Digital PCR (Polymerase Chain Reaction) detection method and kit for copy number variation of candida tropicalis ERG11 gene

Abstract

The invention provides a Candida tropicalis ERG11 gene copy number variation digital PCR (polymerase chain reaction) detection method, the total copy number of ERG11 genes is detected through a group of universal primers, a wild type probe of an A395 site of the ERG11 genes in a detection sample and a mutant type probe of the A395 site of the ERG11 genes, and the total copy number of the ERG11 genes is equal to the sum of the copy number of wild target spots and the copy number of mutant target spots; in the PCR detection method, the copy number of the wild target and the copy number of the mutation target are corrected by using the copy number of two reference genes. Compared with an ERG11 gene mutation and copy number variation detection method based on a fluorescent quantitative PCR method, the accuracy of copy number variation detection is greatly improved, and the problem of pain points of tandem sequence copy number recognition and quantitative detection in the candida tropicalis drug resistance detection process can be effectively solved.

Description

Technical Field

[0001] This invention relates to the field of digital PCR, and in particular to a digital PCR detection method and kit for copy number variation of the ERG11 gene in Candida tropicalis. Background Technology

[0002] In recent years, with the development of medical technology, cases of invasive fungal infections, especially Candida infections, have continued to increase. Azole antibiotics are the most commonly used, economical, and widely applied antifungal drugs for the clinical prevention and treatment of candidiasis. However, along with the widespread use of azole drugs, the proportion of drug-resistant Candida strains in clinical practice is also constantly increasing. According to the latest data from the Invasive Fungal Surveillance Network, the drug resistance rate among invasive infections caused by Candida tropicalis has exceeded 30%, and the vast majority exhibit cross-resistance with fluconazole and voriconazole. The clinical demand for drug resistance identification and testing of similar strains has increased dramatically.

[0003] The target of azole drugs is a key enzyme in the ergosterol synthesis pathway, encoded by the ERG11 gene. Therefore, mutations in the ERG11 gene are the main cause of drug resistance in clinical Candida species. In recent years, the drug resistance rate of Candida tropicalis, especially in the Asia-Pacific region and China, has been significantly higher than in other regions. Studies using whole-genome sequencing analysis have found that the expansion of the AZR (Azole-Resistant) subpopulation of Clade IV (Multiple-Site Sequence Typing) is the direct cause of the surge in resistance rates. This subpopulation is characterized by the widespread presence of mutations at the A395T nucleotide site in the ERG11 gene, accompanied by copy number amplification and tandem duplication of alleles, forming gene clusters with three or more copies, leading to a significant increase in gene expression. The MIC value of fluconazole can reach ≥256 mg / L, far exceeding that of wild-type Candida tropicalis strains.

[0004] The mechanisms of azole resistance are relatively simple, and the copy number variation of the ERG11 gene in *Candida tropicalis* is correlated with the degree of azole resistance. Therefore, good consistency between molecular resistance detection results and phenotypic detection is expected. To more promptly identify highly resistant strains and even conduct epidemiological surveillance of *Candida tropicalis* resistance, a reliable method that can effectively and simultaneously detect both the resistance gene and its copy number variation in *Candida tropicalis* is urgently needed.

[0005] Clinically, methods for detecting ERG11 gene mutations and / or copy number variations in Candida tropicalis include: Sanger sequencing (first-generation sequencing), quantitative real-time PCR (qPCR), whole-genome sequencing (second-generation sequencing), and nanopore sequencing (third-generation sequencing). Sanger sequencing can only obtain gene mutation information and cannot obtain copy number variation information; it also has poor ability to identify heterozygous mutants in diploid Candida tropicalis. Quantitative real-time PCR relies on a standard curve for copy number quantification, and its accuracy is poor in complex samples due to interference, resulting in poor resolution for copy number variations. Whole-genome sequencing is time-consuming and expensive, requiring a strong bioinformatics background for result analysis, making it unsuitable for clinical testing. Nanopore sequencing's read length limitations affect its accuracy in analyzing high copy number variations (e.g., accurate copy number quantification is difficult when a gene has more than 7 tandem repeats).

[0006] Digital PCR (dPCR) technology, as a novel method for nucleic acid detection and quantification, has superior sensitivity, specificity, and accuracy. Its advantages in detecting extremely small amounts of nucleic acid samples and sensitively detecting rare mutations against a background of high abundance of wild-type genes have been widely recognized. Based on the Poisson distribution principle, it can quantify without a standard curve, has stronger anti-interference ability, faster detection, and lower detection cost compared to second- or third-generation sequencing technologies. It has become an important tool in the field of clinical testing. However, long tandem repeat sequences can cause template segmentation problems during digital PCR droplet generation, which can affect the accuracy of copy number variation detection results. Summary of the Invention

[0007] To address the aforementioned problems, this invention provides a digital PCR method for detecting copy number variations in the ERG11 gene of Candida tropicalis. This method utilizes a set of universal primers and both wild-type and mutant probes at the A395 site of the ERG11 gene in the sample to detect the total copy number of the ERG11 gene. The total copy number of the ERG11 gene = wild-type target copy number + Mutant target copy number; the copy numbers of two internal reference genes are used to correct the wild-type target copy number and the mutant target copy number in the PCR detection method; the calculation method for determining the wild-type target copy number and the mutant target copy number based on the mutant type of the ERG11 gene A395 site in the digital PCR detection method is as follows: when the ERG11 gene A395 site is wild-type, homozygous mutant, or heterozygous mutant, the calculation methods for the wild-type target copy number and the mutant target copy number are as follows: wild-type target copy number = (wild-type target gene copy number / (internal reference gene 1 copy number + internal reference gene 2 copy number) / 2) × 2; homozygous mutant target copy number = (mutant target gene copy number / (internal reference gene 1 copy number + internal reference gene 2 copy number) / 2) × 2; and, heterozygous mutant target copy number = (mutant target gene copy number / (wild-type target gene copy number × 2) + mutant target gene copy number / internal reference gene 1 copy number + mutant target gene copy number / The internal reference gene copy number is 2 times the number of copies of the wild-type allele (2 copies) × 2 / 3. Since Candida tropicalis is diploid, the theoretical copy number of the wild-type allele is 1. Therefore, the wild-type target gene copy number in the heterozygous state is multiplied by 2 to correct for the heterozygous allele ratio.

[0008] In one implementation, the two internal reference genes are the Actin-1 gene and the GAPDH gene.

[0009] In one embodiment, the upstream and downstream primers and probes for PCR digital amplification detection of the Actin-1 gene are SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7, respectively, and the upstream and downstream primers and probes for PCR digital amplification detection of the GAPDH gene are SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10, respectively.

[0010] In one embodiment, the upstream and downstream primers and wild-type probe and mutant probe for PCR digital amplification detection of the A395 site of the ERG11 gene are SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0011] In one embodiment, the sample DNA is further fragmented before digital PCR amplification.

[0012] In one embodiment, the concentration of the fragmented enzyme in the fragmentation reaction system is 1-5 U, and the reaction time ranges from 10 to 30 min. In one embodiment, the concentration of the fragmented enzyme in the fragmentation reaction system is 2.5 U, and the reaction time is 20 min.

[0013] In one embodiment, a digital PCR detection kit for use in the above method is provided. The kit includes a primer and probe set for digital PCR amplification detection of the ERG11 gene A395 site and two internal reference gene primer and probe sets for normalization correction. The upstream and downstream primers and wild-type and mutant probes for digital PCR amplification detection of the ERG11 gene A395 site are SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, respectively. The upstream and downstream primers and probes for digital PCR amplification detection of the Actin-1 gene are SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7, respectively. The upstream and downstream primers and probes for digital PCR amplification detection of the GAPDH gene are SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, respectively.

[0014] In one embodiment, the digital PCR kit further includes a fragmentation enzyme for processing the extracted Candida tropicalis DNA template.

[0015] In addition to the A395T nucleotide mutation in the ERG11 gene, copy number amplification and tandem duplication of alleles also occur, forming gene clusters of 3 or more copies, leading to a significant increase in gene expression. Verification, through comprehensive analysis of indicators such as the mean difference, upper and lower limits of deviation, and positive and negative differences, shows that the mean difference calculated by the method of this invention is not significantly different from the theoretical value compared to other groups, has a small lower limit of deviation, and exhibits even smaller positive and negative differences. Therefore, the calculation method of this invention is the optimal method for copy number variation analysis.

[0016] This invention improves the accuracy of copy number variation detection by using a dual internal reference detection system in primer and probe design, and obtains primer and probe sequence sets with better performance through screening.

[0017] The detection method of the present invention includes a Candida tropicalis DNA fragmentation process. The optimized fragmentation process helps to improve the uniformity of template segmentation during droplet generation in digital PCR, thereby improving the accuracy of detecting ERG11 gene copy number variations in the digital PCR platform.

[0018] By establishing and optimizing the method for calculating copy number variation of the mutant gene and the two internal reference genes, the accuracy of detecting copy number variation of the ERG11 gene in Candida tropicalis was further improved.

[0019] This invention represents a significant improvement over existing commonly used detection methods. Compared to methods for detecting ERG11 gene mutations and copy number variations based on quantitative real-time PCR, it greatly enhances the accuracy of copy number variation detection, effectively addressing the critical issue of tandem sequence copy number identification and quantification in the detection of Candida tropicalis drug resistance. Compared to sequencing-based methods for detecting ERG11 gene mutations and copy number variations, this invention reduces the entire detection and reporting process from several days to just a few hours, while also significantly lowering detection costs. Attached Figure Description

[0020] Figure 1 This is a 2D amplification diagram of the first set of primers and probes in Example 2; Figure 2 This is a schematic diagram illustrating different mutations or copy number variations in the ERG11 gene of Candida tropicalis. Figure 3 This is a graph showing the analysis results of 98 clinical isolates using different algorithms. Detailed Implementation

[0021] To enable those skilled in the art to better understand the technical solutions in this application, the present invention will be further described below with reference to embodiments. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this application.

[0022] Example 1: Primer and probe design for the Candida resistance mutation detection system 1. Primer and probe design: Primers and probes for the ERG11 gene A395 wild-type site, A395T mutant site, and the internal reference gene Actin-1 were designed based on the NCBI database using the sequence of the reference strain (MYA-3404) of *Candida tropicalis*. Specific information is shown in Table 1 below. HEX is generally considered equivalent to VIC, but the channel name is usually VIC channel.

[0023] Table 1

[0024] Serial Number Sequence information (5'-3') and information on modified groups Sequence and probe name SEQ ID NO.1 5'-ATACCCATTTGACTACTCCTGT-3' ERG11 upstream primer SEQ ID NO.2 5'-TTAGCAAACTTCTTTTGTTCCAT-3' ERG11 downstream primer SEQ ID NO.3 5'FAM-AGGTGTTATTTATGATTG-3'MGB ERG11 wild-type probe SEQ ID NO.4 5'HEX-AGGTGTTATTTTTGATTG-3'MGB ERG11 mutant probe SEQ ID NO.5 5'-TTGTCTCCAACTGGGATGATA-3' Actin-1 upstream primer SEQ ID NO.6 5'-AACTGGGTGTTCTTCTGGAG-3' Actin-1 downstream primer SEQ ID NO.7 5'CY5-CTGGCACCACACTTTCTACAACG-3'BHQ2 Actin-1 probe .

[0025] 2. Preparation of the basic reaction system, as shown in Table 2 below.

[0026] Table 2 Material Name Original concentration Final concentration Amount added (μL) Tris-HCl 1 M 0.05 M 1.5 glycerin 80% 1% 0.375 dNTPs 75× 1× 0.4 KCl 1 M 0.01 M 0.3 MgCl2 1 M 4 mM 0.12 Taq DNA polymerase 5U / μL 0.067 U / μL 0.4 UNG enzyme 1U / μL 0.005 U / μL 0.15 10× Primer and Probe Set 10× 1× 3 DNA template - - 1 Ultrapure water - - Add to a total volume of 30 μL .

[0027] The final concentrations of primers and probes were all 600 nM and 300 nM, respectively. Four strains of *Candida tropicalis* isolates, including a standard strain and clinical isolates, whose ERG11 gene mutations and copy number variations were confirmed by whole-genome sequencing, were used as test samples (sequencing results were rounded to the nearest integer to calculate the theoretical total copy number). Nucleic acid was extracted from the cultured colonies using the XinYi nucleic acid extraction reagent. The nucleic acid was quantified using a Nanodrop instrument, and the genomic concentration was calculated based on the theoretical genome length, then diluted to approximately 10,000 copies / μL for use as the test sample nucleic acid.

[0028] 3. Digital PCR Detection Process Microdroplet preparation: Using the sample preparation instrument and droplet generation chip from Xinyi Manufacturing Technology (Beijing) Co., Ltd., 30 μL of PCR reaction system was added to the sample well of the droplet generation chip, and 180 μL of microdroplet generation oil was added to the oil well. The chip and the 8-tube array were placed in the preparation instrument, and the adhesive pad was covered to prepare microdroplets.

[0029] PCR amplification: Place the sealed 8-tube array containing the droplets onto a PCR instrument for amplification. Set the amplification conditions as follows: .

[0030] Microdroplet detection: After PCR, the microchip analyzer from NewYi Manufacturing Technology (Beijing) Co., Ltd. was used for detection. The 8-tube array and the droplet detection chip were placed in the fixture, and 430uL and 500uL of detection oil were added to the oil wells respectively. The rubber gasket was then placed on the microchip analyzer for droplet detection.

[0031] Data Analysis: After chip detection, data analysis is performed. Based on the 1D and 2D scatter plots and the detection results of the positive control, a suitable positive region is delineated (generally, the positive region is delineated at the center of the region where the FAM / VIC / Cy5 channel signals are concentrated). The copy number of the target gene or internal reference gene corresponding to each fluorescent channel of FAM, VIC, and Cy5 is obtained by correcting with the Poisson distribution model.

[0032] 4. Analysis of Test Results Total copy number of ERG11 gene = Wild-type target copy number + Mutant target copy number The calculation methods for wild-type and mutant copy numbers are as follows:

[0033] The total copy number of the four strains was analyzed by comparing it with the theoretical total copy number from whole-genome sequencing to assess whether it met expectations. The results are shown in Table 3 below.

[0034] Table 3

[0035] Theoretical total copy number Wild target copy number Mutation target copy number Total copy number of ddPCR Difference (dPCR - Theoretical) Standard strain 2 2.74 0.00 2.74 0.74 isolate 1 2 2.37 0.00 2.37 0.37 isolate 2 2 1.18 1.24 2.42 0.42 isolate 3 2 0.00 2.30 2.30 0.30 .

[0036] The results analysis showed that the difference (dPCR-theoretical) was greater than 0, and the ratio of target to internal reference gene was higher, indicating a slight deviation. One possible reason for this is that the detection value of the internal reference gene was lower, indicating that there is still room for further optimization of this scheme.

[0037] Example 2: Candida Antimicrobial Resistance Mutation Detection System - Multiplex Internal Reference System - Primer and Probe Design and Screening 1. To further improve the accuracy of total copy number quantification of the ERG11 gene, we plan to introduce other internal reference genes, GAPDH and β-tubulin, as alternative internal reference genes for calculating copy number variation, forming two combined internal reference normalization strategies. Similarly, we will use a basic buffer reaction system to screen primer-probe sets with different internal reference combinations. Table 4 below shows the information of candidate primer-probe sets.

[0038] Table 4. Information on multiple primer-probe combinations

[0039] 2. The basic reaction system is prepared as shown in Table 5 below.

[0040] Table 5 Material Name Original concentration Final concentration Amount added (μL) Tris-HCl 1 M 0.05 M 1.5 glycerin 80% 1% 0.375 dNTPs 75× 1× 0.4 KCl 1 M 0.01 M 0.3 <![CDATA[MgCl2]]> 1 M 4 mM 0.12 Taq DNA polymerase 5U / μL 0.067 U / μL 0.4 UNG enzyme 1U / μL 0.005 U / μL 0.15 10× Primer and Probe Set 10× 1× 3 DNA template - - 1 Ultrapure water - - Add to a total volume of 30 μL .

[0041] The final concentrations of primers and probes were all 600 nM and 300 nM, respectively. A total of 25 Candida tropicalis cultures (with sequencing results rounded to the nearest integer to represent the theoretical total copy number of 2 copies each) were used as test samples. Nucleic acid was extracted from the culture colonies using the XinYi nucleic acid extraction reagent. The nucleic acid was quantified using a Nanodrop instrument, and the concentration was calculated based on the theoretical genome length, then diluted to approximately 10,000 copies / μL for use as the test sample nucleic acid.

[0042] 3. Digital PCR Detection Process Microdroplet preparation: Using the sample preparation instrument and droplet generation chip from Xinyi Manufacturing Technology (Beijing) Co., Ltd., 30 μL of PCR reaction system was added to the sample well of the droplet generation chip, and 180 μL of microdroplet generation oil was added to the oil well. The chip and the 8-tube array were placed in the preparation instrument, and the adhesive pad was covered to prepare microdroplets.

[0043] PCR amplification: Place the sealed 8-tube array containing the droplets onto a PCR instrument for amplification. Set the amplification conditions as follows: .

[0044] Microdroplet detection: After PCR, the microchip analyzer from NewYi Manufacturing Technology (Beijing) Co., Ltd. was used for detection. The 8-tube array and the droplet detection chip were placed in the fixture, and 430uL and 500uL of detection oil were added to the oil wells respectively. The rubber gasket was then placed on the microchip analyzer for droplet detection.

[0045] Data Analysis: After chip detection, data analysis is performed. Based on the 1D and 2D scatter plots and the detection results of the positive control, a suitable positive region is delineated (generally, the positive region is delineated at the center of the region where the FAM / VIC / ROX / CY5 channel signals are concentrated). The copy number of the target gene or internal reference gene corresponding to each fluorescent channel of FAM, VIC, ROX, and CY5 is obtained by correcting with the Poisson distribution model.

[0046] 4. Analysis of Detection Results and Screening of Primer and Probe Sets Total copy number of ERG11 gene = Wild-type target copy number + Mutant target copy number The calculation methods for wild-type and mutant copy numbers are as follows:

[0047] The results of the total copy number detection by ddPCR for the above 25 strains are shown in Table 6 below.

[0048] Table 6

[0049]

[0050] The difference between the total copy number and the theoretical value of whole-genome sequencing was further calculated, and the mean was used to assess whether it met expectations. The analysis results are shown in Tables 7 and 8 below.

[0051] Table 7 Difference between combination 1 and theoretical value Difference between combination 2 and theoretical value strain 1 0.05 0.31 strain 2 0.01 0.12 strain 3 0.21 0.21 strain 4 0.06 0.13 strain 5 0.16 0.22 strain 6 0.10 0.19 strain 7 -0.10 -0.03 strain 8 0.15 0.08 strain 9 0.05 0.12 strain 10 0.05 0.31 strain 11 0.31 0.46 strain 12 0.45 0.64 strain 13 0.13 0.26 strain 14 0.15 0.16 strain 15 0.17 -0.06 strain 16 -0.33 -0.02 strain 17 0.07 0.16 strain 18 0.40 0.79 strain 19 0.14 0.68 strain 20 -0.08 0.18 strain 21 0.45 0.65 strain 22 0.21 0.53 strain 23 0.46 0.66 strain 24 -0.02 0.39 strain 25 -0.22 0.19 Table 8

[0052] Mean of difference Deviation (upper limit) Deviation (lower limit) Positive and negative differences (top - bottom) Combination 1 0.13 0.46 -0.33 0.79 Combination 2 0.30 0.79 -0.06 0.86 .

[0053] The results analysis showed that combination 1 had a smaller mean difference from the theoretical value, and a smaller positive and negative difference between the upper and lower limits of deviation, indicating that this internal control primer-probe combination was superior. Therefore, the first group was the optimal primer-probe combination. The 2D image of amplified heterozygous Candida tropicalis is shown below. Figure 1 As shown, Figure 1 The left side of the 2D plot shows the fluorescence intensity of the FAM channel on the horizontal axis and the HEX channel on the vertical axis. The right side of the 2D plot shows the fluorescence intensity of the ROX channel on the horizontal axis and the Cy5 channel on the vertical axis. The primer and probe information is summarized in Table 9 below.

[0054] Table 9 Serial Number Sequence information (5'-3') and information on modified groups Sequence and probe name SEQ ID NO.1 5'-ATACCCATTTGACTACTCCTGTT-3' ERG11 upstream primer SEQ ID NO.2 5'-TTAGCAAACTTCTTTTGTTCCAT-3' ERG11 downstream primer SEQ ID NO.3 5'FAM-AGGTGTTATTTATGATTGT-3'MGB ERG11 wild-type probe SEQ ID NO.4 5'HEX-AGGTGTTATTTTTGATTGT-3'MGB ERG11 mutant probe SEQ ID NO.8 5'-AGCCGGTATCTTGTTGACC-3' GAPDH upstream primer SEQ ID NO.9 5'-CTCTGGTGGAGTAACCGTATTC-3' GAPDH downstream primers SEQ ID NO.10 5'ROX-CAAATTGATCTCCTGGTACGATAA-3'MGB GAPDH probe SEQ ID NO.5 5'-TTGTCTCCAACTGGGATGATAT-3' Actin-1 upstream primer SEQ ID NO.6 5'-AACTGGGTGTTCTTCTGGAG-3' Actin-1 downstream primer SEQ ID NO.7 5'CY5-CTGGCACCACACTTTCTACAACGA-3'BHQ2 Actin-1 probe .

[0055] Example 3: Fragmentation of the ERG11 gene The detection method provided by this invention optimizes the DNA fragmentation conditions of Candida tropicalis and improves the accuracy of ERG11 gene copy number variation detection.

[0056] Mutations and copy number variations in the ERG11 gene of Candida tropicalis can be broadly categorized as follows: homozygous wild-type, heterozygous mutant, homozygous mutant, tandem duplication complex mutant, and tandem repeat homozygous mutant. Figure 2 As illustrated, the presence of tandem repeat sequences in Candida tropicalis DNA templates can lead to tandem repeat genes being encapsulated within the same droplet during digital PCR detection, resulting in decreased accuracy in copy number variation detection. To improve the accuracy of copy number variation detection, DNA fragmentation is necessary.

[0057] Although the amplification product fragments of the ERG11 gene or internal reference gene in this invention are relatively short, the length of DNA fragmentation products usually does not affect the detection results. However, considering the different lengths of amplicones for each target and the differences in base composition of different fragments, fragmentation may still affect the accuracy of copy number variation detection results. For example, insufficient fragmentation may cause tandem repeat sequences to be wrapped in the same droplet, thereby reducing the copy number variation detection results. Excessive fragmentation may cause differences in the copy numbers of the internal reference gene and the ERG11 gene (the ratio may increase or decrease), thus affecting the accuracy of copy number variation calculation.

[0058] In high-copy-number variant strains with tandem repeats, inaccuracies in the total copy number are more easily observed due to insufficient fragmentation. Therefore, in this example, two heterozygous mutant strains of *Candida tropicalis* with a large number of tandem repeat target genes (1:5 and 1:7, i.e., total copy numbers of the ERG11 gene of 1+5=6 and 1+7=8, respectively) verified by next-generation sequencing were selected. The genotypes of the relevant isolates are as follows: Figure 2 (The heterozygous mutant tandem repeat is shown) serves as a template source for fragmentation condition optimization.

[0059] The steps of the detection method are as follows: 1. DNA template extraction: The two isolates were extracted using the magnetic bead nucleic acid extraction and purification reagent from Xinyi Manufacturing Technology (Beijing) Co., Ltd. 3-5 colonies were picked and the elution volume was 70 μL. The extracted nucleic acid was quantified by ultraviolet spectrophotometry (Nanodrop) with a concentration range of 10-200 ng / μL.

[0060] 2. DNA Fragmentation: The fragmentation system was prepared using Hieff® Smearase fragmentation enzyme. The amount of fragmented DNA added was 50-500 ng. Subsequently, nine conditions were optimized for the fragmentation time and fragmentation enzyme dosage according to the present invention, as shown in the table below: .

[0061] 3. Fragmentation product recovery and purification: DNA fragmentation products were recovered using the Tiangen product recovery kit with an elution volume of 30 μL. Considering the strict requirements for input volume in downstream detection, a low-concentration, more accurate fluorescent dye method (qubit, HS-DNA) was used to quantify the recovered DNA. DNA was extracted from two unfragmented strains as controls and recovered simultaneously.

[0062] 4. Digital PCR detection: The recovered DNA was diluted. To avoid exceeding the upper limit of digital PCR detection values, 0.1~0.2 ng of fragmented Candida tropicalis DNA was added per reaction for digital PCR detection. Each experimental group was independently repeated once. The specific procedure is as described in Example 1.

[0063] 5. Results Analysis: In this embodiment, the copy number variation of tandem repeat sequences was used as the evaluation criterion for optimization conditions. The copy number of A395T was confirmed by the calculation method of (A395T copy number / mean internal reference copy number) × 2. The results of second-generation sequencing were used as theoretical values. The difference between the detected copy number under different conditions and the theoretical value (the theoretical total copy number is 6 and 8 respectively) was calculated. The difference between the detected copy number and the theoretical value is not more than 1 and is considered to be an acceptable group. The optimal conditions were selected based on the results. The specific results are shown in Table 10.

[0064] Table 10 .

[0065] The results showed that increasing the amount of enzyme and extending the reaction time would result in more complete fragmentation, improve the copy number variation detection value, and thus solve the problem of template segmentation by droplet encapsulation in digital PCR. However, it was also found that excessively high enzyme amounts or excessively long reaction times may lead to inaccurate detection results. The possible reason is that the internal reference gene is fragmented and degraded, resulting in a detection calculation result that is significantly higher than the theoretical value. The groups in which the difference between the theoretical and theoretical values ​​of the two strains did not exceed 1 were: (1) 10 min, 2.5 U (2) 10 min, 5 U (3) 20 min, 2.5 U (4) 20 min, 5 U (5) 30 min, 1 U (6) 30 min, 2.5 U. Therefore, the reaction time range is 10~30 min, and the enzyme digestion concentration is 1~5 U. The optimal conditions after optimization are: reaction time 20 min, enzyme digestion concentration 2.5 U.

[0066] Example 4: Gene copy number calculated using different methods The calculation method for analyzing the ERG11 gene copy number variation detection results in this invention has been optimized, further improving the accuracy of detecting ERG11 gene copy number variations.

[0067] This invention involves two internal reference genes used to calculate or correct copy number variation detection results. The optimized calculations include: single internal reference analysis; dual internal reference analysis; heterozygous allele comparison; and heterozygous multi-internal reference analysis. The principle involves reducing uncertainty in the detection process of different internal reference genes used for correction through arithmetic mean, thereby improving the accuracy of copy number variation detection. For heterozygous Candida tropicalis, due to the characteristics of its wild-type A395 target (usually with a copy number of 1 in the allele), the detection result of its wild-type target can also be used as a third internal reference gene to correct copy number variation detection results. Therefore, this invention sets up the following five different analytical calculation methods based on three categories: homozygous wild-type, heterozygous mutant, and homozygous mutant, such as... Figure 3 As shown, the accuracy of different calculation methods in detecting copy number variations is evaluated, and the optimal calculation method for analyzing copy number variation results is optimized.

[0068] In the analysis of ERG11 gene copy number variation described in this invention, the sum of wild-type and mutant copy numbers is calculated and compared with the theoretical value of the total copy number of ERG11 gene (rounded) from the next-generation sequencing results (the copy number variation calculation method based on next-generation sequencing described in this invention also uses rounding). When the absolute value of the difference between the detection result and the theoretical value is greater than 0, it is defined as a "difference". The maximum difference and the distribution of the difference between different calculation methods are statistically analyzed to select the optimal calculation method.

[0069] In this embodiment, an additional 98 Candida tropicalis isolates from clinical batches were tested and confirmed by next-generation sequencing to include 66 wild-type isolates, 3 homozygous mutant isolates, and 29 heterozygous mutant isolates.

[0070] The specific steps are as follows: 1. Following the steps of nucleic acid extraction, fragmentation, and digital PCR detection of the Candida tropicalis isolates to be tested in Examples 1, 2, and 3, the dPCR detection results of the above strains were collected, and the specific results are shown in Table 11.

[0071] Table 11

[0072] strain number FAM VIC ROX CY5 Classification 1 5533.3 0 4528.3 4044.8 wild type 2 6729.2 0 5365.9 7032.9 wild type 3 3138.3 0 2600.5 2292.5 wild type 4 1449.2 0 1531.1 1308.1 wild type 5 3279.2 0 3067.9 2635.5 wild type 6 299.5 0 352.4 323.3 wild type 7 393.6 0 319.6 272.1 wild type 8 791.2 0 594.6 398.7 wild type 9 1647.6 0 1192.6 1124.7 wild type 10 181.1 0 176.2 120.6 wild type 11 774.1 4491.6 1166.6 1013.1 Heterozygous mutant 12 1013.2 0 855.8 859 wild type 13 669.6 0 501.2 488 wild type 14 924.2 0 701.6 543.3 wild type 15 136.5 245.2 217.3 167.5 Heterozygous mutant 16 148.6 0 175.7 162.2 wild type 17 578.7 0 467.5 520.6 wild type 18 664.9 0 595.1 429.5 wild type 19 516.3 0 542.6 414.2 wild type 20 995 0 1115.8 954.2 wild type 21 382.3 0 391 306 wild type 22 294.7 1293.6 583.2 449.2 Heterozygous mutant 23 2976.9 0 2346.9 2246.5 wild type 24 549 0 553.8 459.3 wild type 25 2134.9 8043.7 3292.9 2680 Heterozygous mutant 26 257.3 1185.8 549.9 431.7 Heterozygous mutant 27 2911.3 3249.7 5329.1 3986.6 Heterozygous mutant 28 527.8 0 420.4 385.9 wild type 29 438.7 477.3 705.5 693.1 Heterozygous mutant 30 301.7 1681.1 506.7 497.3 Heterozygous mutant 31 61 549.5 240.9 165.3 Heterozygous mutant 32 1936.6 0 1480.2 1428.3 wild type 33 0 509.2 253.2 215.5 homozygous mutant 34 463.4 0 487.8 403.4 wild type 35 1167.5 0 1184 1347.4 wild type 36 782.1 0 765.8 708.8 wild type 37 1457.9 7456.3 2513.8 2237.3 Heterozygous mutant 38 13624 31004.9 10522.2 10052.7 Heterozygous mutant 39 2843.5 13053.2 4149.4 4015.3 Heterozygous mutant 40 1356.7 7074.8 2210 1855.4 Heterozygous mutant 41 2898.2 0 2432.7 2057.5 wild type 42 1196.5 5957 1932.3 1802.2 Heterozygous mutant 43 2243.7 0 1725 1544.9 wild type 44 366.7 0 350 230.3 wild type 45 232.7 1669.3 570.3 483.3 Heterozygous mutant 46 218.8 1416.6 503 323.4 Heterozygous mutant 47 902.2 4406.2 1558.2 1190.3 Heterozygous mutant 48 394.4 3059.6 954.9 731 Heterozygous mutant 49 115.5 695.1 300 176.9 Heterozygous mutant 50 447.4 0 472.3 322.9 wild type 51 238 0 196.2 196.2 wild type 52 2936.1 8354.5 4545.9 3939.2 Heterozygous mutant 53 2345.6 6639.7 3858.1 3350.4 Heterozygous mutant 54 11521.6 0 9156.7 9438.8 wild type 55 3267.2 0 2544.8 2262.8 wild type 56 0 33658.1 4788.7 4389.6 homozygous mutant 57 5095.8 0 4126.3 3813.9 wild type 58 2343.4 0 1901 1770.5 wild type 59 1008.1 0 822.7 731.1 wild type 60 1779.1 0 1711 1392 wild type 61 3833.7 18049.8 5749.8 5377.5 Heterozygous mutant 62 3152.3 0 1923.7 1927.2 wild type 63 771.5 0 550.3 468.7 wild type 64 1815.8 0 1367.8 1168.9 wild type 65 1396.1 0 987.1 774.3 wild type 66 5145 0 4549.1 3680.4 wild type 67 8526.6 42561.2 13119.6 13396.1 Heterozygous mutant 68 5263 0 4327.9 3534.2 wild type 69 3986 0 2983.1 2590 wild type 70 8567.4 0 6950.4 5655.5 wild type 71 8663.6 0 6659.3 5909.6 wild type 72 21219 0 16237.2 15548.7 wild type 73 4290.1 0 3312.5 2734.4 wild type 74 1835.3 0 1743.7 1287.6 wild type 75 1103.1 0 1275.2 967.7 wild type 76 1635 5966.8 3514.4 2303.1 Heterozygous mutant 77 4152.7 0 3793.1 3096.2 wild type 78 2802.1 0 2392.9 1834.9 wild type 79 1200 0 924 655.3 wild type 80 16515.4 0 12361.3 11017.4 wild type 81 1835.6 0 1304.3 996.7 wild type 82 608.2 0 522.6 430.4 wild type 83 2750.9 0 2296.3 2022.3 wild type 84 2289.5 0 1706 1383.5 wild type 85 2150.7 14493.4 3445.3 3075.9 Heterozygous mutant 86 4641.1 0 3470.2 2720.2 wild type 87 2307.7 4939.1 3305.4 2916.1 Heterozygous mutant 88 3202 3315.3 4388.5 4078.8 Heterozygous mutant 89 1717.1 1746.5 2762.9 2190.1 Heterozygous mutant 90 771.9 0 739.6 519.2 wild type 91 833.9 0 777.7 656 wild type 92 753.8 0 640.1 504.9 wild type 93 3555.9 0 2956 2461 wild type 94 1037.5 0 814.2 620.2 wild type 95 567.1 2974.4 924.5 636 Heterozygous mutant 96 2980.7 0 2210.6 1927.5 wild type 97 0 296.1 232.4 196.3 homozygous mutant 98 4931.7 0 4531.1 3933.1 wild type .

[0073] 2. The detection results were analyzed using different established analytical calculation methods. The formula for calculating the total copy number is as follows: Wild type: Total copy number = Wild target copy number Homozygous mutant: Total copy number = homozygous mutant copy number Heterozygous mutant: Total copy number = wild-type target copy number + heterozygous mutant copy number Furthermore, the formulas for calculating the copy number of different targets are as follows.

[0074]

[0075] The copy number of the ERG11 gene of the strain was calculated based on the copy number detected by each fluorescence channel under each calculation method. The total copy number of different calculation methods was then obtained by summing the results and performing a difference analysis by comparing the result with the theoretical value. The specific results are shown in Table 12.

[0076] Table 12 strain number Theoretical value Calculation Method 1 Difference 1 Calculation Method 2 Difference 2 Calculation Method 3 Difference 3 Calculation method 4 Difference 4 Calculation Method 5 Difference 5 1 2 2.444 0.444 2.582 0.582 2.582 0.582 2.582 0.582 2.582 0.582 2 2 2.508 0.508 2.171 0.171 2.171 0.171 2.171 0.171 2.171 0.171 3 2 2.414 0.414 2.566 0.566 2.566 0.566 2.566 0.566 2.566 0.566 4 2 1.893 -0.107 2.042 0.042 2.042 0.042 2.042 0.042 2.042 0.042 5 2 2.138 0.138 2.300 0.300 2.300 0.300 2.300 0.300 2.300 0.300 6 2 1.700 -0.300 1.773 -0.227 1.773 -0.227 1.773 -0.227 1.773 -0.227 7 2 2.463 0.463 2.661 0.661 2.661 0.661 2.661 0.661 2.661 0.661 8 2 2.661 0.661 3.186 1.186 3.186 1.186 3.186 1.186 3.186 1.186 9 2 2.763 0.763 2.844 0.844 2.844 0.844 2.844 0.844 2.844 0.844 10 2 2.056 0.056 2.441 0.441 2.441 0.441 2.441 0.441 2.441 0.441 11 7 5.177 -1.823 8.650 1.650 7.223 0.223 8.877 1.877 9.704 2.704 12 2 2.368 0.368 2.363 0.363 2.363 0.363 2.363 0.363 2.363 0.363 13 2 2.672 0.672 2.708 0.708 2.708 0.708 2.708 0.708 2.708 0.708 14 2 2.635 0.635 2.970 0.970 2.970 0.970 2.970 0.970 2.970 0.970 15 3 2.385 -0.615 3.655 0.655 3.215 0.215 3.746 0.746 4.011 1.011 16 2 1.692 -0.308 1.759 -0.241 1.759 -0.241 1.759 -0.241 1.759 -0.241 17 2 2.476 0.476 2.343 0.343 2.343 0.343 2.343 0.343 2.343 0.343 18 2 2.235 0.235 2.596 0.596 2.596 0.596 2.596 0.596 2.596 0.596 19 2 1.903 -0.097 2.158 0.158 2.158 0.158 2.158 0.158 2.158 0.158 20 2 1.783 -0.217 1.923 -0.077 1.923 -0.077 1.923 -0.077 1.923 -0.077 21 2 1.955 -0.045 2.194 0.194 2.194 0.194 2.194 0.194 2.194 0.194 22 5 3.229 -1.771 5.928 0.928 5.531 0.531 6.004 1.004 6.240 1.240 23 2 2.537 0.537 2.592 0.592 2.592 0.592 2.592 0.592 2.592 0.592 24 2 1.983 -0.017 2.168 0.168 2.168 0.168 2.168 0.168 2.168 0.168 25 6 3.739 -2.261 6.142 0.142 5.197 -0.803 6.315 0.315 6.874 0.874 26 5 3.092 -1.908 5.804 0.804 5.657 0.657 5.854 0.854 5.952 0.952 27 2 1.702 -0.298 2.538 0.538 2.366 0.366 2.572 0.572 2.675 0.675 28 2 2.511 0.511 2.618 0.618 2.618 0.618 2.618 0.618 2.618 0.618 29 2 1.920 -0.080 2.513 0.513 2.343 0.343 2.527 0.527 2.620 0.620 30 7 4.509 -2.491 7.477 0.477 6.774 -0.226 7.525 0.525 7.900 0.900 31 8 2.787 -5.213 6.843 -1.157 9.609 1.609 7.340 -0.660 6.206 -1.794 32 2 2.617 0.617 2.663 0.663 2.663 0.663 2.663 0.663 2.663 0.663 33 4 4.022 0.022 4.346 0.346 4.346 0.346 4.346 0.346 4.374 0.374 34 2 1.900 -0.100 2.080 0.080 2.080 0.080 2.080 0.080 2.080 0.080 35 2 1.972 -0.028 1.845 -0.155 1.845 -0.155 1.845 -0.155 1.845 -0.155 36 2 2.043 0.043 2.122 0.122 2.122 0.122 2.122 0.122 2.122 0.122 37 7 4.126 -2.874 7.063 0.063 6.342 -0.658 7.131 0.131 7.526 0.526 38 8 5.536 -2.464 6.539 -1.461 4.924 -3.076 7.428 -0.572 8.680 0.680 39 7 4.516 -2.484 7.047 0.047 5.984 -1.016 7.188 0.188 7.790 0.790 40 7 4.429 -2.571 7.597 0.597 6.550 -0.450 7.749 0.749 8.349 1.349 41 2 2.383 0.383 2.582 0.582 2.582 0.582 2.582 0.582 2.582 0.582 42 7 4.321 -2.679 7.115 0.115 6.260 -0.740 7.200 0.200 7.670 0.670 43 2 2.601 0.601 2.745 0.745 2.745 0.745 2.745 0.745 2.745 0.745 44 2 2.095 0.095 2.528 0.528 2.528 0.528 2.528 0.528 2.528 0.528 45 8 3.743 -4.257 7.477 -0.523 8.057 0.057 7.529 -0.471 7.264 -0.736 46 7 3.686 -3.314 7.783 0.783 7.533 0.533 8.015 1.015 8.256 1.256 47 7 3.986 -3.014 7.120 0.120 6.197 -0.803 7.294 0.294 7.843 0.843 48 8 4.030 -3.970 8.354 0.354 8.693 0.693 8.448 0.448 8.325 0.325 49 7 3.087 -3.913 6.860 -0.140 6.987 -0.013 7.139 0.139 7.215 0.215 50 2 1.895 -0.105 2.251 0.251 2.251 0.251 2.251 0.251 2.251 0.251 51 2 2.426 0.426 2.426 0.426 2.426 0.426 2.426 0.426 2.426 0.426 52 4 3.130 -0.870 4.876 0.876 4.230 0.230 4.972 0.972 5.343 1.343 53 4 2.937 -1.063 4.649 0.649 4.132 0.132 4.714 0.714 5.004 1.004 54 2 2.517 0.517 2.478 0.478 2.478 0.478 2.478 0.478 2.478 0.478 55 2 2.568 0.568 2.718 0.718 2.718 0.718 2.718 0.718 2.718 0.718 56 12 14.057 2.057 14.669 2.669 14.669 2.669 14.669 2.669 14.696 2.696 57 2 2.470 0.470 2.567 0.567 2.567 0.567 2.567 0.567 2.567 0.567 58 2 2.465 0.465 2.553 0.553 2.553 0.553 2.553 0.553 2.553 0.553 59 2 2.451 0.451 2.595 0.595 2.595 0.595 2.595 0.595 2.595 0.595 60 2 2.080 0.080 2.293 0.293 2.293 0.293 2.293 0.293 2.293 0.293 61 6 4.473 -1.527 7.140 1.140 6.086 0.086 7.278 1.278 7.874 1.874 62 2 3.277 1.277 3.274 1.274 3.274 1.274 3.274 1.274 3.274 1.274 63 2 2.804 0.804 3.028 1.028 3.028 1.028 3.028 1.028 3.028 1.028 64 2 2.655 0.655 2.863 0.863 2.863 0.863 2.863 0.863 2.863 0.863 65 2 2.829 0.829 3.170 1.170 3.170 1.170 3.170 1.170 3.170 1.170 66 2 2.262 0.262 2.501 0.501 2.501 0.501 2.501 0.501 2.501 0.501 67 7 4.544 -2.456 7.147 0.147 6.278 -0.722 7.231 0.231 7.707 0.707 68 2 2.432 0.432 2.678 0.678 2.678 0.678 2.678 0.678 2.678 0.678 69 2 2.672 0.672 2.861 0.861 2.861 0.861 2.861 0.861 2.861 0.861 70 2 2.465 0.465 2.719 0.719 2.719 0.719 2.719 0.719 2.719 0.719 71 2 2.602 0.602 2.757 0.757 2.757 0.757 2.757 0.757 2.757 0.757 72 2 2.614 0.614 2.670 0.670 2.670 0.670 2.670 0.670 2.670 0.670 73 2 2.590 0.590 2.838 0.838 2.838 0.838 2.838 0.838 2.838 0.838 74 2 2.105 0.105 2.422 0.422 2.422 0.422 2.422 0.422 2.422 0.422 75 2 1.730 -0.270 1.967 -0.033 1.967 -0.033 1.967 -0.033 1.967 -0.033 76 5 2.628 -2.372 5.064 0.064 4.774 -0.226 5.200 0.200 5.413 0.413 77 2 2.190 0.190 2.411 0.411 2.411 0.411 2.411 0.411 2.411 0.411 78 2 2.342 0.342 2.651 0.651 2.651 0.651 2.651 0.651 2.651 0.651 79 2 2.597 0.597 3.039 1.039 3.039 1.039 3.039 1.039 3.039 1.039 80 2 2.672 0.672 2.826 0.826 2.826 0.826 2.826 0.826 2.826 0.826 81 2 2.815 0.815 3.191 1.191 3.191 1.191 3.191 1.191 3.191 1.191 82 2 2.328 0.328 2.553 0.553 2.553 0.553 2.553 0.553 2.553 0.553 83 2 2.396 0.396 2.548 0.548 2.548 0.548 2.548 0.548 2.548 0.548 84 2 2.684 0.684 2.964 0.964 2.964 0.964 2.964 0.964 2.964 0.964 85 8 5.455 -2.545 9.354 1.354 8.058 0.058 9.511 1.511 10.238 2.238 86 2 2.675 0.675 2.999 0.999 2.999 0.999 2.999 0.999 2.999 0.999 87 4 2.891 -1.109 4.218 0.218 3.624 -0.376 4.322 0.322 4.672 0.672 88 2 2.215 0.215 2.850 0.850 2.548 0.548 2.903 0.903 3.081 1.081 89 2 1.875 -0.125 2.636 0.636 2.404 0.404 2.679 0.679 2.816 0.816 90 2 2.087 0.087 2.453 0.453 2.453 0.453 2.453 0.453 2.453 0.453 91 2 2.145 0.145 2.327 0.327 2.327 0.327 2.327 0.327 2.327 0.327 92 2 2.355 0.355 2.633 0.633 2.633 0.633 2.633 0.633 2.633 0.633 93 2 2.406 0.406 2.626 0.626 2.626 0.626 2.626 0.626 2.626 0.626 94 2 2.549 0.549 2.893 0.893 2.893 0.893 2.893 0.893 2.893 0.893 95 7 4.444 -2.556 8.076 1.076 6.699 -0.301 8.465 1.465 9.348 2.348 96 2 2.697 0.697 2.881 0.881 2.881 0.881 2.881 0.881 2.881 0.881 97 2 2.548 0.548 2.763 0.763 2.763 0.763 2.763 0.763 2.783 0.783 98 2 2.177 0.177 2.331 0.331 2.331 0.331 2.331 0.331 2.331 0.331 .

[0077] Subsequently, the difference between the total copy number and the theoretical value under different calculation methods was analyzed, and the optimal calculation method was selected. The results are shown in Table 13.

[0078] Table 13 Mean of difference Deviation (upper limit) Deviation (lower limit) Positive and negative differences (top - bottom) Calculation Method 1 -0.36 2.06 -5.21 7.27 Calculation Method 2 0.53 2.67 -1.46 4.13 Calculation Method 3 0.38 2.67 -3.08 5.74 Calculation method 4 0.58 2.67 -0.66 3.33 Calculation Method 5 0.67 2.70 -1.79 4.50 .

[0079] As verified, such as Figure 3 As shown in the table above, a comprehensive analysis of indicators such as the mean difference, upper and lower limits of deviation, and positive and negative differences shows that the mean difference of calculation method 4 is not significantly different from the theoretical value (not exceeding 1) compared with other groups, and the lower limit of deviation is small and has a smaller "positive and negative difference". Therefore, calculation method 4 is selected as the optimal copy number variation analysis calculation method of this invention.

[0080] It should be understood that the disclosed invention is not limited to the specific methods, schemes, and substances described, as these are all subject to variation. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims.

[0081] Those skilled in the art will also recognize, or be able to identify, many equivalents of the specific embodiments of the invention described herein using no more than conventional experiments. These equivalents are also included in the appended claims.

Claims

1. A digital PCR method for detecting copy number variation in the ERG11 gene of Candida tropicalis, characterized in that, The method uses a set of universal primers and wild-type and mutant probes at the A395 site of the ERG11 gene in the detection sample to detect the total copy number of the ERG11 gene. The total copy number of the ERG11 gene = wild-type target copy number + mutant target copy number. In the PCR detection method, two internal reference gene copy numbers are used to correct the wild-type and mutant target copy numbers. The calculation method for determining the wild-type and mutant target copy numbers based on the mutant type of the ERG11 gene at the A395 site in the digital PCR detection method is as follows: When the ERG11 gene at the A395 site is wild-type, homozygous, or heterozygous, the calculation methods for the wild-type and mutant target copy numbers are as follows: Wild-type target copy number = (wild-type target gene copy number / (internal reference gene 1 copy number + internal reference gene 2 copy number) / 2) × 2; Copy number of homozygous mutant target gene = (copy number of mutant target gene / (copy number of internal reference gene 1 + copy number of internal reference gene 2) / 2) × 2; The copy number of the heterozygous mutant target gene = (copy number of mutant target gene / (copy number of wild-type target gene × 2) + copy number of mutant target gene / copy number of internal reference gene 1 + copy number of mutant target gene / copy number of internal reference gene 2) × 2 / 3.

2. The method according to claim 1, characterized in that, The two internal reference genes are Actin-1 and GAPDH.

3. The method according to claim 2, characterized in that, The upstream and downstream primers and probes for PCR digital amplification detection of the Actin-1 gene are SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7, respectively, and the upstream and downstream primers and probes for PCR digital amplification detection of the GAPDH gene are SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10, respectively.

4. The method according to claim 1, characterized in that, The upstream and downstream primers and wild-type and mutant probes for PCR digital amplification detection of the A395 site of the ERG11 gene are SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, respectively.

5. The method according to claim 1, characterized in that, Before performing digital PCR amplification, the sample DNA is also fragmented.

6. The method according to claim 5, characterized in that, The concentration of the fragmentation enzyme in the fragmentation reaction system is 1~5U, and the reaction time ranges from 10 to 30 minutes.

7. The method according to claim 6, characterized in that, The concentration of the fragmentation enzyme in the fragmentation reaction system was 2.5 U, and the reaction time was 20 min.

8. The digital PCR detection kit used in any one of the methods described in claims 1-7, characterized in that, The kit includes a primer and probe set for PCR digital amplification detection of the A395 site of the ERG11 gene and two internal reference gene primer and probe sets for normalization correction. The upstream and downstream primers and wild-type probes and mutant probes for PCR digital amplification detection of the ERG11 gene A395 site are SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, respectively; the upstream and downstream primers and probes for PCR digital amplification detection of the Actin-1 gene are SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7, respectively; and the upstream and downstream primers and probes for PCR digital amplification detection of the GAPDH gene are SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, respectively.

9. The digital PCR detection kit according to claim 8, characterized in that, The digital PCR kit also includes a fragmentation enzyme for processing the extracted Candida tropicalis DNA template.

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