A novel MNP marker site of cryptococcus, primer composition, kit and application thereof

Abstract

The application discloses a new MNP marker site of Cryptococcus neoformans, a primer composition, a kit and application thereof, wherein the MNP marker site refers to a genomic region screened on a genome of the Cryptococcus neoformans, which is distinguished from other species and has multiple nucleotide polymorphisms in the species, and at least one of the marker sites of MNP-1 to MNP-3; the primer is shown as SEQ ID NO. 1 to SEQ ID NO. 6. The primer pair combination is a multiplex PCR amplification primer designed to amplify the MNP marker in one reaction; the kit integrates multiplex PCR amplification and next-generation sequencing technology, performs sequence analysis on multiple MNP markers of the Cryptococcus neoformans in the sample in one reaction, and has the technical advantages of high throughput, multiple targets, high sensitivity, high precision, culture-free in the application of epidemic prevention monitoring, scientific research and variation monitoring of the Cryptococcus neoformans.

Description

TECHNICAL FIELD

[0001] The embodiment of the present application relates to the field of biotechnology, in particular to a new MNP marker site of Cryptococcus neoformans, primer composition, kit and application thereof. BACKGROUND

[0002] Cryptococcus neoformans is a common saprophyte in soil, pigeons, cow milk, fruits, etc., and can also exist in human oral cavity, and can invade humans and animals. The bacteria are mostly transmitted through respiratory tract, causing mild inflammation in the lungs, or latent infection, and can also be transmitted through damaged skin and intestinal tract. When the immune function of the body decreases, it can spread to the whole body, mainly invading the central nervous system, causing fungal meningitis, encephalitis, brain granuloma, etc., and can also invade the skeleton, muscle, lymph node, skin mucosa to cause chronic inflammation and abscess. The identity identification and genetic variation detection of Cryptococcus neoformans are of great significance for the monitoring of pathogenic bacteria. Traditional fungal detection mainly includes smear staining and microscopic examination, pathogenic bacteria isolation and culture identification, and nucleic acid detection technology based on PCR and metagenomic sequencing developed in recent years. These technologies have advantages, but also have one or more limitations in time, operation complexity, detection throughput, detection accuracy and sensitivity of variation, and cost. For example, the operation of fungal isolation and identification is complex and time-consuming, and cannot detect variation; the PCR detection technology detects only one to two markers of one fungus in one reaction, which is low in efficiency, and is prone to detection failure due to variation in the primer region, and cannot detect sequence variation. The metagenomic sequencing technology often includes a large amount of host sequencing data, and the genome of the fungus is large, so when detecting a sample with low pathogen load, deep sequencing is required, resulting in high data sequencing cost.

[0003] Therefore, how to provide multiple new molecular markers with high polymorphism for Cryptococcus neoformans, and how to realize efficient, accurate and sensitive identification and variation monitoring of Cryptococcus neoformans in one detection are currently urgent technical problems to be solved. SUMMARY

[0004] The purpose of the present application is to provide a new MNP marker site specific to Cryptococcus neoformans, primer composition, kit and application thereof, which can be used for qualitative identification and variation detection of Cryptococcus neoformans, and has the effects of multi-target, high throughput and high sensitivity.

[0005] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0006] In a first aspect of the present application, a new Cryptococcus neoformans MNP marker combination is provided, which comprises at least one of three marker sites MNP-1 to MNP-3, for screening a genomic region with multiple nucleotide polymorphisms that are distinguished from other species and within the species on the genome of Cryptococcus neoformans, and the nucleotide sequences are shown in SEQ ID NO. 7 to SEQ ID NO. 9.

[0007] In the above technical solution, the specific MNP-1 to MNP-3 marker sites are shown in Table 1 of the specification, and the start and end positions of the MNP markers marked in Table 1 are determined based on the reference sequence corresponding to the same row in Table 1.

[0008] In a second aspect of the present application, a multiplex PCR primer combination for detecting the MNP marker sites is provided, which comprises at least one of three pairs of primers, and the nucleotide sequences of the three pairs of primers are shown in SEQ ID NO. 1 to SEQ ID NO. 2, SEQ ID NO. 3 to SEQ ID NO. 4, and SEQ ID NO. 5 to SEQ ID NO. 6, respectively.

[0009] In the above technical solution, the primers of each MNP marker site comprise an upper primer and a lower primer, and the specific primers are shown in Table 1 of the specification.

[0010] In a third aspect of the present application, a detection kit for detecting the Cryptococcus neoformans MNP marker sites is provided, and the kit comprises the primer combination.

[0011] Further, the kit further comprises a multiplex PCR premix.

[0012] In a fourth aspect of the present application, the Cryptococcus neoformans MNP marker sites, the multiplex PCR primer combination, or the detection kit is applied in the identification of Cryptococcus neoformans.

[0013] In a fifth aspect of the present application, the Cryptococcus neoformans MNP marker sites, the multiplex PCR primer combination, or the detection kit is applied in the variation monitoring and database construction of Cryptococcus neoformans.

[0014] In the above applications, the specific operation steps are as follows:

[0015] Firstly, total DNA of a sample to be detected is obtained; the total DNA and a blank control are subjected to first-round multiplex PCR amplification by using the kit, and the number of cycles is not more than 25; after the amplification product is purified, sample labeling and second-generation sequencing adapter addition based on second-round PCR amplification are performed; the second-round amplification product is purified and quantified; when multiple strains are detected, the second-round amplification products are mixed in equal amounts and subjected to high-throughput sequencing; the sequencing results are compared to reference sequences of the Cryptococcus neoformans to obtain the number of detection sequences of the Cryptococcus neoformans in the total DNA and genotype data of the Cryptococcus neoformans. According to the number of sequencing sequences of the Cryptococcus neoformans and the number of detected MNP sites obtained in the total DNA and the blank control, data quality control and data analysis are performed on the sequencing data of the total DNA to obtain the number of detected MNP sites of the Cryptococcus neoformans, the number of sequencing sequences covering each MNP site and the genotype data of the MNP site.

[0016] When used for Cryptococcus neoformans identification, according to the number of sequencing sequences of the Cryptococcus neoformans and the number of detected MNP sites detected in the sample to be detected and the blank control, it is determined whether the sample to be detected contains nucleic acid of the Cryptococcus neoformans after quality control. The quality control scheme and determination method are to use DNA of the Cryptococcus neoformans with known copy number as a detection sample to evaluate the sensitivity, accuracy and specificity of the kit in detecting the Cryptococcus neoformans, and to develop a quality control scheme and determination method for detecting the Cryptococcus neoformans by using the kit.

[0017] When used for constructing a Cryptococcus neoformans variation monitoring and DNA fingerprint database, the genotype data of the MNP site of the Cryptococcus neoformans identified from the sample is recorded in a database file to form a DNA fingerprint database of the Cryptococcus neoformans; when different samples are identified, whether the Cryptococcus neoformans in the sample and a strain in the database have a difference in the main genotype (a genotype supported by more than 50% of sequencing fragments at one MNP site) at the MNP site is identified by comparison with the DNA fingerprint database of the Cryptococcus neoformans; the Cryptococcus neoformans having a difference in the main genotype at at least one MNP site is a new variation type, and the genotype is recorded in the DNA fingerprint database.

[0018] The one or more technical solutions in the embodiments of the present application have at least the following technical effects or advantages:

[0019] The application provides a new Cryptococcus MNP marker site, primer composition, kit and application thereof, the application analyzes the genomic sequence of the new Cryptococcus, a total of 3 MNP marker sites are screened, and a multiplex primer composition is designed according to the sequence information of the 3 MNP marker sites; the multiplex primer composition is used for multiplex PCR amplification, and a second-generation sequencing platform is used for sequencing of the amplification product, so that the detection of the 3 MNP marker sites of the new Cryptococcus can be realized at one time, the limitation of the prior art that one marker of the new Cryptococcus is detected at one time and parallel detection of standard samples is relied on is broken through, high-throughput, high-efficiency and high-accuracy detection of the new Cryptococcus is realized, and technical support is provided for identification and variation monitoring of the new Cryptococcus. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0021] Figure 1 The MNP marker polymorphism principle schematic diagram provided for the embodiments of the present application;

[0022] Figure 2 The screening and primer design flowchart of the new Cryptococcus MNP marker site provided for the embodiments of the present application;

[0023] Figure 3 The detection flowchart of the MNP marker site provided for the embodiments of the present application. DETAILED DESCRIPTION

[0024] The advantages and various effects of the embodiments of the present application will be more clearly presented by the following specific embodiments and examples. Those skilled in the art should understand that these specific embodiments and examples are used to illustrate the embodiments of the present application, rather than limit the embodiments of the present application.

[0025] Throughout the specification, unless otherwise specifically indicated, the terms used herein are understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as generally understood by those skilled in the art to which the embodiments of the present application belong. If there is a contradiction, the present specification is preferred.

[0026] Unless otherwise specified, the various raw materials, reagents, instruments and equipment used in the embodiments of the present application can be purchased from the market or can be prepared by existing methods.

[0027] The technical solutions of the embodiments of the present application are as follows to solve the above technical problems:

[0028] The embodiments of the present application provide an MNP marker site of Cryptococcus neoformans, which is a genomic region with species specificity and multiple nucleotide polymorphisms within the species screened on the genome of Cryptococcus neoformans; the MNP marker site includes at least one of the three marker sites of MNP-1 to MNP-3.

[0029] Based on one general inventive concept, the embodiments of the present application provide a multiplex PCR primer composition for detecting the MNP marker site, which includes at least one of the three pairs of primers, and the nucleotide sequences of the three pairs of primers are respectively shown in SEQ ID NO. 1-SEQ ID NO. 2, SEQ ID NO. 3-SEQ ID NO. 4, and SEQ ID NO. 5-SEQ ID NO. 6.

[0030] The primers do not conflict with each other, and can be efficiently amplified by multiplex PCR.

[0031] In the embodiments of the present application, the composition of the specific multiplex amplification primer composition for detecting the MNP marker site is refined, which can perform full-coverage detection on Cryptococcus neoformans, thereby realizing high-throughput, high-efficiency, high-accuracy, and high-sensitivity detection and variation monitoring of Cryptococcus neoformans.

[0032] The multiplex PCR primer composition is designed based on the above MNP marker site, and the specific composition and region of the MNP marker site can be referred to the above embodiments. Since the multiplex PCR primer composition adopts part or all of the technical solutions of the above embodiments, it at least has all the beneficial effects brought by the technical solutions of the above embodiments, which will not be repeated here.

[0033] The multiplex PCR primer composition can be used as a detection kit for the MNP marker site of Cryptococcus neoformans, and the kit provided by the present application can accurately identify Cryptococcus neoformans.

[0034] Based on one general inventive concept, the embodiments of the present application provide a kit for detecting the MNP marker site, which includes the multiplex PCR primer composition.

[0035] The kit is realized based on the above multiplex PCR primer composition, and the specific sequence information of the multiplex PCR primer composition can be referred to the above embodiments. Since the kit adopts part or all of the technical solutions of the above embodiments, it at least has all the beneficial effects brought by the technical solutions of the above embodiments, which will not be repeated here.

[0036] Based on a general inventive concept, the application provides a use of the MNP marker site, the primer composition or the kit in the preparation of a Cryptococcus neoformans identification product.

[0037] In the application, when used for the identification of Cryptococcus neoformans, the number of sequencing sequences of Cryptococcus neoformans detected in the sample to be tested and the blank control and the number of MNP markers detected are used to determine whether the sample to be tested contains the nucleic acid of the Cryptococcus neoformans after quality control; wherein the quality control scheme and determination method are to use known Cryptococcus neoformans DNA as a detection sample to evaluate the sensitivity, accuracy and specificity of the kit in detecting Cryptococcus neoformans, and to develop a quality control scheme and determination method for detecting Cryptococcus neoformans.

[0038] In the reproducibility test of the application, the difference in the logarithm of the MNP marker main genotype between different libraries of each sample and between different library construction batches is 0, the reproducibility rate r is 100%, and the accuracy a is 100%.

[0039] The application is based on the above-mentioned MNP marker site, and the specific composition and region of the MNP marker site can refer to the above-mentioned embodiments. Since the application uses part or all of the technical solutions of the above-mentioned embodiments, it at least has all the beneficial effects brought by the technical solutions of the above-mentioned embodiments, which will not be repeated here.

[0040] The MNP marker site of Cryptococcus neoformans, primer composition, kit and application thereof of the application will be described in detail below with reference to examples, comparative examples and experimental data.

[0041] Example 1, screening of Cryptococcus neoformans MNP marker site and design of multiplex PCR amplification primer

[0042] 1. Screening of Cryptococcus neoformans MNP marker site:

[0043] The specific steps of the above screening are as follows:

[0044] The genomic sequence of a representative strain of the Cryptococcus neoformans is selected as a reference genome, and the genomic sequences of other strains of the Cryptococcus neoformans are compared with the reference genome to obtain single nucleic acid polymorphic sites between different representative strains of the Cryptococcus neoformans;

[0045] On the reference genome, a window of 100-300 bp is translated by 1 bp as a step to screen a plurality of candidate MNP marker site regions, wherein the candidate MNP marker site region requires containing ≥2 single nucleotide variation sites, and the sequences of 30 bp at both ends do not contain the single nucleic acid polymorphic sites;

[0046] At least 5 sites with high discrimination DP value in the candidate MNP marker site region are selected; wherein the selection needs to meet:

[0047] DP = d / t,

[0048] In the formula, t is the comparison number of all genomes in the candidate MNP marker site region, and d is the number of at least two single nucleic acid polymorphism differences in the genome in the candidate MNP marker site region.

[0049] The window translation stage can also use other steps, not limited to 1 bp, and the use of 1 bp by the present application is beneficial to the comprehensive screening of the fungal genome polymorphism site.

[0050] After obtaining the MNP marker site of the Cryptococcus neoformans, sequence alignment is performed in the public database, and at least 5 sites specific to the Cryptococcus neoformans are selected as candidate MNP marker sites.

[0051] 2. Design of multiplex PCR amplification primers:

[0052] As shown in Figure 2 , the multiplex PCR amplification primers of the candidate MNP marker are designed by primer design software, and the primer design follows the principle that the primers do not interfere with each other, and all primers can be combined into a primer pool for multiplex PCR amplification, that is, all designed primers can be normally amplified in one amplification reaction.

[0053] 3. Detection efficiency evaluation of primer combination:

[0054] The MNP marker detection kit is used to detect Cryptococcus neoformans positive samples, a total of 3 repeats; sequencing data is analyzed, and 3 pairs of highly compatible and product specific primer combinations are screened out for specific identification of 3 MNP markers of Cryptococcus neoformans. As shown in Figure 1 , based on the publicly available genome sequence of Cryptococcus neoformans, through sequence alignment, 3 MNP marker sites of Cryptococcus neoformans are screened out as shown in Table 1.

[0055] Table 1-Starting position of the MNP marker site on the reference sequence and sequence of the detection primer

[0056]

[0057] Example 2, performance evaluation and threshold setting of MNP marker and primer identification of Cryptococcus neoformans

[0058] 1. The detection of Cryptococcus neoformans has technical stability, high specificity and sensitivity as low as 10 copies / reaction

[0059] Positive samples of Cryptococcus neoformans were prepared, with the copy number of Cryptococcus neoformans being 1 copy per reaction, 10 copies per reaction and 100 copies per reaction, and an equal volume of sterile water was set as a blank control. Each sample was detected for 3 repeated libraries, and was continuously detected for 4 days, that is, 12 groups of sequencing data were obtained for each sample; according to the data analysis results of each sample shown in Table 2, the reproducibility and accuracy of the detection method were evaluated, a threshold for detecting pollution and target Cryptococcus neoformans was formulated, and the detection process of the MNP labeling site was as shown in Figure 3

[0060] Table 2- Sensitivity and stability analysis table of detecting Cryptococcus neoformans

[0061]

[0062]

[0063] As shown in Table 2, in the detection of positive samples of 10 copies per reaction and 100 copies per reaction, the three MNP labeling sites of Cryptococcus neoformans were detected, and the sequence-specific alignment was on the reference sequence of the Cryptococcus neoformans, indicating that the primer and the detection method for detecting the Cryptococcus neoformans have technical stability, high specificity and a sensitivity as low as 10 copies per reaction.

[0064] 2. Reproducibility and accuracy evaluation of the MNP labeling detection kit for detecting Cryptococcus neoformans:

[0065] Based on whether the genotypes of the commonly detected sites in the two repeated experiments can be reproduced, the reproducibility and accuracy of the MNP labeling detection method for detecting Cryptococcus neoformans were evaluated. Specifically, the genotypes of each MNP site generated by the 12 groups of data of the positive sample of 100 copies per reaction were compared in pairs, and the results are shown in Table 3.

[0066] Table 3- Reproducibility and accuracy evaluation of the MNP labeling detection kit for detecting genotypes

[0067] Repeat 1 Repeat 2 Number of common sites Number of repeatable sites Reproducibility r Accuracy a S-1 S-2 3 3 100％ 100％ S-1 S-3 3 3 100％ 100％ S-1 S-4 3 3 100％ 100％ S-1 S-5 3 3 100％ 100％ S-1 S-6 3 3 100％ 100％ S-1 S-7 3 3 100％ 100％ S-1 S-8 3 3 100％ 100％ S-1 S-9 3 3 100％ 100％ S-1 S-10 3 3 100％ 100％ S-1 S-11 3 3 100％ 100％ S-1 S-12 3 3 100％ 100％

[0068] As shown in Table 3, the number of MNP labels with different main genotypes is 0; according to the principle that the reproducible genotypes between the two repeated experiments are considered to be accurate, the accuracy a = 1-(1-r) / 2 = 0.5+0.5r, and r represents the reproducibility rate, that is, the ratio of the number of main genotypes that can be reproduced to the total number of sites. In the reproducibility test of the present application, the difference in the main genotypes of the MNP labels between different libraries of each sample and between different library construction batches is 0, the reproducibility rate r = 100%, and the accuracy a = 100%.

[0069] ​In the positive samples of 10 copies per reaction and 100 copies per reaction, 3 MNP markers of Cryptococcus neoformans can be stably detected, and at most 1 MNP marker can be detected in the blank control; therefore, the determination standard of the Cryptococcus neoformans positive sample in the present application is: when the detected Cryptococcus neoformans is not less than 2 MNP marker sites in the sample and the sequence abundance (i.e. the ratio of the number of sequences detected in the sample to the number of total sequences in the sample) is at least 10 times that in the blank control, it is determined that the nucleic acid of the Cryptococcus neoformans is detected in the sample.

[0070] Example 3, application of MNP marker sites, kits and methods in identification of Cryptococcus neoformans

[0071] The MNP marker site detection kit was used to detect 6 nucleic acid samples provided by the Hubei CDC, and the samples were named S1-S6 (S1 was a Cryptococcus neoformans negative sample, and S2-S6 were Cryptococcus neoformans positive samples). The results are shown in Table 4.

[0072] Table 4 - sample detection analysis table

[0073] Sample number Fungus with sequencing sequence detected Number of MNP markers detected Determination result S1 Cryptococcus neoformans 0 Not detected S2 Cryptococcus neoformans 3 Detected S3 Cryptococcus neoformans 3 Detected S4 Cryptococcus neoformans 3 Detected S5 Cryptococcus neoformans 3 Detected S6 Cryptococcus neoformans 3 Detected

[0074] As can be seen from the results of Table 4, the kit and method accurately detected 3 MNP marker sites in the Cryptococcus neoformans contained in each sample in one reaction. The same detection, the method of detecting one marker based on fluorescent PCR needs to be detected for 3 times, indicating that the kit and method for detecting Cryptococcus neoformans are accurate and efficient.

[0075] Example 4, application of MNP marker sites, kits and methods in genetic variation detection of Cryptococcus neoformans

[0076] The kit and MNP marker combination detection method were used to detect 4 copies of a Cryptococcus neoformans strain collected, and the samples were named S-1-S-4 in turn. All 3 MNP markers of Cryptococcus neoformans can be detected, and the average coverage of each MNP marker is up to 3600 times (Table 5). The genotypes of the 4 strains at the 3 markers were compared in pairs, and the results are shown in Table 5. There is a main genotype difference between 1 sample (S-2) and 3 Cryptococcus neoformans detected together in the same batch (Table 5), indicating that there is genetic variation.

[0077] Table 5 - detection analysis of 4 copies of a Cryptococcus neoformans strain

[0078]

[0079] As can be seen from Table 5, the kit can identify genetic variations among strains by detecting MNP labeled sequences, can be used to find variations among strains in epidemic prevention monitoring, and can be used in scientific research to ensure the genetic consistency of strains with the same name in different laboratories, thereby ensuring the comparability of research results. Therefore, it is of great significance for the prevention and control of pathogens, precision treatment and scientific research.

[0080] The one or more technical solutions in the embodiments of the application have at least the following technical effects or advantages:

[0081] (1) The three MNP marker sites of the new Cryptococcus provided in the embodiments of the application have high specificity in identifying the new Cryptococcus.

[0082] (2) The multiplex PCR amplification primer composition of the new Cryptococcus provided in the embodiments of the application can satisfy amplification of the three MNP marker sites in one reaction, and can be fused to a second-generation sequencing platform for sequencing of the amplification products, so as to realize sequence analysis of the three marker sites of the new Cryptococcus in one reaction. Compared with the method based on fluorescent PCR, which only uses one pair of primers to detect one site and detects a fluorescent signal, the multiplex primer pair provided in the application provides technical support for efficient and accurate identification of the new Cryptococcus based on sequences.

[0083] (3) The new Cryptococcus MNP marker site detection kit provided in the embodiments of the application can satisfy detection of all three MNP marker sites in one reaction. In the reproducibility test, the three marker sites can be stably detected, indicating the high stability of the kit in detection. The difference in logarithm of the MNP marker main genotype between different libraries of each sample and between different library construction batches detected by the kit is 0, the reproducibility r = 100%, and the accuracy a = 100%, indicating the high accuracy and stability of the kit in detection of the new Cryptococcus.

[0084] (4) The method provided in the embodiments of the application detects samples by high-throughput sequencing, and by adding a unique tag to each sample, the detection of hundreds or thousands of samples at a time can be realized, and the detection efficiency is further improved.

[0085] (5) The method provided in the embodiments of the application detects samples by high-throughput sequencing, and the identification of fungi is realized by detecting the base sequences of the MNP markers, without the need for parallel detection of standard samples. The method based on fluorescent PCR detects fluorescent signals and relies on parallel experiments of standard samples.

[0086] (6) The method provided by the embodiment of the present application detects the sample through high-throughput sequencing, obtains the base sequence of the detected MNP marker, and can not only identify the fungus in one reaction, but also detect the genetic variation of the pathogen among samples. Finally, it should be noted that the terms "comprising", "including", or any other variant thereof are intended to cover non-exclusive inclusion, so that the process, method, article or equipment including a series of elements not only includes those elements, but also includes other elements not explicitly listed or inherent to such process, method, article or equipment.

[0087] Although preferred embodiments of the embodiments of the present application have been described, those skilled in the art who are familiar with the basic inventive concept can make further changes and modifications to the embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications falling within the scope of the embodiments of the present application.

[0088] Obviously, those skilled in the art can make various modifications and variations to the embodiments of the present application without departing from the spirit and scope of the embodiments of the present application. Thus, if these modifications and variations of the embodiments of the present application fall within the scope of the claims of the embodiments of the present application and their equivalent technologies, the embodiments of the present application also intend to include these modifications and variations.

Claims

1. A novel MNP marker combination for Cryptococcus, characterized in that, The MNP marker combination includes three MNP markers, with nucleotide sequences shown in SEQ ID NO.7 to SEQ ID NO.

9.

2. A multiplex PCR primer composition for detecting the novel Cryptococcus MNP marker combination of claim 1, characterized in that, The multiplex PCR primer composition comprises three pairs of primers, the nucleotide sequences of which are shown in SEQ ID NO.1–SEQ ID NO.2, SEQ ID NO.3–SEQ ID NO.4, and SEQ ID NO.5–SEQ ID NO.6, respectively.

3. A detection kit for detecting the novel Cryptococcus MNP marker combination of claim 1, characterized in that, The kit includes the primer composition of claim 2.

4. The detection kit according to claim 3, characterized in that, The kit also includes a multiplex PCR premix.

5. The use of the MNP marker combination of Cryptococcus neoformans according to claim 1, the primer composition according to claim 2, or the detection kit according to any one of claims 3-4 in the preparation of an identification product for Cryptococcus neoformans.

6. The application of the MNP marker combination of Cryptococcus neoformans according to claim 1, the primer composition according to claim 2, or the detection kit according to any one of claims 3-4 in the non-diagnostic monitoring of Cryptococcus neoformans variants.

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