments of the present invention, the sample to be tested is any one of whole blood, serum, plasma, cervical swab, ear canal swab, oropharyngeal swab, and nasopharyngeal swab.

[0038] This invention also provides the application of the above-mentioned primer-probe combination and kit in the simultaneous detection of Candida auris and its drug-resistant mutations, and in the identification of drug-resistant mutation targets.

[0039] Beneficial effects of the present invention

[0040] Compared with existing technologies, the present invention has the following beneficial effects: Based on real-time quantitative PCR technology, a method is provided to simultaneously detect Candida auris and the ERG11 gene Y132F mutation target (corresponding to the ERG11 gene mutation site A395T), K143R mutation target (corresponding to the ERG11 gene mutation site A428G), and FKS1 gene S639F mutation target (corresponding to the FKS1 gene mutation site C1916T). By detecting the sample, it is possible to quickly determine whether the sample is positive for Candida auris and whether Candida auris drug resistance mutation has occurred. At the same time, it can identify the drug resistance mutation target, which can meet clinical needs and realize precision medicine.

[0041] The detection reagent in this invention is a lyophilized reagent, which eliminates the need for preparation of the detection system and allows direct detection of nucleic acids extracted from the sample. It features simple operation, good stability, and wide applicability. Currently, the commonly used real-time PCR method for detecting Candida auris takes approximately 2 hours, while this invention's detection time is only 1 hour. Furthermore, the detection reagent exhibits good specificity and sensitivity; the detection limit for Candida auris, ERG11 gene A395T, A428G drug-resistant mutants, and FKS1 gene C1916T drug-resistant mutants can all reach 250 copies / mL. At a wild-type template concentration of 10... 6 At copies / mL, the detection rate of drug resistance mutations A395T and A428G in the ERG11 gene and C1916T in the FKS1 gene reached 0.1%. Attached Figure Description

[0042] Figure 1 The amplification curves of Candida auris, ERG11 mutant, FKS1 mutant and human internal standard primer and probe combination in Example 1 of the present invention are shown; wherein, blue is primer and probe combination 1, green is primer and probe combination 2, red is primer and probe combination 3 and yellow is primer and probe combination 4.

[0043] Figure 2 The results of the primer-probe combination test for wild-type tolerance to ERG11 gene A395T, A428G mutation and FKS1 gene C1916T mutation are shown; blue represents primer-probe combination 1, green represents primer-probe combination 2, and red represents primer-probe combination 3.

[0044] Figure 3 The graph shows the results of PCR reaction reagent amplification efficiency detection; green represents reagent 1, and red represents reagent 2.

[0045] Figure 4 The graph shows the results of specific detection of PCR reaction reagents;

[0046] Figure 5The diagram shows the limit of detection for Candida auris and three drug-resistant mutations, along with amplification curves for human internal standards.

[0047] Figure 6 Amplification curves at different mutation rates for three mutation sites are shown;

[0048] Figure 7 Amplification curves for three clinical samples are shown;

[0049] Figure 8 The experimental results of amplification efficiency stability are shown in the figure. Detailed Implementation

[0050] The following examples are used to illustrate preferred embodiments of the invention. The techniques disclosed in the examples represent technologies discovered by the inventors that can be used to implement the invention, and therefore can be considered preferred embodiments for implementing the invention. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein, still yielding the same or similar results, without departing from the spirit or scope of the invention.

[0051] 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 this invention pertains, and all materials disclosed herein and cited therein are incorporated herein by reference. Many equivalent techniques of specific embodiments of the invention described herein will be recognized or can be understood by ordinary experimentation by those skilled in the art. These equivalents will be included in the claims.

[0052] The technical solution of this application will be further described in detail below with reference to specific embodiments.

[0053] Example 1 Primer and probe design and screening

[0054] The sequences of Candida auris ITS2, ERG11, FKS1 and human actin genes were searched in the NCBI database. Conserved regions of each sequence were identified by sequence alignment. Primers and probes were designed for each conserved region, resulting in four sets of primers and probes, as shown in Table 1.

[0055] Table 14 sets of primer and probe information

[0056]

[0057] Four primer-probe combinations were designed to screen for Candida auris, the A395T and A428G mutations in the ERG11 gene, the C1916T mutation in the FKS1 gene, and the human actin gene. Specifically, the mutation sites A395T (corresponding to the target mutation Y132F) and A428G (corresponding to the target mutation K143R) in the ERG11 gene, and the C1916T mutation site (corresponding to the target mutation S639F) in the FKS1 gene (a site of resistance to echinocandin), were selected. The results are as follows: Figure 1 As shown. Based on the screening results, use 10 7 Wild-type tolerance tests were performed on three primer-probe combinations for the ERG11 gene A395T and A428G mutations and the FKS1 gene C1916T mutation, using ERG11 and FKS1 wild-type and mutant plasmids at copies / mL, respectively. The results are as follows: Figure 2 As shown.

[0058] The results show: Figure 1 , 2 The amplification curve of primer-probe combination 1 is the most linear, and the wild-type tolerance is the best. Therefore, primer-probe combination 1 is used as the primer-probe combination in this application.

[0059] The specific information of the primer and probe sequences of the selected Candida auris, the Candida auris drug-resistant mutations: ERG11 gene Y132F mutation and K143R mutation, FKS1 gene S639F mutation, and human internal standard is shown in Table 2 as SEQ ID NO. 1 to SEQ ID NO. 13.

[0060] Table 2 Primer and probe information

[0061]

[0062] Example 3 Screening of PCR detection reagents

[0063] Amplification efficiency and specificity were tested using PCR detection reagent 1: 2×Hieff Unicon® Multiplex ARMSqPCR Mix reagent produced by Yisheng Biotechnology Co., Ltd.; and PCR detection reagent 2: 2×High-Specificity Probe qPCR SuperMix (UDG) reagent produced by Nearshore Protein Co., Ltd.

[0064] In the amplification efficiency test, the templates used were Candida auris and three drug-resistant mutant plasmids. The amplification efficiency results are as follows: Figure 3 As shown. In the specificity test, no positive template was added; sterile, nuclease-free water was used instead. The test results are as follows. Figure 4 As shown.

[0065] Figure 3 and Figure 4 The results showed that there was no significant difference in amplification efficiency between the two PCR detection reagents, but PCR detection reagent 2 exhibited nonspecific amplification.

[0066] The PCR detection reagent used in this invention is the 2×Hieff Unicon® Multiplex ARMS qPCR Mix reagent produced by Yisheng Biotechnology Co., Ltd.

[0067] Example 3: Detection method for Candida auris and its drug-resistant mutation sites

[0068] 1. Preparation of primer and probe working solutions

[0069] The primer and probe powder was diluted to 20 μM with 1×TE Buffer, and the calculation method is as follows:

[0070] Dilute according to the number of nmoles: Add 1×TE Buffer per tube = number of nmoles × 50 (μL); after adding 1×TE Buffer, vortex for about 30 seconds to homogenize, and then briefly centrifuge at low speed.

[0071] Prepare primer and probe working solutions according to Table 3 below. Vortex the prepared solutions and label them.

[0072] Table 3. Preparation of Primer and Probe Working Solutions

[0073]

[0074] 2. Preparation of the reaction system

[0075] Prepare the reaction system according to Table 4.

[0076] The freeze-drying protectants are: 4% sucrose, 3% trehalose, 5% mannitol, 0.04% BSA, 0.15% Tween-80, and the balance is DEPC water.

[0077] Table 4. Reaction System Preparation Table

[0078]

[0079] Add the prepared reagent to the eight-tube bundle at a rate of 15 μL / well and freeze-dry according to the procedure in Table 5.

[0080] Table 5 Freeze-drying process

[0081]

[0082] 3. PCR detection

[0083] (1) Nucleic acid extraction (PCR II laboratory)

[0084] Extract the sample to be tested according to the instructions of the nucleic acid extraction or purification reagent (magnetic bead method) and prepare nucleic acid.

[0085] (2) Sample loading (PCR II room)

[0086] Take out the lyophilized reagent, briefly centrifuge the eight tubes, add the nucleic acid prepared in (1) to the reaction system at 20 μL / well, and after the sample is added, transfer it to the PCR III room for instrument detection.

[0087] (3) On-machine testing (PCR III room)

[0088] Place the reaction tubes into the fluorescence PCR detector and set the PCR reaction program according to Table 6. The fluorescence signals were collected as FAM, VIC, ROX, and CY5, and data acquisition was performed at 58℃. The reaction volume for each well was 20 μL.

[0089] Table 6 PCR reaction procedures

[0090]

[0091] 4. Results Analysis

[0092] After the reaction, the instrument automatically saves the results. After adjusting the "Amplification Curve Algorithm" in the program parameter settings to "Absolute Fluorescence Method," analyze the image and adjust the Start, End, and Threshold values ​​of the Baseline (these can be set to default, adjusted manually, or the Start value can be between 3 and 15, and the End value between 5 and 20). Analyze the sample detection results according to the judgment information recorded in Table 7.

[0093] Table 7 Result Judgment Information

[0094]

[0095] Note: *ΔCt value = Ct value of the corresponding fluorescence channel - Ct value of the fluorescence channel corresponding to the Candida auris gene

[0096] Example 4: Detection Limit Test

[0097] Plasmids containing Candida auris, ERG11 gene A395T, A428G resistance mutations, and FKS1 gene C1916T resistance mutations were selected as simulant samples for testing the limit of detection (LOD). The specific protocol was as follows: plasmids with known copy numbers were diluted with negative samples to 500 copies / mL, 250 copies / mL, and 125 copies / mL, respectively. Each plasmid was extracted and tested 20 times, and the lowest concentration with a 95% positive detection rate was taken as the LOD. The detection results are shown in Table 8. The LOD and amplification curves of human internal standard for Candida auris, ERG11 gene A395T, A428G resistance mutations, and FKS1 gene C1916T resistance mutations are shown in Table 8. Figure 5 As shown.

[0098] Table 8. Detection results of the minimum detection limit

[0099]

[0100] The results showed that the limit of detection for Candida auris, the A395T and A428G resistance mutations of the ERG11 gene, and the C1916 resistance mutation of the FKS1 gene was 250 copies / mL.

[0101] Example 5: Mutation Detection Rate Test

[0102] Candida auris plasmids were mixed with ERG11 gene A395T and A428G drug-resistant mutants and FKS1 gene C1916T drug-resistant mutant plasmids in a certain proportion to test the mutation detection rate. The specific protocol is as follows: Candida auris plasmids of known concentration were diluted with negative nucleic acid to a concentration of 10T of Candida auris. 6 copies / mL, dilute the mutant plasmid of known concentration to 10. 3 copies / mL (i.e., mutation rate of 0.1%) and 5×10 3 copies / mL (i.e., mutation rate of 0.5%), each mixed plasmid was extracted and tested 20 times, and the lowest mutation rate with a positive detection rate of 95% was taken as the mutation detection rate.

[0103] The amplification curves for mutation rates of 0.02%, 0.1%, and 0.5% are shown below. Figure 6 As shown in Table 9, the results of the mutation detection rate are as follows.

[0104] Table 9. Mutation Detection Rate Results

[0105]

[0106] The results showed that the wild-type template concentration was 10. 6At a template concentration of 10 copies / mL, the detection results for templates with a mutation rate of 0.1% all showed a positive detection rate of 95%. Therefore, at a wild-type template concentration of 10... 6 At copies / mL, the detection rate of drug resistance mutations A395T and A428G in the ERG11 gene and C1916T in the FKS1 gene reached 0.1%.

[0107] Example 6 Clinical Sample Testing

[0108] Three clinical samples were selected: Sample 1, Sample 2, and Sample 3. These samples were tested using the methods described in this invention. The Ct values ​​are shown in Table 10, and the amplification curves are shown below. Figure 7 As shown.

[0109] Table 10 Clinical Sample Detection Results

[0110]

[0111] Example 7: Freeze-drying stability test

[0112] The stability of the lyophilized reagents was verified by storing the lyophilized reagents at room temperature for 0 days, 6 months, and 12 months, and then testing the detection limit, amplification efficiency, wild-type tolerance, and mutation detection rate of the lyophilized reagents.

[0113] (1) Samples of Candida auris with drug-resistant mutants of ERG11 gene A395T, A428G and FKS1 gene C1916T were diluted to 250 copies / mL and subjected to limit of detection stability tests using lyophilized reagents stored at room temperature for 0 days, 6 months and 12 months, respectively. The test results are shown in Table 11.

[0114] Table 11 Stability test results for the lowest detection limit

[0115]

[0116] (2) Dilute known concentrations of Candida auris with ERG11 gene A395T, A428G drug-resistant mutants and FKS1 gene C1916T drug-resistant mutant plasmids to 10. 7 copies / mL, 10 6 copies / mL, 10 5 copies / mL, 10 4 Samples of plasmids at copies / mL were subjected to amplification efficiency and stability tests using lyophilized reagents stored at room temperature for 0 days, 6 months, and 12 months. The Ct values ​​are shown in Table 12. The amplification efficiency curves for the stability experiment are shown below. Figure 8 As shown.

[0117] Table 12 Results of Ct values ​​in the amplification efficiency and stability experiment.

[0118]

[0119] (3) Three wild-type Candida auris samples were taken and subjected to tolerance stability tests using lyophilized reagents stored at room temperature for 0 days, 6 months, and 12 months, respectively. The Ct values ​​of the tolerance stability test are shown in Table 13.

[0120] Table 13 Results of wild-type tolerance stability

[0121]

[0122] (4) Dilute the known concentrations of the ERG11 gene A395T and A428G drug resistance mutants and the FKS1 gene C1916T drug resistance mutant plasmids to 10. 3 The mutation detection rate was measured at 0 copies / mL (i.e., a mutation rate of 0.1%), and the stability was tested using lyophilized reagents stored at room temperature for 0 days, 6 months, and 12 months. The results of the stability test are shown in Table 14.

[0123] Table 14 Mutation Detection Rate and Tolerance Stability Results

[0124]

[0125] The results showed that there was no significant difference in the data of the lyophilized reagent after 0 days, 6 months and 12 months of storage at room temperature, the amplification curve morphology did not change significantly and all performance remained stable. The detection reagent of the present invention can be stored at room temperature for at least 1 year.

[0126] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by this application.

Claims

1. A primer-probe combination for detecting Candida auris and its drug-resistant mutation sites, characterized in that, The Candida auris drug resistance mutation sites include azole resistance mutation sites: ERG11 gene mutation sites A395T and A428G, and echinocandin resistance mutation site: FKS1 gene mutation site C1916T. The primer-probe combination includes: (1) Primer pairs for targeted amplification of Candida auris: the forward primer is shown in SEQ ID NO.1, the reverse primer is shown in SEQ ID NO.3, and the probe for detecting Candida auris is shown in SEQ ID NO.2; (2) Targeted amplification of anti-azole drug resistance mutation sites: Primer pairs for the mutation sites A395T and A428G of the ERG11 gene: forward primers are shown in SEQ ID NO. 4, reverse primers are shown in SEQ ID NO. 6 and 7, and probes for detecting the A395T and A428G mutation sites are shown in SEQ ID NO. 5; (3) Targeted amplification of the anti-echinocandin resistance mutation site: Primer pair for the C1916T mutation site of the FKS1 gene: forward primer as shown in SEQ ID NO. 8, reverse primer as shown in SEQ ID NO. 10, and probe for detecting the C1916T mutation site as shown in SEQ ID NO. 9; and (4) Primer pairs for targeted amplification of human internal standard: forward primer as shown in SEQ ID NO. 11, reverse primer as shown in SEQ ID NO. 13, and probe for detecting human internal standard as shown in SEQ ID NO.

12.

2. The primer-probe combination for detecting Candida auris and its drug-resistant mutation sites according to claim 1, characterized in that: In the primer-probe combination, the 5' end of the probe is provided with a fluorescent group and the 3' end is provided with a quenching group. The fluorescent groups on the probes detecting the same target are the same, and the fluorescent groups on the probes detecting different targets are different. The fluorescent group is one of FAM, HEX, VIC, ROX and CY5, and the quenching group is one of BHQ1, MGB and BHQ2.

3. A detection kit for Candida auris and its drug-resistant mutation sites, characterized in that: The kit includes the primer-probe combination as described in any one of claims 1-2.

4. The detection kit according to claim 3, characterized in that: The primer-probe combination was configured into a primer-probe working solution. After adding the PCR reaction reagent and lyophilization protectant at a ratio of 10:3:2, the solution was lyophilized to prepare a lyophilized reagent. The lyophilization program was as follows: (1) Pre-freezing: Plate temperature -40℃, cooling time 30min, hold for 90min; (2) Sublimation: Plate temperature -30℃, heating time 30min, holding time 360min, pressure 10Pa; (3) Analysis: Plate temperature 30℃, heating time 60min, holding time 240min, pressure 5Pa.

5. The detection kit according to claim 4, characterized in that: In the primer and probe working solution, the final concentration of primers for Candida auris, mutation site A395T, mutation site A428G, and mutation site C1916T is 1.85-2.15 μM; the final concentration of probes for Candida auris, mutation site A395T, and mutation site A428G is 1.07-1.08 μM; the final concentration of probe for mutation site C1916T is 0.85-0.86 μM; the final concentration of primers for human internal standard is 0.7-0.72 μM; and the final concentration of probe for human internal standard is 0.35-0.36 μM.

6. The detection kit according to claim 4, characterized in that: The freeze-drying protectant It includes the following raw materials: sucrose with a mass-volume concentration of 4%~5%, trehalose with a mass-volume concentration of 2%~3%, mannitol with a mass-volume concentration of 4%~5%, BSA with a mass-volume concentration of 0.02~0.05%, Tween-80 with a volume concentration of 0.05%~0.2%, and the balance being DEPC water.

7. A method for detecting Candida auris and its drug-resistant mutation sites for non-diagnostic and non-therapeutic purposes using the kit according to any one of claims 3-6, characterized in that, Includes the following steps: S1. Extract nucleic acid from the sample to be tested; S2. Add the nucleic acid of the sample to be tested directly into the lyophilization reagent, vortex to mix, centrifuge, and then put it into the PCR instrument for detection. S3. Analysis of test results.

8. The method according to claim 7, characterized in that, The method for analyzing the detection results is as follows: (1) If the human internal standard has no Ct value in the fluorescence channel, and the Ct value of the Candida auris fluorescence channel is ≤36, then Candida auris is positive; and if a. If the Ct value of the fluorescence channel corresponding to the three mutations A395T, A428G, and C1916T is ≤36, and the difference between the Ct value of the fluorescence channel corresponding to the mutation and the fluorescence channel of Candida auris is ≤10, then the corresponding drug resistance site mutation will occur simultaneously. b. If any one of the following does not meet the requirements in a, then the resistance site corresponding to the mutations A395T, A428G, and C1916T, or the difference between the fluorescence channel Ct value corresponding to the mutation and the fluorescence channel Ct value of Candida auris, then the resistance site corresponding to the mutation that does not meet the requirements in a has not mutated. (2) If the Ct value of the human internal standard fluorescence channel is ≤31, and the Ct value of the Candida auris fluorescence channel is >36, and other fluorescence channels have no Ct value, then Candida auris is negative; (3) If the Ct value of the human internal standard fluorescence channel is >31 and the Ct value of the Candida auris fluorescence channel is >36, and other fluorescence channels have no Ct value, then the sample is considered invalid and needs to be extracted or sampled again.

9. The method according to claim 7, characterized in that, In the PCR detection, the PCR reaction program is as follows: 45-50℃ for 5 min, 1 cycle; 90-95℃ for 2 min, 1 cycle; 90-95℃ for 3 s, 50-54℃ for 5 s, 70-72℃ for 5 s, 5 cycles; 90-95℃ for 5 s, 55-58℃ for 10 s, 40 cycles.

10. The method according to claim 7, characterized in that, The sample to be tested can be any one of whole blood, serum, plasma, cervical swab, ear canal swab, oropharyngeal swab, or nasopharyngeal swab.

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