Mnp marker site of pichia kudriavzevii, primer composition, kit and application thereof

Abstract

The application discloses a MNP marker site of Pichia kudriavzevii, a primer composition, a kit and application thereof. The MNP marker site refers to a genomic region screened on a genome of the Pichia kudriavzevii, 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 Pichia kudriavzevii in a sample in one reaction, and has technical advantages of high throughput, multiple targets, high sensitivity, high precision, culture-free in application of epidemic prevention monitoring, scientific research and variation monitoring of the Pichia kudriavzevii.

Description

TECHNICAL FIELD

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

[0002] Pichia kudriavzevii, also known as Candida krusei, is commonly used in the brewing process of vinegar and wine, but the strains isolated from the environment are also a high-risk pathogenic bacteria, which often infects people with low or defective immunity, such as organ transplant recipients or AIDS patients. The identity identification and genetic variation detection of Pichia kudriavzevii are of great significance for monitoring the pathogenicity of pathogenic bacteria and the performance of industrial 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 their own advantages, but there is 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 at the same time. 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 pathogenic load, deep sequencing is required, resulting in high data sequencing cost.

[0003] Therefore, how to provide multiple new molecular markers with high polymorphism for Pichia kudriavzevii and achieve efficient, accurate and sensitive detection and variation monitoring of Pichia kudriavzevii in one detection is a technical problem to be solved at present. SUMMARY

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

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

[0006] In a first aspect of the present application, a MNP marker combination of Pichia kudriavzevii 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 distinguishing from other species and within the species on the genome of Pichia kudriavzevii, and the nucleotide sequence is 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 MNP marker sites of Pichia kudriavzevii 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 MNP marker sites of Pichia kudriavzevii, or the multiplex PCR primer combination, or the detection kit is applied in the identification of Pichia kudriavzevii.

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

[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 then subjected to high-throughput sequencing; the sequencing results are compared with reference sequences of the fungi to obtain the number of detected sequences and genotype data of the total DNA. According to the number of sequencing sequences and the number of detected MNP sites of the fungi obtained from the total DNA and the blank control, data quality control and data analysis are performed on the sequencing data of the total DNA.

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

[0017] When used for constructing a Pichia kudriavzevii variation monitoring and DNA fingerprint database, the genotype data of the MNP sites of Pichia kudriavzevii identified from the sample are recorded in a database file to form a DNA fingerprint database of Pichia kudriavzevii; when different samples are identified, whether Pichia kudriavzevii in the sample is different from strains in the database in the main genotype (a genotype supported by more than 50% of sequencing fragments in one MNP site) at the MNP sites is identified by comparison with the DNA fingerprint database of Pichia kudriavzevii; Pichia kudriavzevii with a difference in the main genotype at at least one MNP site is a new variation type, and the genotype is included 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 MNP marker site of Pichia kudriavzevii, a primer composition, a kit and application thereof, the application analyzes the genome sequence of Pichia kudriavzevii, and a total of 3 MNP marker sites are screened, and a multiplex primer composition is designed according to sequence information of the 3 MNP marker sites; multiplex PCR amplification is carried out by using the designed multiplex primer composition, and a second-generation sequencing platform is used for sequencing of the amplification product, so that one-time detection of the 3 MNP marker sites of Pichia kudriavzevii can be met, the limitation that one marker of Pichia kudriavzevii is detected at one time in the prior art is broken through, parallel detection of standard samples is relied on, and high-throughput, high-efficiency and high-accuracy detection of Pichia kudriavzevii is realized, thereby providing technical support for Pichia kudriavzevii identification and variation monitoring. 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 A MNP marker polymorphism principle schematic diagram provided for the embodiments of the present application;

[0022] Figure 2 A screening and primer design flowchart of the MNP marker site of Pichia kudriavzevii provided for the embodiments of the present application;

[0023] Figure 3 A 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 the 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 specifically indicated, the various materials, reagents, instruments and equipment used in the embodiments of the application are commercially available or are readily prepared by known methods.

[0027] The technical solutions of the embodiments of the application are to solve the above technical problems, and the general idea is as follows:

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

[0029] Based on one general inventive concept, the embodiments of the application provide a multiplex PCR primer composition for detecting the MNP marker site, the primer composition includes at least one of 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 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 Pichia kudriavzevii, so as to realize high-throughput, high-efficiency, high-accuracy and high-sensitivity detection and variation monitoring of Pichia kudriavzevii.

[0032] The multiplex PCR primer composition is designed 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 multiplex PCR primer composition adopts 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.

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

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

[0035] The kit is realized based on the above-mentioned multiplex PCR primer composition. The specific sequence information of the multiplex PCR primer composition can be referred to the above-mentioned embodiments. Since the kit adopts 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.

[0036] Based on the overall inventive concept, the application provides an application of the MNP marker site or the primer composition or the kit in preparing an identification product of P. kudriavzevii.

[0037] In the embodiments of the application, when used for identification of P. kudriavzevii, according to the number of sequencing sequences of P. kudriavzevii detected in the sample to be tested and the blank control and the number of MNP markers detected, whether the nucleic acid of the P. kudriavzevii is contained in the sample to be tested is determined after quality control; wherein, the quality control scheme and determination method are to use the known P. kudriavzevii DNA as a detection sample to evaluate the sensitivity, accuracy and specificity of the kit in detecting P. kudriavzevii, and to formulate the quality control scheme and determination method when the kit is used to detect P. kudriavzevii.

[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 realized based on the above-mentioned MNP marker site. The specific composition and region of the MNP marker site can be referred to the above-mentioned embodiments. Since the application adopts 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, primer composition, kit and application of P. kudriavzevii of the application will be described in detail below in combination with examples, comparative examples and experimental data.

[0041] Example 1

[0042] I. Screening of P. kudriavzevii MNP marker site and design of multiplex PCR amplification primer

[0043] 1. Screening of P. kudriavzevii MNP marker site

[0044] The specific steps of the above-mentioned screening are as follows:

[0045] The genome sequence of a representative strain of the fungus is selected as a reference genome, and the other genome sequences of the fungus are compared with the reference genome to obtain the single nucleic acid polymorphism sites of the fungus;

[0046] On the reference genome, a window of 100bp to 300bp is used for window translation with a step size of 1bp to screen and obtain regions of multiple candidate MNP marker sites. The candidate MNP marker site regions are required to contain ≥2 of the single nucleotide variant sites and there are no single nucleotide polymorphism sites on the sequences at both ends of 30bp.

[0047] At least five sites with high DP (discrimination) values ​​in the candidate polynucleotide polymorphism (PNP) regions were selected; the selection criteria included the following:

[0048] DP=d / t,

[0049] In the formula, t is the number of comparisons when all genomes are compared pairwise in the candidate polynucleotide polymorphism site region, and d is the number of genome pairs with at least two single nucleotide polymorphism differences in the candidate polynucleotide polymorphism site region.

[0050] Other step sizes can also be used in the window translation stage, not limited to 1 bp, but the use of 1 bp in this application is beneficial for the comprehensive screening of polymorphic sites on the fungal genome.

[0051] After obtaining the MNP marker sites of the fungus, sequence alignment was performed in a public database to screen at least 5 fungus-specific sites as candidate MNP marker sites.

[0052] 2. Design of primers for multiplex PCR amplification:

[0053] like Figure 2 As shown, multiplex PCR amplification primers for the candidate MNP marker were designed using primer design software. The primer design followed the principle that 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 amplified normally in one amplification reaction.

[0054] 3. Evaluation of the detection efficiency of primer combinations:

[0055] Positive samples of Pichia kudriezwiye were detected using the aforementioned MNP marker detection kit, with a total of 3 replicates. Sequencing data were analyzed, and 3 highly compatible and product-specific primer combinations were selected for the specific identification of 3 MNP markers in Pichia kudriezwiye.

[0056] like Figure 1As shown, based on the publicly available genome sequence of P. kudriavzevii, three MNP markers screened from P. kudriavzevii are shown in Table 1 by sequence alignment.

[0057] Table 1 - The start position of the MNP marker sites on the reference sequence and the sequence of the detection primer

[0058]

[0059] Example 2, Performance evaluation and threshold setting of MNP markers and primers for P. kudriavzevii

[0060] 1. The detection of P. kudriavzevii has technical stability, high specificity and sensitivity as low as 10 copies / reaction

[0061] The samples of P. kudriavzevii were prepared, with the copy number of P. kudriavzevii being 1 copy / reaction, 10 copies / reaction and 100 copies / reaction, and an equal volume of sterile water was set as a blank control. Each sample was detected for 3 repeated libraries, and the detection was performed continuously for 4 days, i.e. 12 sets 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, the threshold for detecting contamination and target P. kudriavzevii was formulated, and the detection process of the MNP marker sites is shown in Figure 3 .

[0062] Table 2 - Sensitivity and stability analysis table for detecting P. kudriavzevii

[0063] As shown in Table 2, in the detection of positive samples of 10 copies / reaction and 100 copies / reaction, the three MNP marker sites of P. kudriavzevii were detected, and the sequence-specific alignment was on the reference sequence of the fungus, indicating that the primers and the detection method had technical stability, high specificity and sensitivity as low as 10 copies / reaction for detecting the fungus.

[0064] 2. Reproducibility and accuracy evaluation of MNP marker detection kit for detecting P. kudriavzevii:

[0065] Based on whether the genotypes of the commonly detected sites in the two repeated experiments were reproducible, the reproducibility and accuracy of the MNP marker detection method for detecting P. kudriavzevii were evaluated. Specifically, the genotypes of each MNP site generated by the 12 sets of data of the positive sample of 100 copies / reaction were compared pairwise, and the results are shown in Table 3.

[0066] Table 3 - reproducibility and accuracy evaluation of MNP marker detection kit for genotyping

[0067]

[0068] As shown in Table 3, the number of MNP markers with different main genotypes is 0; according to the principle that the genotypes reproducible between two repeated experiments are considered to be accurate, the accuracy a = 1 - (1 - r) / 2 = 0.5 + 0.5r, r represents the reproducibility rate, i.e. the ratio of the number of main genotypes reproducible sites to the total number of sites. In the reproducibility test of the present application, the difference in main genotypes of MNP markers 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 / reaction and 100 copies / reaction, 3 MNP markers of P. kudriavzevii can be stably detected, and at most 1 MNP marker can be detected in the blank control; therefore, the determination standard of P. kudriavzevii positivity of the present application is: when at least 2 MNP marker sites of P. kudriavzevii are detected in the sample and the sequence abundance (i.e. the ratio of the number of sequences detected in the sample and aligned to the MNP marker site of P. kudriavzevii to the total number of sequences in the sample) is at least 10 times that in the blank control, it is determined that the nucleic acid of P. kudriavzevii is detected in the sample.

[0070] Example 3, Application of MNP marker sites, kit and method in identifying P. kudriavzevii

[0071] The MNP marker site detection kit was used to detect 6 nucleic acid samples provided by the Hubei CDC, and the sample names were S-1-S-6 (S-1 was a P. kudriavzevii negative sample, and S-2-S-6 were P. kudriavzevii positive samples). The detection results are shown in Table 4.

[0072] Table 4 - P. kudriavzevii detection and analysis in samples

[0073]

[0074] As shown in Table 4, the kit and method accurately detected 3 MNP marker sites in P. kudriavzevii in each sample in one reaction. The same detection based on fluorescence PCR requires 3 times of detection for one marker, indicating that the kit and method for detecting P. kudriavzevii are accurate and efficient.

[0075] Example 4, Application of MNP marker sites, kits and methods in genetic variation detection of Pichia ciferrii

[0076] Using the kit and MNP marker combination detection method, four copies of a Pichia ciferrii strain collected were detected, and the samples were named S-1-S-4 in turn. All three MNP markers of Pichia ciferrii can be detected, and the average coverage of sequencing of each MNP marker reaches 5420 times (Table 5). Comparing the genotypes of the four strains at the three markers, the results are shown in Table 5 REF _Ref74838347 \h \* MERGEFORMAT. One (S-1) and three new Pichia ciferrii detected at the same time have main genotype differences (Table 5), indicating that there is genetic variation.

[0077] Table 5 - Detection analysis of four copies of one new Cryptococcus strain

[0078]

[0079] As can be seen from Table 5, the kit can identify genetic variation between strains by detecting the sequences of MNP markers, can be used to find variation between strains in epidemic prevention monitoring, and can be used to ensure the genetic consistency of strains with the same name in different laboratories in scientific research, thereby ensuring the comparability of research results. Therefore, this is of great significance for the prevention and control of pathogens, precise 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 Pichia ciferrii provided in the embodiments of the application have high specificity in identifying Pichia ciferrii.

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

[0083] (3) The Pichia kudriavzevii MNP marker site detection kit provided by the embodiment of the present application can meet the 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 detection. The difference in logarithm of the MNP marker main genotype between different libraries and different library construction batches of each sample 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 detecting Pichia kudriavzevii.

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

[0085] (5) The method provided by the embodiment of the present application detects samples by high-throughput sequencing, and the fungi are identified by the detected base sequence of the MNP marker, without the parallel detection of standard samples. The method based on fluorescent PCR detects fluorescent signals and relies on the parallel experiment of standard samples.

[0086] (6) The method provided by the embodiment of the present application detects samples by high-throughput sequencing, and the base sequence of the detected MNP marker is obtained. In one reaction, not only the fungi can be identified, but also the genetic variation of pathogens between samples can be detected. Finally, it should be noted that the terms “include”, “contain” or any other variant thereof are intended to cover non-exclusive inclusion, so that the process, method, article or device 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 device.

[0087] Although the preferred embodiments of the embodiments of the present application have been described, those skilled in the art can make further changes and modifications to the embodiments once they know the basic inventive concept. 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 combination of MNP markers of Pichia kudriavzevii, characterized in that, The MNP marker combination includes three marker sites, MNP-1 to MNP-3, with nucleotide sequences as shown in SEQ ID NO.7 to SEQ ID NO.

9.

2. A multiplex PCR primer composition for detecting the Pichia kudriavzevii 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 Pichia kudriavzevii 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 Pichia kudriezwiez as described in claim 1, the primer composition as described in claim 2, or the detection kit as described in any one of claims 3-4 in the preparation of an identification product of Pichia kudriezwiez.

6. The use of the MNP marker combination of Pichia kudriezwiez as described in claim 1, the primer composition of claim 2, or the detection kit of any one of claims 3-4 in the non-diagnostic variation monitoring of Pichia kudriezwiez.

7. The use of the MNP marker combination of Pichia kudriezwiez as described in claim 1, the primer composition of claim 2, or the detection kit of any one of claims 3-4 in constructing a DNA fingerprint database of Pichia kudriezwiez.

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