Candida auricula and drug-resistant mutation site detection primer probe combination, kit and method thereof

Abstract

The invention discloses candida auricula and a primer probe combination for detecting drug-resistant mutation sites of the candida auricula. The drug-resistant mutation sites of the candida auricula comprise an anti-azole drug-resistant mutation site, a mutation site A395T and a mutation site A428G of an ERG11 gene and an anti-echinocandin drug-resistant mutation site, namely a mutation site C1916T of an FKS1 gene; the primer probe combination comprises primer and probe sequences as shown in SEQ ID NO. 1-13. The invention also provides a corresponding kit and a detection method. The primer and probe combination is prepared into a freeze-drying reagent, the preparation process of a detection system is omitted during use, nucleic acid extracted from a sample is directly detected, the method has the advantages of being easy and convenient to operate, good in stability and wide in application range, whether the sample is positive of candida auricula or not and whether candida auricula drug-resistant mutation occurs or not can be rapidly determined by detecting the sample, and the detection accuracy is high. Meanwhile, drug-resistant mutation targets can be determined, clinical requirements can be met, and precise medical treatment is achieved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a primer-probe combination, a kit and a method for detecting Candida auris and drug-resistant mutation sites thereof. Background Art

[0002] Candida auris is a new type of multidrug-resistant fungus isolated from the external auditory canal of a patient in Japan. Candida auris is susceptible to infecting immunocompromised patients and can cause bloodstream infections, urinary tract infections, and skin infections. It mainly breaks out in the form of hospital infections, of which bloodstream infections are the main type of infection. The mortality rate of fungemia caused by Candida auris is 49% to 68%. Candida auris is called a "super fungus" because of its rapid transmission speed, multiple invasive infections, wide range of infected populations, difficulty in identification, and high mortality rate. It is also prone to produce highly resistant mutant strains. Therefore, Candida auris poses a serious threat to global health security.

[0003] Data show that the resistance rate of Candida auris to azoles is about 90%, and the multidrug resistance rate and multi-azole resistance rate are 25% and 13%, respectively. The resistance mechanism of Candida auris to azole drugs: 1. Mutation and overexpression of the ERG11 gene: The main drug target, lanosterol 14-α-demethylase, is encoded by the ERG11 gene. ERG11 mutations prevent the drug from recognizing and binding to the target normally. ERG11 mutations may also affect the catalytic efficiency of proheme; 2. Overexpression of efflux pumps: Increased expression of the CDR1 gene in efflux proteins; 3. Formation of biofilms, etc. The resistance mechanism of Candida auris to echinocandin: Mutations in the hotspot region of the FKS1 gene can lead to reduced sensitivity of β-(1,3)-D-glucan synthase to echinocandin drugs, resulting in resistance.

[0004] Traditional clinical detection methods include microscopic examination, histopathological examination, culture and serology. However, Candida auris is similar to other Candida species in phenotype and various genetic traits. The above methods have disadvantages such as difficulty in identification, long traditional culture time and easy misjudgment. In recent years, rDNA sequencing based on the internal transcribed spacer or D1 / D2 region and matrix-assisted laser desorption ionization time-of-flight mass spectrometry have been gradually applied to the identification of Candida auris. However, these detection methods are costly, the instruments are expensive, professional detection personnel are required, the scope of use is limited, and they are not suitable for early rapid screening.

[0005] Fluorescence PCR technology is a widely used identification method for Candida auris detection. It is fast, simple, low-cost, highly sensitive, and has good specificity. It can efficiently detect the presence of relevant drug-resistant genes and mutations. At present, there have been relevant research reports on the application of fluorescence quantitative PCR technology in the detection of Candida auris, but there are fewer studies on the detection of drug-resistant mutation sites in Candida auris strains. At the same time, there is a lack of detection reagents on the market that are stable, easy to operate, and can be stored at room temperature. Therefore, it is necessary to develop a kit and detection method for detecting drug-resistant mutation sites in Candida auris strains to meet clinical needs. Summary of the invention

[0006] In order to solve at least one of the above problems, the present invention provides a primer-probe combination, a kit and a method for detecting Candida auris and drug-resistant mutation sites thereof.

[0007] In order to achieve the above object, the present invention adopts the following technical means: The first aspect of the present invention provides a Candida auris and a primer-probe combination for detecting drug-resistant mutation sites thereof, wherein the drug-resistant mutation sites of Candida auris include azole-resistant mutation sites: mutation sites A395T and A428G of the ERG11 gene and echinocandin-resistant mutation sites: mutation site C1916T of the FKS1 gene; The primer-probe combination comprises: (1) Primer pair for targeted amplification of Candida auris: the forward primer is shown in SEQ ID NO. 1, the reverse primer is shown in 3, and the probe for detecting Candida auris is shown in SEQ ID NO. 2; (2) Targeted amplification of anti-azole resistance mutation sites: Primer pair for the mutation sites A395T and A428G of the ERG11 gene: the forward primer is shown in SEQ ID NO. 4, the reverse primers are shown in SEQ ID NO. 6 and 7, and the probe for detecting the A395T and A428G mutation sites is shown in SEQ ID NO. 5; (3) Targeted amplification of anti-echinocandin resistance mutation site: Primer pair for the mutation site C1916T of the FKS1 gene: the forward primer is shown in SEQ ID NO.8, the reverse primer is shown in SEQ ID NO.10, and the probe for detecting the C1916T mutation site is shown in SEQ ID NO.9.

[0008] Furthermore, the primer-probe composition also includes: (4) a primer pair for targeted amplification of a human internal standard: 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 the human internal standard is shown in SEQ ID NO. 12.

[0009] In some embodiments of the present invention, in the primer-probe combination, the 5' end of the probe is provided with a fluorescent group, 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; 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, BHQ2.

[0010] In a specific embodiment of the present invention, the fluorescent group modified at the 5' end of the probe of Candida auris is VIC, and the quenching group modified at the 3' end is BHQ1; the fluorescent group modified at the 5' end of the probe of 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 of 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 of the human internal standard is CY5, and the quenching group modified at the 3' end is BHQ2.

[0011] The second aspect of the present invention provides a kit for detecting Candida auris and drug-resistant mutation sites thereof, wherein the kit comprises the primer-probe combination described in the first aspect.

[0012] In some embodiments of the present invention, the primer-probe combination is configured into a primer-probe working solution, and after adding PCR reaction reagents and freeze-dried protection solution in a ratio of PCR reaction reagents: primer-probe working solution: freeze-dried protection agent = 10:3:2, a freeze-dried reagent is prepared under a freeze-drying procedure, and the freeze-drying procedure is: (1) Prefreezing: plate temperature -40°C, cooling time 30 minutes, hold time 90 minutes; (2) Sublimation: plate temperature -30°C, heating time 30 min, hold time 360 ​​min, pressure 10 Pa; (3) Analysis: Plate temperature 30°C, heating time 60 min, hold time 240 min, pressure 5 Pa.

[0013] In some embodiments of the present invention, in the primer-probe working solution, the final concentrations of primers for Candida auris, mutation site A395T, mutation site A428G, and mutation site C1916T are 1.85-2.15 μM, and the final concentrations of probes for Candida auris, mutation site A395T, and mutation site A428G are 1.07-1.08 μM; the final concentration of probes for mutation site C1916T is 0.85-0.86 μM; the final concentration of primers for human internal standards is 0.7-0.72 μM, and the final concentration of probes for human internal standards is 0.35-0.36 μM.

[0014] In some embodiments of the present invention, the lyoprotectant comprises the following raw materials: sucrose with a mass volume concentration of 4% to 5%, trehalose with a mass volume concentration of 2% to 3%, mannitol with a mass volume concentration of 4% to 5%, BSA with a mass volume concentration of 0.02% to 0.05%, Tween-80 with a volume concentration of 0.05% to 0.2%, and the remainder is DEPC water.

[0015] The third aspect of the present invention provides a method for detecting Candida auris and its drug-resistant mutation sites using the kit described in the second aspect, comprising the following steps: S1. Extracting nucleic acid from the sample to be tested; S2, adding the sample nucleic acid to be tested into the freeze-dried reagent, vortexing to mix, centrifuging and placing in a PCR detector for testing; S3. Analysis of test results.

[0016] In some embodiments of the present invention, the detection result analysis method is as follows: (1) If the fluorescence channel of the human internal standard has no Ct value and the Ct value of the Candida auris fluorescence channel is ≤36, then Candida auris is positive; at the same time, if a. If the Ct values ​​of the fluorescence channels corresponding to the three mutations A395T, A428G, and C1916T are ≤36, and the difference between the Ct values ​​of the fluorescence channels corresponding to the mutations and the Ct values ​​of the fluorescence channels of Candida auris is ≤10, then the corresponding drug resistance site mutations occur simultaneously; b. If any one of the Ct values ​​of the fluorescence channels corresponding to the three mutations A395T, A428G, and C1916T and the difference between the Ct values ​​of the fluorescence channels corresponding to the mutations and the fluorescence channels of Candida auris does not meet the requirements in a, then the drug-resistant sites that do not meet the requirements in a have not mutated; (2) If the Ct value of the fluorescence channel of the human internal standard is ≤31, and the Ct value of the fluorescence channel of Candida auris is >36, and there is no Ct value in other fluorescence channels, then Candida auris is negative; (3) If the Ct value of the fluorescence channel of the human internal standard is greater than 31 and the Ct value of the fluorescence channel of Candida auris is greater than 36, and there is no Ct value in other fluorescence channels, the sample is considered invalid and needs to be re-extracted or sampled.

[0017] In some embodiments of the present invention, the PCR detection reagent is 2×HieffUnicon® Multiplex ARMS qPCR Mix reagent produced by Yisheng Biotechnology Co., Ltd.

[0018] In some embodiments of the present invention, during the PCR detection process, the reaction procedure of PCR is; 45-50°C 5min, 1 cycle; 90-95°C 2min, 1 cycle, 90-95°C 3s 50-54°C 5s 70-72°C 5s, 5 cycles; 90-95°C 5s 55-58°C 10s, 40 cycles.

[0019] In a specific embodiment of the present invention, the reaction program of PCR is; 50°C 5min, 1 cycle; 95°C 2min, 1 cycle, 95°C 3s 54°C 5s 72°C 5s, 5 cycles; 95°C 5s 58°C 10s, 40 cycles.

[0020] In some embodiments of the present invention, 20 μL of the sample nucleic acid to be tested is added to the lyophilized reagent prepared from the 15 μL system.

[0021] In some embodiments of the present invention, the temperature for data acquisition is 58° C., and the reaction volume of each well is 20 μL.

[0022] 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.

[0023] The present invention also provides the use of the primer-probe combination and the kit in simultaneously detecting Candida auris and its drug-resistant mutation and determining the drug-resistant mutation target.

[0024] Beneficial effects of the present invention Compared with the prior art, the present invention has the following beneficial effects: based on fluorescent quantitative PCR technology, a method is provided for simultaneously detecting Candida auris and drug-resistant genes ERG11 gene Y132F mutation target (corresponding to the mutation site A395T of the ERG11 gene), K143R mutation target (corresponding to the mutation site A428G of the ERG11 gene) and FKS1 gene S639F mutation target (corresponding to the mutation site C1916T of the FKS1 gene). By detecting the sample, it can be quickly determined whether the sample is positive for Candida auris and whether a Candida auris resistance mutation occurs. At the same time, the drug-resistant mutation target can be identified, which can meet clinical needs and realize precision medicine.

[0025] The detection reagent in the present invention is a lyophilized reagent. When used, the preparation process of the detection system can be omitted, and the nucleic acid extracted from the sample can be directly detected. It has the characteristics of simple operation, good stability, and a wide range of applications. The detection time of the currently commonly used fluorescent quantitative PCR method for detecting Candida auris is about 2 hours, while the detection time of the present invention is only 1 hour. In addition, the specificity and sensitivity of the detection reagent are good, and the minimum detection limit of Candida auris, ERG11 gene A395T, A428G resistance mutations and FKS1 gene C1916T resistance mutation pathogens can reach 250 copies / mL. When the wild template concentration is 10 6 When the concentration of 50 copies / mL was 40, the detection rate of A395T and A428G resistance mutations in ERG11 gene and C1916T resistance mutation in FKS1 gene reached 0.1%. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The amplification curves of the primer-probe combination of Candida auris, ERG11 mutation, FKS1 mutation and human internal standard in Example 1 of the present invention are shown; wherein the blue is the primer-probe combination 1, the green is the primer-probe combination 2, the red is the primer-probe combination 3, and the yellow is the primer-probe combination 4; Figure 2 The results of the wild-type tolerance test of primer-probe combinations for ERG11 gene A395T, A428G mutations, and FKS1 gene C1916T mutations are shown; blue is primer-probe combination 1, green is primer-probe combination 2, and red is primer-probe combination 3; Figure 3 The figure shows the result of the PCR reaction reagent amplification efficiency detection; green is detection reagent 1, and red is detection reagent 2; Figure 4 Shown is a graph of PCR reaction reagent specificity detection results; Figure 5 The minimum detection limit of Candida auris and three drug-resistant mutations and the amplification curve of human internal standards are shown; Figure 6 Amplification curves of three mutation sites with different mutation rates are shown; Figure 7 Amplification curves of three clinical samples are shown; Figure 8 A graph showing the results of an amplification efficiency stability experiment. DETAILED DESCRIPTION

[0027] The following examples are used to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present invention and therefore can be considered as preferred embodiments of the present invention. However, it will be appreciated by those skilled in the art based on this specification that many modifications may be made to the specific embodiments disclosed herein and still achieve the same or similar results without departing from the spirit or scope of the present invention.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs, and the materials cited herein and those cited by them are incorporated by reference. Those skilled in the art will recognize or will learn through routine experimentation that there are many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0029] The technical solution of the present application is further described in detail below in conjunction with specific implementation methods.

[0030] Example 1 Primer probe design and screening The sequences of Candida auris ITS2, ERG11, FKS1 and human actin were searched in the NCBI database, and the conserved regions of each sequence were searched by sequence alignment. Primers and probes were designed in each conserved region, and 4 sets of primers and probes were obtained, as shown in Table 1.

[0031] Table 14 Primer probe information

[0032] The designed four sets of primer-probe combinations were used to screen Candida auris, ERG11 gene A395T, A428G mutations, FKS1 gene C1916T mutation and human actin gene, among which the mutation sites A395T (corresponding mutation target Y132F), A428G (corresponding mutation target K143R) of ERG11 gene and the mutation site C1916T (corresponding mutation target S639F) of anti-echinocandin resistance mutation site FKS1 gene were screened. Figure 1 According to the screening results, 10 7 The wild-type tolerance test of ERG11 and FKS1 wild-type and mutant plasmids with a concentration of 100 copies / mL was performed on three primer-probe combinations of ERG11 gene A395T, A428G mutations, and FKS1 gene C1916T mutations. The results are as follows: Figure 2 shown.

[0033] 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 of the present application.

[0034] The specific information of the primer probe sequences of the screened Candida auris, Candida auris drug-resistant mutations: ERG11 gene Y132F mutation and K143R mutation, FKS1 gene S639F mutation and human internal standard are shown in SEQ ID NO. 1 to SEQ ID NO. 13 in Table 2.

[0035] Table 2 Primer probe information

[0036] Example 3 PCR detection reagent screening Amplification efficiency test and specificity test were performed using PCR detection reagent 1: 2×Hieff Unicon® Multiplex ARMSqPCR Mix reagent produced by Yisheng Biotechnology Co., Ltd.; PCR detection reagent 2: 2×High-Specificity Probe qPCR SuperMix (UDG) reagent produced by Jinan Protein Co., Ltd.

[0037] In the amplification efficiency test, the detection templates were Candida auris and three drug-resistant mutant plasmids. The amplification efficiency results were as follows: Figure 3 In the specificity test, no positive template was added, and sterile nuclease-free water was used instead. The test results are shown in Figure 4 shown.

[0038] 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 had non-specific amplification.

[0039] The PCR detection reagent used in the present invention is 2×Hieff Unicon®Multiplex ARMS qPCR Mix reagent produced by Yisheng Biotechnology Co., Ltd.

[0040] Example 3 Candida auris and its drug-resistant mutation site detection method 1. Preparation of primer and probe working solution Primer probe powder is diluted to 20 μM with 1×TE Buffer. The calculation method is as follows: Dilute according to the number of nmoles: 1×TE Buffer added to each tube = number of nmoles × 50 (μL); after adding 1×TE Buffer, vortex for about 30 seconds to evenly mix, and briefly centrifuge at low speed.

[0041] Prepare primer and probe working solutions according to Table 3 below, vortex mix the prepared solutions and label them.

[0042] Table 3 Preparation of primer and probe working solution

[0043] 2. Preparation of reaction system The reaction system was prepared according to Table 4.

[0044] The freeze-drying protective agents are: sucrose with a mass volume concentration of 4%, trehalose with a mass volume concentration of 3%, mannitol with a mass volume concentration of 5%, BSA with a mass volume concentration of 0.04%, Tween-80 with a volume concentration of 0.15%, and the balance is DEPC water.

[0045] Table 4 Reaction system preparation table

[0046] Add the prepared reagent to the eight-tube strips at 15 μL / well and perform freeze-drying according to the process in Table 5.

[0047] Table 5 Freeze-drying procedure

[0048] 3. PCR testing (1) Nucleic acid extraction (PCR II room) Extract the sample to be tested and prepare nucleic acid according to the instructions of the nucleic acid extraction or purification reagent (magnetic bead method).

[0049] (2) Sample addition (PCR II chamber) Take out the freeze-dried reagent, briefly centrifuge the eight-tube strip, add the nucleic acid prepared in (1) to the reaction system at 20 μL / well, and transfer to the PCR III chamber for on-machine testing after the sample addition is completed.

[0050] (3) On-machine testing (PCR III room) Place the reaction tube in a fluorescent PCR detector and set the PCR reaction program for detection according to Table 6. The collection of fluorescent signals is set to FAM, VIC, ROX and CY5, the data collection is set at 58°C, and the reaction volume of each well is 20 μL.

[0051] Table 6 PCR reaction program

[0052] 4. Results Analysis After the reaction is completed, the instrument automatically saves the results, adjusts the "amplification curve algorithm" in the program parameter setting to "absolute fluorescence method", analyzes the image, and adjusts the Start value, End value and Threshold value of the Baseline (can be set by default, can be adjusted by yourself, or the Start value can be between 3-15, and the End value can be between 5-20). Analyze the sample test results according to the judgment information recorded in the judgment information table 7.

[0053] Table 7 Result judgment information

[0054] Note: *ΔCt value = Ct value of the corresponding fluorescence channel - Ct value of the fluorescence channel corresponding to the Candida auris gene Example 4 Detection limit test Candida auris, ERG11 gene A395T, A428G resistance mutations and FKS1 gene C1916T resistance mutation plasmids were selected as simulated samples for detection limit testing. The specific scheme is as follows: the plasmids with known copy numbers were diluted to 500 copies / mL, 250 copies / mL and 125 copies / mL respectively with negative samples, and each plasmid was extracted and tested 20 times repeatedly. The lowest concentration with a positive detection rate of 95% was taken as the minimum detection limit. The test results are shown in Table 8. The minimum detection limit of Candida auris, ERG11 gene A395T, A428G resistance mutations, FKS1 gene C1916T resistance mutations and the amplification curves of the human internal standard are shown in Table 8. Figure 5 shown.

[0055] Table 8 Minimum detection limit test results

[0056] The results showed that the minimum detection limit of Candida auris, ERG11 gene A395T, A428G resistance mutations and FKS1 gene C1916 resistance mutation was 250 copies / mL.

[0057] Example 5 Mutation Detection Rate Test Candida auris was mixed with ERG11 gene A395T, A428G resistance mutation plasmid and FKS1 gene C1916T resistance mutation plasmid in proportion as a simulated sample for mutation detection rate test. The specific scheme is as follows: a known concentration of Candida auris plasmid was diluted with negative nucleic acid to 10 6 copies / mL, and the mutant plasmids with known concentrations were diluted to 10 3 copies / mL (i.e. mutation rate 0.1%) and 5×10 3copies / mL (i.e., mutation rate 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.

[0058] The amplification curves of 0.02%, 0.1% and 0.5% mutation rates are shown in Figure 2. Figure 6 The detection results of mutation detection rate are shown in Table 9.

[0059] Table 9 Mutation detection rate results

[0060] The results showed that the wild template concentration was 10 6 When the concentration of wild template is 10 copies / mL, the detection results of 0.1% mutation rate template have a positive detection rate of 95%. 6 When the concentration of 50 copies / mL was 40, the detection rate of A395T and A428G resistance mutations in ERG11 gene and C1916T resistance mutation in FKS1 gene reached 0.1%.

[0061] Example 6 Clinical sample testing Three clinical samples were selected: Sample 1, Sample 2, and Sample 3 were tested using the methods of the present invention. The Ct values ​​are shown in Table 10, and the amplification curves are shown in Figure 7 shown.

[0062] Table 10 Clinical sample test results

[0063] Example 7 Freeze-dried stability test The stability of the lyophilized reagent was verified by storing the lyophilized reagent at room temperature for 0 days, 6 months, and 12 months, and the detection limit, amplification efficiency, wild-type tolerance, and mutation detection rate of the lyophilized reagent were tested respectively.

[0064] (1) The plasmids of Candida auris and ERG11 gene A395T, A428G resistance mutations and FKS1 gene C1916T resistance mutations were diluted to 250 copies / mL plasmid samples, and the minimum detection limit stability test was performed using freeze-dried reagents stored at room temperature for 0 days, 6 months, and 12 months. The test results are shown in Table 11.

[0065] Table 11 Minimum detection limit stability test results

[0066] (2) Dilute the known concentration of Candida auris and the ERG11 gene A395T, A428G resistance mutation plasmid and the FKS1 gene C1916T resistance mutation plasmid to 10 7copies / mL, 10 6 copies / mL, 10 5 copies / mL, 10 4 The samples of 1000 copies / mL plasmid were tested for amplification efficiency stability using freeze-dried reagents stored at room temperature for 0 days, 6 months, and 12 months. The Ct value results are shown in Table 12. The amplification efficiency curve of the stability experiment is shown in Figure 8 shown.

[0067] Table 12 Amplification efficiency stability test Ct value results

[0068] (3) Three wild-type samples of Candida auris were taken and subjected to tolerance stability tests using freeze-dried reagents stored at room temperature for 0 days, 6 months, and 12 months, respectively. The Ct value results of the tolerance stability test are shown in Table 13.

[0069] Table 13 Wild type tolerance stability results

[0070] (4) Dilute the plasmids with known concentrations of ERG11 gene A395T, A428G resistance mutations and FKS1 gene C1916T resistance mutations to 10 3 The tolerance stability test was performed using freeze-dried reagents stored at room temperature for 0 days, 6 months, and 12 months. The results of the mutation detection rate tolerance stability test are shown in Table 14.

[0071] Table 14 Mutation detection rate tolerance stability results

[0072] The results showed that there was no significant difference in the data of the freeze-dried reagent stored at room temperature for 0 days, 6 months, and 12 months, the amplification curve morphology did not change significantly, and various properties remained stable. The detection reagent of the present invention can be stored at room temperature for at least 1 year.

[0073] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as references separately. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined in the present application.

Claims

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

12.

2. The primer-probe combination for detecting Candida auris and drug-resistant mutation sites thereof according to claim 1, characterized in that: In the primer-probe combination, a fluorescent group is provided at the 5' end of the probe, and a quenching group is provided at the 3' end. 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 kit for detecting Candida auris and its drug-resistant mutation sites, characterized in that: The kit comprises the primer-probe combination according to any one of claims 1 to 2.

4. The detection kit according to claim 3, characterized in that: The primer-probe combination is configured into a primer-probe working solution, and after adding the PCR reaction reagent and the freeze-dried protection solution in a ratio of PCR reaction reagent: primer-probe working solution: freeze-dried protection agent = 10:3:2, the freeze-dried reagent is prepared under a freeze-drying procedure, and the freeze-drying procedure is: (1) Prefreezing: plate temperature -40°C, cooling time 30 min, hold time 90 min; (2) Sublimation: plate temperature -30°C, heating time 30 min, hold time 360 ​​min, pressure 10 Pa; (3) Analysis: Plate temperature 30°C, heating time 60 min, hold time 240 min, pressure 5 Pa.

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

6. The detection kit according to claim 4, characterized in that: The lyoprotectant The invention comprises the following raw materials: sucrose with a mass volume concentration of 4% to 5%, trehalose with a mass volume concentration of 2% to 3%, mannitol with a mass volume concentration of 4% to 5%, BSA with a mass volume concentration of 0.02% to 0.05%, Tween-80 with a volume concentration of 0.05% to 0.2%, and the balance is DEPC water.

7. A method for detecting Candida auris and its drug-resistant mutation sites using the kit according to any one of claims 3 to 6, characterized in that: The steps include: S1. Extracting nucleic acid from the sample to be tested; S2, directly add the sample nucleic acid to be tested into the freeze-dried reagent, vortex mix, centrifuge and place in the PCR detector for detection; S3. Analysis of test results.

8. The method according to claim 7, characterized in that The test result analysis method is as follows: (1) If the fluorescence channel of the human internal standard has no Ct value and the Ct value of the Candida auris fluorescence channel is ≤36, then Candida auris is positive; and if a. If the Ct values ​​of the fluorescence channels corresponding to the three mutations A395T, A428G, and C1916T are ≤36, and the difference between the Ct values ​​of the fluorescence channels corresponding to the mutations and the Ct values ​​of the fluorescence channels of Candida auris is ≤10, then the corresponding drug resistance site mutations occur simultaneously; b. If any one of the Ct values ​​of the fluorescence channels corresponding to the three mutations A395T, A428G, and C1916T and the difference between the Ct values ​​of the fluorescence channels corresponding to the mutations and the fluorescence channels of Candida auris does not meet the requirements in a, then the drug-resistant sites that do not meet the requirements in a have not mutated; (2) If the Ct value of the fluorescence channel of the human internal standard is ≤31, and the Ct value of the fluorescence channel of Candida auris is >36, and there is no Ct value in other fluorescence channels, then Candida auris is negative; (3) If the Ct value of the fluorescence channel of the human internal standard is greater than 31 and the Ct value of the fluorescence channel of Candida auris is greater than 36, and there is no Ct value in other fluorescence channels, the sample is considered invalid and needs to be re-extracted or sampled.

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

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

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