Methods and kits for detecting fungicidal pathogens with resistance

Abstract

A method for detecting a fungicidal pathogen having drug resistance is disclosed. The method comprises detecting a single nucleotide polymorphism genotype of XYR1 a gene of the fungicidal pathogen and SNQ2 a gene of the fungicidal pathogen. A kit for detecting a fungicidal pathogen having drug resistance is disclosed. The kit comprises a lysis buffer for lysing the fungicidal pathogen and primers for detecting a single nucleotide polymorphism genotype of XYR1 a gene of the fungicidal pathogen and / or SNQ2 a gene of the fungicidal pathogen.

Description

[0001] Cross-references to related applications This invention claims priority to U.S. Provisional Application No. 63 / 550,611, filed February 7, 2024, the contents of which are incorporated herein by reference. Background Technology 1. Technical Field This invention contains a sequence list electronically submitted in .XML file format, the entire text of which is hereby incorporated by reference. This .XML copy (created on January 22, 2025) is named NHR-P0029-PUS-SequencingListing.xml and has a file size of 45,519 bytes.

[0003] This invention relates to methods and kits for detecting drug-resistant fungal pathogens, and particularly to methods for detecting drug-resistant Candida tropicalis with the clade 4 genotype. Candidatropicalis ).

[0004] 2. Description of existing technology Fungal infections cause approximately 2.5 million deaths annually, posing a serious public health problem, especially as drug-resistant pathogens increase with widespread drug use. The number of fungal infection cases is underestimated due to diagnostic difficulties. Preliminary estimates in the United States indicate that fungal infections cost approximately $11.5 billion in 2019, with medical expenses accounting for $7.5 billion.

[0005] Candida spp. Candida species; abbreviation Candida Candida species (spp.) are generally harmless eukaryotic symbiotic yeasts found in various environments and in human and other mammalian sources. In humans, Candida plays a vital role in the microbiota of the skin, oral cavity, vaginal mucosa, and gastrointestinal tract. However, in cases of severe immune impairment, Candida can transform into opportunistic pathogens, leading to illness and even death. Drug-resistant Candida strains are spreading in many countries. Currently, whole genome sequencing or traditional biochemical identification is commonly used to identify major drug-resistant genotypes of Candida, but these methods are time-consuming and costly.

[0006] In view of this, there remains an unmet need in this technical field to develop rapid detection methods to identify major drug-resistant genotypes of Candida. Therefore, this information can assist healthcare professionals in determining appropriate antifungal drugs for treating fungal infections. Summary of the Invention

[0007] In view of the above problems, the present invention provides a method for detecting drug-resistant fungal pathogens. The method includes: detecting the fungal pathogen... XYR1 Genes and SNQ2 Single nucleotide polymorphism (SNP) genotype of a gene.

[0008] The present invention also provides a kit for detecting drug-resistant fungal pathogens. The kit comprises: a lysis buffer for lysing the fungal pathogen; and primers for detecting the fungal pathogen. XYR1 Genes and / or SNQ2 Gene.

[0009] The subject matter of this invention will undoubtedly become readily understood by those skilled in the art upon reading the following detailed description of the embodiments and various drawings. Attached Figure Description

[0010] The patent or application documents must contain at least one color drawing. A color drawing copy of the published text of the patent or patent application may be provided by the Office upon the applicant's request and payment of the necessary fees.

[0011] Figure 1 Two illustrations according to an embodiment of the present invention XYR1 A schematic diagram of a gene.

[0012] Figure 2 Two illustrations according to an embodiment of the present invention XYR1 Box plots of intra-genotype and inter-genotype distances (boxes represent interquartile ranges, with lines extending into the data; points outside the range are minor outliers, with values ​​greater than 1.5 times the upper bound of the interquartile range; "**" indicates p-value < 0.01).

[0013] Figure 3A This image shows an amplification of a target gene by multiplex polymerase chain reaction (PCR) according to an embodiment of the present invention (the template for multiplex PCR is ATCC750; the nine amplicons are divided into three groups according to the size of the PCR product, including group 1 (blue; approximately 750 bp): ICL1 , MDR1 , SAPT2 , SAPT4 , ZWF1a and SNQ2 Group 2 (green; approx. 1 kb): CTRG_05978 and GTRG_05993; and Group 3 (orange, approx. 1.5 kb): ERG11 ).

[0014] Figure 3BImages showing the results of multiplex PCR at two bonding temperatures (60°C (channels 1 and 2) and 61°C (channels 3 and 4) according to an embodiment of the present invention (negative control group shown in channels 2 and 4).

[0015] Figure 4 A bar chart showing the percentages of nine amplicon reads in seven isolates according to an embodiment of the present invention (the percentages were calculated using Samtools to identify the individual reads).

[0016] Figure 5A This displays the target gene (6 multilocus sequence typing; MLST) in 96 samples according to an embodiment of the present invention. ICL1, MDR1 , SAPT2 , SAPT4 , XYR1 , ZWF1a , ERG11 , SNQ2 Box plots of the percentage distribution of readings for each gene (the box plot represents the interquartile range of the percentage of readings for each gene, with the median indicated by a line inside the box; it must extend to the minimum and maximum values ​​of the data, and individual outliers must be plotted).

[0017] Figure 5B This invention demonstrates that, according to an embodiment of the present invention, multiplex PCR-MinION sequencing was used to screen 96 single nucleotide polymorphisms (SNPs) at four single nucleotide polymorphism (SNP) screening thresholds. C. tropicalis A bar graph showing the sequencing success rate of the six MLST genes from the isolates (the bars represent the percentage of accuracy using bcftools compared to existing sequencing results in the field; four SNP screening thresholds include: Screening 1: a minimum reading of 40% for all MLST genes, excluding...). XYR1 (20%) excluding; Screening 2: The minimum reading for all MLST genes is 30%, except XYR1 (15%) except; Screening 3: The minimum reading for all MLST genes is 25%, except XYR1 (10%) outside; Screening 4: Minimum reading of all MLST genes is 10%).

[0018] Figure 5C A bar graph showing optimized readouts for correctly assembled multiplex PCR-MinION sequencing according to an embodiment of the present invention (96 readouts selected using a consistent 10% SNP screening threshold). C. tropicalis The accuracy of detecting six MLST gene fragments in isolates was measured using different reading thresholds (5,000, 7,000, 10,000, 12,000).

[0019] Figure 6 The left half of the diagram shows the allele grouping of CTRG_05978 according to an embodiment of the present invention, which shows alleles of GA / GA homozygotes (red group in the lower right corner), GA / AA heterozygotes (green group in the middle), and AA / AA homozygotes (blue group in the upper left corner). The strains in the green group in the middle correspond to the IV genotype (GA / AA) of CTRG_05978. Six strains are located in the green group in the middle, including YM140156, YM180233, YM180351, YM180473, YM180681, and YM180969.

[0020] Figure 6 The right half shows an embodiment of the present invention. SNQ2 A2977G shows the allele distribution diagram for G / G homozygotes (bottom right red group), G / A heterozygotes (middle green group), and A / A homozygotes (top left blue group). (Eight strains are shown.) SNQ2 Mutation; by binding CTRG_05978 and SNQ2 The genotyping results, using TaqMan genotyping, can directly identify CTRG_05978 type IV and... SNQ2 A2977G).

[0021] Figure 7 This demonstrates, according to an embodiment of the present invention, the detection of CTRG_05978 SNP and SNQ2 A2977G mutation for the diagnosis and management of the fourth branch C. tropicalis The schematic flowchart (SNPs are marked with red arrows; red yeast represents the main antibiotic resistance fourth branch genotype, and other colors represent different genotypes). Detailed Implementation

[0022] The following embodiments are provided to illustrate the present invention in detail. Those skilled in the art will readily understand the advantages and effects of the present invention upon reading the disclosure of this specification, and it can be implemented or applied in other different embodiments. Therefore, the following embodiments for carrying out the present invention can be modified and / or changed without departing from their different aspects and scope of application, and any element or method within the scope of the present invention can be combined with any other element or method disclosed in any embodiment of the present invention.

[0023] To make this invention easier to understand, specific terminology is first defined. Furthermore, it must be noted that whenever a parameter's numerical value or range is described, it should be understood that all values ​​and ranges between those values ​​also fall within the scope of this invention.

[0024] As stated herein, the singular articles “a,” “one,” and “the” also include plural forms, unless otherwise expressly stated herein. The term “or” is used interchangeably with “and / or”, unless otherwise expressly stated herein.

[0025] As described herein, the terms “comprising,” “including,” “having,” “containing,” and any other variations thereof are intended to cover non-exclusive inclusions. For example, when describing something as “comprising” a limitation, it may additionally include other ingredients, elements, components, structures, regions, parts, alleles, locations, steps, or connections, unless otherwise stated, and should not exclude other limitations.

[0026] In at least one embodiment of the present invention, XYR1 The single nucleotide polymorphism genotype of a gene may include the sequence ATRA, and R may be A or G. In some embodiments, the sequence ATRA may be... XYR1 The first 4 nucleotides of a gene.

[0027] In at least one embodiment of the present invention, SNQ2 The single nucleotide polymorphism genotype of a gene can include the substitution of nucleotide A at position 2977 with nucleotide G (A2977G).

[0028] In at least one embodiment of the present invention, the fungal pathogen may be a non-Candida genus (non- albicans Candida species). In some embodiments, non-Candida spp. may be selected from Candida tropicalis (species). Candidatropicalis ), Candida parapsilosis ( Candidaparapsilosis ), Candida glabrata ( Candidaglabrata ), Candida auris ( Candida auris ), Candida albicans ( Candida guilliermondii ) and Candida krusei ( Candidakrusei At least one of the groups consisting of Candida albicans. In some embodiments, non-Candida albicans may be Candida tropicalis ( Candidatropicalis In some embodiments, Candida tropicalis (…) Candidatropicalis It can include the fourth branch genotype.

[0029] In at least one embodiment of the present invention, a drug-resistant fungal pathogen may be resistant to drugs containing azoles.

[0030] In at least one embodiment of the present invention, fungal pathogens XYR1 Genes and SNQ2Single nucleotide polymorphisms (SNPs) of genes can be detected using high-throughput assays. In some embodiments, high-throughput assays may include a combination of multiple polymerase chain reaction (MLCR) and MinION nanopore sequencing.

[0031] In at least one embodiment of the present invention, fungal pathogens XYR1 Genes and SNQ2 The genotype of a gene with single nucleotide polymorphisms can be determined using the TaqMan genotyping assay.

[0032] In at least one embodiment of the present invention, the kit may further comprise DNA polymerase; and PCR buffer, comprising Mg 2+ .

[0033] In some embodiments, Candida species found in humans ( Candida In spp.), Candida albicans ( Candida albicans Candida albicans is the most commonly isolated species in hospital environments and causes nosocomial infections. However, in recent years, more treatment-resistant non-Candida species have gradually emerged, such as Candida tropicalis (Candida). Candidatropicalis ), Candida parapsilosis ( Candidaparapsilosis ), Candida glabrata ( Candidaglabrata ) and Candida krusei ( Candidakrusei The prevalence of these fungal species can vary depending on geographical location. In some regions, such as Asia and Latin America, tropical Candida (…) C. tropicalis It stands out among non-Candida albicans species, becoming the second or even the most common pathogenic yeast.

[0034] In some embodiments, for Candida tropicalis ( C. tropicalis Molecular epidemiological surveys of antibiotic-resistant strains (ARS) play a crucial role in identifying prevalent genotypes, potential transmission routes, genetic composition of resistant strains, habitat characteristics, and population structure. Multilocus sequence typing (MLST) has been developed to characterize population structure within strains and differentiate geographical origins. Allele combinations at different locations produce unique diploid sequence types (DSTs), allowing for the differentiation and classification of genetic associations between strains. Currently, [the text abruptly ends here, likely due to an incomplete sentence or missing information]. C. tropicalis The MLST method uses six genes, including ICL1 , MDR1 , SAPT2 , SAPT4 , XYR1 and ZWF1aFurthermore, MLST has been widely used in evaluation C. tropicalis The clonality of isolates was assessed, and various characteristics, such as fluconazole sensitivity, hospital origin, and anatomical origin, were identified. Finally, by using the online MLST database, allele profiles of isolates could be compared to explore different geographical origins, drug resistance transmission, and patterns of genetic variation.

[0035] In some embodiments, MLST is also used to reveal fluconazole resistance. C. tropicalis The strains (isolated strains) had the same genotype, i.e., the fourth clade, emphasizing the MLST typing as a way to differentiate drug resistance. C. tropicalis The practicality of the strain method. Although MLST is useful in determining strains... C. tropicalis While the genotyping aspect offers simple applications, rapid identification and more comprehensive characterization of drug-resistant strains (isolated isolates) remain important in diagnostics and antifungal management. Therefore, this invention illustrates one such method. XYR1 Gene (CTRG_05978) and located 25 kb upstream of CTRG_05978 SNQ2 Genotype, similar to Candida glabrata ( Candidaglabrata ) and Candida auris ( Candida auris The correlation between drug resistance and the ability to rapidly identify fluconazole resistance is crucial. C. tropicalis Potential markers to help prevent further spread of the main fourth branch strain (isolated strain). Example

[0036] Exemplary embodiments of the present invention are further illustrated in the following examples, which should not be construed as limiting the scope of the invention.

[0037] Materials and Methods C. tropicalis Isolation of strains Separation from the environment C. tropicalisThe methods are detailed in the China Taiwan Surveillance of Antimicrobial Resistance of Yeasts (TSARY) (Yang, YL, Lin, CC, Chang, TP, Lauderdale, TL, Chen, HT, Lee, CF, Hsieh, CW, Chen, PC and Lo, HJ (2012) Comparison of human and soil Candida tropicalis isolates with reduced susceptibility to fluconazole. PloS one, 7, e34609; and Zhou, ZL, Lin, CC, Chu, WL, Yang, YL and Lo, HJ (2016) The distribution and drug susceptibilities of clinical Candidaspecies in TSARY 2014. Diagnostic microbiology and infectious disease, 86, 399-404). All yeast strains were identified by rDNA sequencing (Leaw, SN, Chang, HC, Barton, R., Bouchara, JP and Chang, TC (2007) Identification of medically important Candida and non-Candida yeast species by an oligonucleotide array. Journal of clinical microbiology, 45, 2220-2229). To ensure... C. tropicalis Representative groups (at least 10 strains) were analyzed, with 37 strains from 11 of the most common groups having complete genome data. A total of 120 strains were preliminarily analyzed. C. tropicalis Strains, including ATCC750 and 119 strains from TSARY (see Table 5), were used to identify potential markers. For validation, 161 strains were further analyzed, including all fourth-branch whole-genome data from the NCBI database, as well as 10 other populations containing the most DST members.

[0038] DNA extraction and C. tropicalis MLST analysis of strains Each C. tropicalis Genomic DNA from the strain (isolated strain) was extracted using a yeast genomic DNA kit (Geneaid) according to the manufacturer's instructions, and quantified using a Qubit fluorescence quantification system (ThermoFisher Scientific). DNA fragments of six genes were extracted, containing... ICL1 , MDR1 , SAPT2 , SAPT4 , XYR1 and ZWF1a Amplification and sequencing were performed using previous research methods (Yang, YL, Lin, CC, Chang, TP, Lauderdale, TL, Chen, HT, Lee, CF, Hsieh, CW, Chen, PC and Lo, HJ (2012) Comparison of human and soil Candida tropicalis isolates with reduced susceptibility tofluconazole. PloS one, 7, e34609; and Zhou, ZL, Tseng, KY, Chen, YZ,Tsai, DJ, Wu, CJ, Chen, YC, Peng, HL, Yang, YL, Hsieh, LY, Chen,CH et al. (2022) Genetic relatedness among azole-resistant Candida tropicalis Clinical strains in Taiwan, China from 2014 to 2018. International Journal of Antimicrobial Agents, 59, 106592. The primers used are shown below (see Table 1). The obtained sequences were aligned using BioNumerics 3.0 (Applied Maths, Kortrijk, Belgium) and compared with... C. tropicalis The sequences were compared with those in the database (http: / / pubmlst.org / website) to determine the degree of sequence identity (DST).

[0039] Table 1: Primers used in this invention ("*" indicates that the translation start position of ATG is defined as +1; "F" indicates forward primer; and "R" indicates reverse primer).

[0040]

[0041] Whole genome sequencing High molecular weight DNA was used to construct multiplex Nanopore sequencing libraries. Oxford Nanopore Technologies (ONT) ligation sequencing kit (SQK-LSK109) and barcode-free amplification kit (Native Barcoding Expansion Kit) (EXP-NBD104) were used, following the ONT instructions. A standard 72-hour sequencing script was executed on MinKNOW; after collecting raw reads, base-called and demultiplexed reads were performed using the standalone application Guppy.

[0042] Through Nanopore survey XYR1 17 C. tropicalis Raw readings of the strains (isolated strains) were aligned to [the specified values] using minimap2 (Li 2018). XYR1 Gene (XM_002546469.1) to generate containing XYR1 The readings were then analyzed using SMARTdenovo (version 1.0) (Liu et al. 2021). XYR1 The readings are reassembled (de novo) to generate contigs.

[0043] The assembled package includes XYR1 The overlapping groups were further corrected using Medaka (version 1.5.0), and the original readings were corrected to match the sequence. C. tropicalis The MYA-3404 genome scaffold supercont3.11 (NW_003020040.1) is in the same orientation. This is for 17 genomes containing... XYR1 Sequence analysis of overlapping groups showed that, compared with strain MYA-3404, the two XYR1 Genes exhibit similar schematic structures.

[0044] Multiple sequence alignment and evolutionary distance After designing primer sequences for sequencing partial sequences of CTRG_05978 and CTRG_05993, 120 primers were analyzed. C. tropicalisThe strains included ATCC750 and 119 strains from clinical trials. Multiple sequence alignment was performed using ClustaIW from MEGA 10. XYR1 DNA sequences were aligned. Paired genetic distances were divided into intraspecific and interspecific parameters and calculated using MEGA10 to characterize intraspecific and interspecific variation between the CTRG_05978 coding DNA sequence (CDS) and the CTRG_05993 CDS. Statistical significance (p-value), Wilcoxon Rank Sum and WHM test, and Signed Rank Test were determined using R software version 4.1.2 (Wilcox.test kit).

[0045] TaqMan SNP Genotyping TaqMan SNP genotyping was performed using the TaqMan Genotyping Test Kit (Applied Biosystems) and the ABI QuantStudio™ 6Flex System (Applied Biosystems), following the manufacturer's instructions. The thermal cycling conditions for CTRG_05978 were as follows: one cycle at 95°C for 10 minutes; 40 cycles of 95°C for 15 seconds, 54°C for 20 seconds, and then 60°C for 1 minute; and one cycle at 60°C for 1 minute. SNQ2 The thermal cycling conditions are as follows: 1 cycle at 95°C for 10 minutes; 40 cycles at 95°C for 15 seconds, 60°C for 1 minute, and 1 cycle at 60°C for 1 minute.

[0046] Statistical analysis Data analysis was performed using SPSS software (Windows version, 12.0). The null hypothesis that the frequencies of the MLST genotype and the CTRG_05978 start codon region type are equal was examined using the chi-square (χ²) test. For categorical variables, the chi-square test or Fisher's precision test was used with one-tailed correction. For factors with a median less than 0.05 in univariate analyses, logistic regression was applied to assess their independent effects. A p-value less than 0.05 was considered statistically significant.

[0047] Multiplex PCR Nine target gene fragments were amplified by multiplex PCR using KOD-Multi & Epi-™ DNA polymerase (Toyobo, Osaka, Japan) in a 50 μl reaction volume. ICL1 , MDR1 , SAPT2, SAPT4 , XYR1 , ZWF1a , ERG11 andSNQ2 The reaction mixture contains 25 μl of 2× PCR buffer containing dNTPs (dATP, dGTP, dCTP, and dTTP) and 2.0 mM final concentration of Mg. 2+ In addition, the mixture contains 10 μl of primer mixture, 1 μl of KOD-Multi & Epi enzyme, anti-KOD DNA polymerase antibody (to neutralize the polymerase and inhibit its 3ʹ→5ʹ exonuclease activity), 5 μl of yeast lysis buffer (dissolved in 0.02 N NaOH), and 14 μl of nuclease-free water. The primer mixture contains... ICL1 , MDR1 , SAPT2 , SAPT4 , XYR1 , ZWF1a , ERG11 and SNQ2 Primers were prepared at concentrations of 0.2 μM, 0.1 μM, 0.2 μM, 0.15 μM, 0.15 μM, 0.2 μM, 0.15 μM, and 0.1 μM. The primer sequences are detailed in Table 2. PCR conditions were as follows: initial denaturation was performed at 94°C for 2 minutes, followed by 35 cycles: denaturation at 98°C for 10 seconds, adhesion at 60°C for 30 seconds, extension at 68°C for 1 minute and 45 seconds, and a final extension at 68°C for 10 minutes.

[0048] Table 2: Oligonucleotides and probes used in this invention ("*" indicates the position number, indicating the position of the gene fragment based on the position of the ATG translation start position of each gene, with the start position designated as +1; "F" indicates the forward primer; and "R" indicates the reverse primer).

[0049]

[0050] Multiplex PCR analysis The Rapid Barcode Kit 96 (SQK-RBK114.96) was used to rapidly barcode 96 samples. Each sample was mixed with 5 μl of multiplex PCR product, 2.5 μl of nuclease-free water, and 2.5 μl of rapid barcode. The mixture was incubated at 30°C for 2 minutes, followed by incubation at 80°C for 2 minutes. All 96 barcoded DNA samples were pooled, and 120 μl of the pooled DNA was mixed with an equal volume of solid phase reversible immobilization (SPRI) magnetic beads. After incubation for 5 minutes using a hula mixer, the barcoded DNA was washed twice with 240 μl of freshly prepared 0% ethanol and eluted with 15 μl of elution buffer (EB). 800 ng of the barcoded DNA was taken and adjusted to a total volume of 11 μl using EB. Add 1 μl of diluted rapid adaptor F to the barcoded DNA and incubate the mixture at room temperature for 5 minutes. Add the BSA primer mixture to optimize sequencing performance.

[0051] Pre-sequencing mix (PSM) was prepared by mixing 37.5 μl of sequencing buffer and 25.5 μl of library beads (LIB) immediately before use in a 12 μl DNA library. PSM was added to a Primed Flow Cell (FLO-MIN114) via the SpotON port for sequencing. Base reading and multiplexing were performed in real-time on a desktop PC equipped with an NVIDIA RTX3090 graphics card and 24 GB of RAM using the MinKNOW GUI with GPU Gupp to generate high-accuracy FASTQ format reads. MinKNOW's default output was set to a FASTQ archive containing 10,000 reads. This was used for assembling the MLST gene... ERG11 and SNQ2 The FASTQ archive containing the readings was optimized by Medaka, and heterojunction sites with variation sites ranging from 10% to 90% were identified from these 9 alleles.

[0052] Assemble the MLST gene To assess the accuracy of the common sequences generated from multiplex PCR-MinION sequencing data, the common sequences were compared with existing sequencing results in the field. A custom Python script was used to process Nanopore FASTQ archives, and the impact of different single nucleotide polymorphism (SNP) selection criteria was analyzed based on substitution allele ratios. The script used Minimap2 for sequence alignment, Medaka for preservation reads, and Bcftools for variant reads, generating a VCF archive containing SNPs and their alt / ref ratios.

[0053] The script executes via appropriate command-line parameters to specify the input FASTQ archive, the reference FASTQ archive, and the output directory. The output is compared, including common sequences, screened VCF archives, and SNP ratio summaries, to evaluate the effectiveness of different SNP screening and sequence modification criteria. The correctness of the common sequence generated by the script is evaluated by aligning the common sequence with the reference sequence and comparing SNP positions, base substitutions, and overall sequence consistency.

[0054] result XYR1 Of the six genes used for MLST analysis (i.e.) ICL1 , MDR1 , SAPT2 , SAPT4 , XYR1 and ZWF1a It has the highest fractal efficiency among them. Because the MLST method can distinguish the major drug-resistant population, i.e., the fourth clade, members with the highest genotyping efficiency become particularly valuable candidate genes for further analysis. Among the six gene fragments used in MLST, according to... C. tropicalis MLST database ICL1 49 polymorphic sites were identified in the sample. MDR1 There are 73 in the middle. SAPT2 There are 93 in the middle. SAPT4 There are 106 in the middle. XYR1 There are 63 in the middle, and ZWF1a There are 49 in total. XYR1 This demonstrated the highest genotyping efficiency, capable of distinguishing 230 genotypes per polymorphism at 63 polymorphic positions (Table 3). Subsequent analysis... XYR1 Comprehensive analysis of the sequences showed the highest genotyping efficiency in MLST analysis.

[0055] Table 3: Six for MLST Candida tropicalis Excerpt

[0056] Two were detected on one chromosome. XYR1 Gene See Figure 1 Two were found on chromosome 6. XYR1 The genes (CTRG_05978 and CTRG_05993; GenBank accession number: NW_003020040.1) are approximately 30 kb apart and are in opposite directions. The direction of the arrows indicates the two genes. XYR1 The direction of genes. Because of these two... XYR1 Genes are highly identical, in order to distinguish two XYR1 The MLST fragment sequence of the gene was obtained using primer sets targeting CTRG_05978 and CTRG_05993, respectively, HJL2808 / HJL3097 and HJL2808 / HJL3098. Analysis of 17 previously collected different DST strains and ATCC750 showed that they (e.g., MYA-3404) all possessed two... XYR1 Gene.

[0057] two XYR1 Genes may be duplicates from ancestors Two were detected on chromosome 6 XYR1 The genes (CTRG_05978 and CTRG_05993; GenBank accession number: NW_003020040.1) are approximately 30 kb apart and are in opposite directions. Figure 1 Distance matrix analysis was performed using MEGA X software (Kumar, S., Stecher, G., Li, M., Knyaz, C. and Tamura, K. (2018) MEGA X: Molecular Evolutionary Genetics Analysis across ComputingPlatforms). Molecular biology and evolution(35, 1547-1549), including inter-genotypic and intra-genotypic distances for CTRG_05978 and CTRG_05993. Box plots of distance indices were generated for the intra-genotypic populations of CTRG_05978 (median = 0.003; mean ± standard deviation = 0.004 ± 0.005), CTRG_05993 (median = 0; mean ± standard deviation = 0.002 ± 0.001), and inter-genotypic populations (median = 0.005; mean ± standard deviation = 0.009 ± 0.007). Notably, the largest distances were observed in inter-genotypic comparisons (0–0.022), while the distances within the intra-genotypic populations of CTRG_05993 showed lower values ​​(0–0.005). Figure 2 ).

[0058] The intergenotypic distance is greater than the intragenotypic distance of CTRG_05978 and CTRG_05993, indicating that these two alleles may be ancestral duplications.

[0059] SNPs in the translation start codon of CTRG_05978 and major fourth-branch azole resistance C. tropicalis Genotype-related Due to the high diversity of CTRG_05978, its sequence was further analyzed. Multiple SNPs were found in the translation start codon of CTRG_05978 (Tables 4 and 5). One SNP in the start codon had a G substitution for A, resulting in a loss-of-function mutation. Subsequently, a more comprehensive analysis of the first 60 nucleotides of the CTRG_05978 codon sequence was performed on all 120 strains (isolated isolates). Notably, this invention revealed that approximately 82.5% (99 out of 120) of the strains (isolated isolates) possessed at least one allele of CTRG_05978 with a loss-of-function mutation in Xyr1.

[0060] Table 4: The first 4 nucleotide sequences of CTRG_05978 and CTRG_05993

[0061] Table 5: CTRG_05978 and SNQ2 The SNPs (DST stands for diploid sequence type; WT stands for wild type; Hom stands for homozygote; and Het stands for heterozygote)

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068] Furthermore, the first four nucleotides of the open reading frame of CTRG_05978 can be classified into seven different sequence classes: ATAA, ATGA, ATGT, ATGW, ATRA, ATRW, and ATRW (Table 4). Interestingly, an association was observed between the CTRG_05978 sequence and the DST in MLST (Tables 6 and 5). Among the fourth clade strains tested, most strains, except for YM180542 (DST506) and YM180921 (DST508), exhibited genotype IV (ATRA) sequence (36 / 48 vs. 2 / 72; p < 0.001). The significant association between the CTRG_05978 sequence and the fourth clade genotype was further validated by logistic regression analysis (p < 0.001; OR = 96.92) (Table 7).

[0069] Table 6: 120 items C. tropicalis Association between CTRG_05978 genotype and sequence class in isolates

[0070] Table 7: Logistic Regression Analysis of Category IV and Branch 4 (OR represents the odds ratio; and CI represents the confidence interval)

[0071] SNQ2 The SNPs in this sequence are associated with the SNPs in sequence class CTRG_05978, and their combination can effectively distinguish the genotypes of the fourth branch. Because the fourth clade was identified as the dominant azole resistance genotype, further investigations were initiated to determine the potential involvement of other genes associated with resistance. This invention discloses... SNQ2 The missense mutation I993V, caused by SNP (A2977G), is located 25 kb upstream of CTRG_05978 and is associated with Candida glabrata (…). Candidaglabrata ) and Candida auris ( Candida aurisThis is associated with drug sensitivity. It has been shown that... SNQ2 The mutations in the sequence may be associated with the sequence type of CTRG_05978 (Tables 8 and 5).

[0072] Table 8: 120 items C. tropicalis In the isolates SNQ2 Association between genotype and SNP at position 2977

[0073] Observed SNQ2 A2977G is highly associated with genotype IV of CTRG_05978 (35 / 43 vs. 13 / 77; p < 0.001; phi = 0.631) and with genotype of the fourth branch (36 / 43 vs. 2 / 77; p < 0.001; phi = 0.820). SNQ2 The combination of A2977G and CTRG_05978 genotype IV distinguishes the fourth branch. C. tropicalis The strains appear to be effective (35 / 35 vs. 3 / 85; p < 0.001), and this association shows a significantly high correlation (phi = 0.943).

[0074] To gain a more comprehensive understanding SNQ2 Can the SNP of CTRG_05978 be effectively detected? C. tropicalis The fourth branch expands the scope to include 161 databases from the NCBI database (https: / / www.ncbi.nlm.nih.gov / sra). C. tropicalis A comprehensive analysis of the whole genome data of the strains. These strains cover geographical variation, with the majority originating from Asia (76%), followed by Oceania (15%), Europe (5%), and North America (3%).

[0075] In general, the present invention discloses SNQ2 The A2977G and CTRG_05978 genotype IV were significantly prevalent in the fourth branch strains (Table 9). SNQ2 CTRG_05978 can indeed be used as a fourth branch for detection. C. tropicalis The markers (95 / 96 vs. 22 / 185; p < 0.001; phi = 0.838).

[0076] Table 9: 281 C. tropicalis In the isolates SNQ2 Association between CTRG_05978 genotype and SNP

[0077] Combining the SNP of CTRG_05978 and SNQ2 SNPs can effectively distinguish the fourth branch. C. tropicalis Compared with other strains, the specificity of the strain improved from 0.877 to 0.991, thus enabling better differentiation of the fourth branch. C. tropicalis Strains (Table 10).

[0078] Table 10: SNPs and / or using CTRG_05978 SNQ2 SNP detection fourth branch C. tropicalis Susceptibility and specificity of strains

[0079] Sensitivity (true positive rate or recall) is calculated as follows: Sensitivity = True Positives (TP) / (True Positives (TP) + False Negatives (FN)). Specificity (true negative rate) is the proportion of actual negative cases that are correctly classified, calculated as follows: Specificity = True Negatives (TN) / (True Negatives (TN) + False Positives (FP)).

[0080] Establishing multiplex PCR for nine genes To better understand and manage C. tropicalis For infection, a single rapid diagnosis is insufficient; efficient, cost-effective, and high-throughput methods are needed for comprehensive surveillance. Multiplex PCR was performed using 17 specific primers targeting nine genes, including six MLST genes. SNQ2 CTRG_05978 and ERG11 (Table 2). See below. Figure 3A and Figure 3B As shown, these nine genes can be amplified independently or simultaneously. When these genes are amplified independently, they are divided into three groups based on DNA fragment length. Figure 3A Although the predicted binding temperature of all primers exceeded 61°C, multiplex PCR showed only two major groups on the DNA electrophoresis gel instead of the expected three. Figure 3B (The third one), and about 1.5 Kb ERG11 Fragment missing. However, when the bonding temperature was lowered to 60°C, three sets of fragments were successfully amplified ( Figure 3B (Channel 1). Importantly, whether all nine target genes were successfully amplified, and the relative abundance of each gene compared to the others, needs to be verified using MinION sequencing.

[0081] MinION's capabilities in molecular subtyping MinION nanopore sequencing was used to assess the quality of multiplex PCR and to determine the optimal primer concentrations for typing these nine genes (Table 11). Readings of all nine genes from seven different strains were analyzed, and comparisons were made of eight genes (due to copy number variation (CNV) effects). ERG11 Except for) the proportion of the total readings of each of the seven strains ( Figure 4 The results showed that the optimal primer concentrations for obtaining the most balanced readings were as follows: ICL1 , SAPT2 , ZWF1a It is 0.2 μM; MDR1 , SAPT4 CTRG_05978, CTRG_05993 and ERG11 It is 0.15 μM; and SNQ2 It is 0.1 μM.

[0082] Table 11: Optimization of multiplex PCR using MinION sequencing data (testing six experimental conditions to optimize multiplex PCR reactions, including changes in primer concentration, cycle number, and binding temperature).

[0083] For MLST genotyping, 96 individual FASTQ output archives were generated, with the total number of reads per sample ranging from 11,211 to 169,720 (combined with a total of 8,172,043 reads and an average of 85,126 reads per sample). The average read depth for each target gene is as follows: ICL1 (6,061.3) MDR1 (10,594.5) SAPT2 (10,762.3) SAPT4 (15,074.4) XYR1 (12,843.1) ZWF1a (4,605.9) ERG11 (10,667.7) and SNQ2 (7,709.5) Figure 5AThe target gene was assembled using Medaka, and variant reads were processed using Bcftools (https: / / samtools.github.io / bcftools / bcftools.html) (Danecek and McCarthy, 2017). The study examined how different SNP selection criteria and read depths affected the consistency of the generated common sequences with existing sequencing results in the field. Initially, the correctness of the MLST gene sequence was evaluated using all reads from a single MinION sequencing flow cell at different SNP selection thresholds, including: Screening 1: MLST gene content is 40% ( XYR1 Except for 20%; Screening 2: MLST gene is 30% ( XYR1 Except for 15%; screening 3: MLST gene is 25% ( XYR1 Except for 10%); Screening 4: All MLST genes are 10%.

[0084] The results showed that when the SNP screening threshold was set to 10%, all MLST locations ( ICL1 , MDR1 , SAPT2 , SAPT4 , XYR1 , ZWF1a The sequencing results showed 100% consistency with existing sequencing data in the field. However, a higher SNP screening threshold (>10%) reduced the similarity at specific locations, particularly... XYR1 At a screening threshold of 40%, only 77.08% consistency was observed. Figure 5B ).

[0085] Next, a 10% SNP screening threshold was applied, while varying the readings (5000, 7000, 10,000, and 12,000) to determine the minimum number of reads required to achieve 100% accuracy. The results showed that at least 10,000 reads were needed to ensure that the generated common sequence was completely consistent with existing sequencing results in the field. Figure 5C ).

[0086] In summary, setting the SNP screening threshold to 10% and using at least 10,000 readings ensures the correct assembly of common sequences and achieves complete consistency with existing sequencing results in the field.

[0087] TaqMan SNP genotyping is useful for detecting CTRG_05978 class IV and... SNQ2 A2977G is helpful. To rapidly detect the main azole resistance fourth branch C. tropicalis A TaqManSNP genotyping method was established for the strain to detect CTRG_05978 class IV and... SNQ2 The A2977G genotype. The specificity of the primers and probes is achieved using samples from 18 different... C. tropicalis Purified genomic DNA of the strains was tested, including ATCC 750 and 17 strains from the clinical environment (Table 12). The primers used are listed in Table 13.

[0088] Table 12: TaqMan genotyping can rapidly detect CTRG_05978 class IV and... SNQ2 A2977G (*: Based on the first 4 nucleotides of CTRG_05978, these strains are classified into 7 categories: Category I is ATAA; Category II is ATGA; Category III is ATGT; Category IV is ATRA; Category V is ATGW; Category VI is ATRW; Category VII is ATWA; and **: CTRG_05979 detects SNPs for GA and AA)

[0089] Table 13: Primers used for TaqMan SNP genotyping (*: ATG translation start position is defined as +1)

[0090] For the CTRG_05978 genotype, all class IV strains (YM140156, YM180233, YM180351, YM180681, and YM180969) are located in the heterozygote region (green dots), while other strains are located in the homozygote region (red and blue dots). Figure 6 (The left half). SNQ2 In the A2977G test, it also specifically detects 7 mutant strains (blue dot and green dot). Figure 6 (The right half). Overall, the results of TaqMan SNP genotyping were consistent with those of Sanger sequencing or whole-genome sequencing, demonstrating that TaqMan SNP genotyping is a rapid and convenient method for detecting SNPs in CTRG_05978 and SNQ2 SNPs are used to identify the fourth branch of major azole resistance. C. tropicalis genotype.

[0091] Summary of Implementation Examples A method for detecting potential markers has been developed and tested in embodiments of the present invention for the rapid detection of azole resistance with a fourth branch genotype. C. tropicalisPotential markers involve only a few nucleotide variations and can be detected using designed capture probes. SNQ2 The invention provides a TaqMan SNP detection method for rapidly detecting the SNPs of CTRG_05978 and the first four nucleotides of CTRG_05978. While real-time PCR, ELISA-like assays, or microarray analysis may also be suitable for this purpose, an easy-to-use and convenient detection method as described in this invention is more practical for clinical settings. This invention provides a TaqMan SNP genotyping method for rapid detection of the SNPs of CTRG_05978 and... SNQ2 Methods in the SNP of A2977G. Some fourth branches. C. tropicalis The isolates do not belong to CTRG_05987 category IV population and / or are not carriers. SNQ2 The A2977G mutation was present, while a non-fourth clade strain belonged to group IV of the CTRG_05987 class and carried the mutation. SNQ2 A2977G mutation. To improve the rapid detection of azole resistance in the fourth branch. C. tropicalis To verify the correctness of this invention, it is recommended that, in addition to checking CTRG_05987 and SNQ2 In addition to genotype, it should also be determined whether these strains carry [a specific trait]. ERG11 mutation.

[0092] SNPs of CTRG_05978 and SNQ2 The SNP of A2977G is an important tool for rapid detection of fourth branch genotypes. Figure 7 This invention can significantly advance the development of targeted diagnostics, thereby providing the correct prescription for treatment. In summary, this invention can provide a method for monitoring and managing a fourth branch. C. tropicalis Promising technologies for the transmission of infection could provide potential tools for mitigating significant health risks to vulnerable patient populations.

[0093] Effective management Candida tropicalis Infections require rapid and scalable diagnostic solutions. To address this issue, this invention provides two innovative approaches. First, a rapid detection marker system utilizes three selected SNPs to detect major drug-resistant genotypes. This simplified approach enables rapid screening and is well-suited for point-of-care testing facilities such as hospitals and clinics. Furthermore, this system aims to optimize existing testing kits, enhancing their scalability and ease of use. Secondly, embodiments of this invention provide a high-throughput approach combining multiplex PCR and MinION sequencing. This synergistic combination enables large-scale routine testing, helping core and institutional laboratories efficiently process large volumes of samples. In summary, these two approaches represent a fourth branch of surveillance and management. C. tropicalisThis provides a promising strategy for the spread of the virus, meeting the needs for rapid, targeted testing as well as large-scale epidemiological surveillance.

[0094] Those skilled in the art will readily recognize that various modifications and alterations can be made to the method and kit without departing from the teachings of this invention. Therefore, the above disclosure should only be interpreted as being within the scope of the appended claims.

Claims

1. A method for detecting drug-resistant fungal pathogens, comprising: Detection of fungal pathogens XYR1 Genes and SNQ2 Single nucleotide polymorphism (SNP) genotype of a gene.

2. The method as described in claim 1, characterized in that, Should XYR1 The genotype of this single nucleotide polymorphism of the gene includes the sequence ATRA, where R is either A or G.

3. The method as described in claim 2, characterized in that, The sequence ATRA is for this XYR1 The first 4 nucleotides of a gene.

4. The method as described in claim 1, characterized in that, Should SNQ2 The genotype of this single nucleotide polymorphism in the gene includes the substitution of nucleotide A at position 2977 for nucleotide G (A2977G).

5. The method as described in claim 4, characterized in that, Should XYR1 The genotype of this single nucleotide polymorphism of the gene includes the sequence ATRA, where R is either A or G.

6. The method as described in claim 5, characterized in that, The sequence ATRA is for this XYR1 The first 4 nucleotides of a gene.

7. The method as described in claim 1, characterized in that, The pathogen of this fungus is not Candida albicans.

8. The method as described in claim 7, characterized in that, This non-Candida albicans species was selected from Candida tropicalis ( Candida tropicalis ), Candida parapsilosis ( Candida parapsilosis ), Candida glabrata ( Candida glabrata ), Candida auris ( Candida auris ), Candida albicans ( Candida guilliermondii ) and Candida krusei ( Candida krusei At least one of the groups consisting of ).

9. The method as described in claim 8, characterized in that, The non-algae Candida species is the tropical Candida ( Candida tropicalis ).

10. The method as described in claim 9, characterized in that, This tropical Candida ( Candida tropicalis This includes the fourth branch genotype.

11. The method as described in claim 1, characterized in that, This drug-resistant fungal pathogen is resistant to drugs, including azoles.

12. The method as described in claim 1, characterized in that, The fungal pathogen XYR1 Genes and this SNQ2 The single nucleotide polymorphism genotype of the gene was detected by high-throughput assay, which included a combination of multiplex polymerase chain reaction (MLCR) technology and MinION nanopore sequencing technology.

13. The method as described in claim 1, characterized in that, The fungal pathogen XYR1 Genes and this SNQ2 The genotype of this single nucleotide polymorphism of the gene was determined by TaqMan genotyping.

14. A kit for detecting drug-resistant fungal pathogens, comprising: Lysis buffer, used to lyse the fungal pathogen; as well as Primers, used to detect the fungal pathogen. XYR1 Genes and / or SNQ2 Gene.

15. The kit of claim 14, further comprising: DNA polymerase; as well as PCR buffer, including Mg 2+ .

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