Yarrowia lipolytica microsatellite site as well as detection method and application thereof
By providing an understanding of Yarrow's lipolytic microsatellite sites and detection methods, using fluorescently labeled primer sets for PCR amplification and capillary electrophoresis detection, the problem of insufficient molecular marker polymorphism in the prior art was solved. , realizes efficient and low-cost detection and analysis, and has important clinical and research significance.
Patent Information
- Application Number
- CN202411294836.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-14
- Publication Date
- 2024-12-13
AI Technical Summary
The existing molecular markers used in Yarrow's lipolytica have low intraspecies polymorphisms and cannot meet the needs of inter-strain kinship identification, molecular traceability, hospital infection monitoring and population genetics.
Provides understanding of Yarrow's lipolytic microsatellite sites and detection methods, uses fluorescently labeled primer sets to perform PCR amplification reaction, and detects the polymorphisms of PCR amplification products through capillary electrophoresis to achieve high polymorphisms of Yarrow's lipolytica Morbidity, specificity and low cost detection.
The high polymorphism and specific detection of Yarrowia lipolytica is achieved, which is of great significance in molecular epidemiology and population genetic research. It can effectively perform molecular typing, molecular traceability, hospital infection monitoring and systematic evolution analysis. .
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Figure CN119120757A_ABST
Abstract
Description
Technical Field
[0001] The present disclosure relates to the field of biotechnology, and in particular to a Yarrowia lipolytica microsatellite locus, a primer set for amplifying the Yarrowia lipolytica microsatellite locus, a kit for detecting the Yarrowia lipolytica microsatellite locus, applications of the Yarrowia lipolytica microsatellite locus and a detection method thereof in molecular typing, molecular tracing, hospital infection monitoring, population genetics and phylogenetic analysis of Yarrowia lipolytica, and a detection method for Yarrowia lipolytica for non-diagnostic purposes. Background Art
[0002] At present, the only molecular markers used for Yarrowia lipolytica are ITS and D1D2 markers. However, these molecular markers have low intraspecific polymorphism and cannot meet the needs of inter-strain relationship identification, molecular traceability, hospital infection monitoring, and population genetics of Yarrowia lipolytica.
[0003] Microsatellite, also known as simple sequence repeats (SSR) or short tandem repeats (STR), refers to short tandem repeats on genomic DNA. The repeat unit is generally 1-5bp and the number of repetitions is 5-40 times. Genetic polymorphism analysis can be performed by detecting the length of the repeat region through PCR. Since microsatellite alleles have the characteristics of fast mutation rate, this detection method has the advantages of high polymorphism, good specificity, high throughput and low cost, and has been widely used in biological genetic mapping, population genetic research, and identification of kinship between individuals. However, it is currently less used in pathogens.
[0004] Therefore, it is of great clinical significance to propose molecular markers of highly polymorphic microsatellite loci within species and their detection methods. Summary of the invention
[0005] The present invention aims to provide a Yarrowia lipolytica microsatellite locus, a primer set for amplifying the Yarrowia lipolytica microsatellite locus, a kit for detecting the Yarrowia lipolytica microsatellite locus, applications of the Yarrowia lipolytica microsatellite locus and a detection method thereof in molecular typing, molecular tracing, hospital infection monitoring, population genetics and phylogenetic analysis of Yarrowia lipolytica, and a detection method for Yarrowia lipolytica for non-diagnostic purposes.
[0006] In order to achieve the above-mentioned object, the present disclosure provides a Yarrowia lipolytica microsatellite locus in a first aspect. The Yarrowia lipolytica microsatellite locus is any one of the nucleotide sequences shown in SEQ ID NOs. 1-4.
[0007] Optionally, the Yarrowia lipolytica microsatellite locus is a combination of any two, three or four of the nucleotide sequences shown in SEQ ID NOs. 1-4.
[0008] A second aspect of the present disclosure provides a method for detecting a microsatellite locus of Yarrowia lipolytica, the detection method comprising the following steps: S1, extracting total DNA of Yarrowia lipolytica; S2, using the total DNA as a template and using a fluorescently labeled primer set to perform a PCR amplification reaction in a PCR reaction system; S3, performing polymorphism detection on the fluorescently labeled DNA fragments in the PCR amplification products; The Yarrowia lipolytica microsatellite locus is the Yarrowia lipolytica microsatellite locus described in the first aspect; The primer set comprises forward primers with nucleotide sequences as shown in SEQ ID NOs. 5-8 and reverse primers with nucleotide sequences as shown in SEQ ID NOs. 9-12.
[0009] Optionally, each 20 μL PCR reaction system contains 2 μL amplification buffer, 1.6-4 μL dNTP mixture, 0.5-1 μL Taq DNA polymerase, 1-3 μL gDNA, 0.2-0.4 μL forward primer, 0.2-0.4 μL reverse primer and balance deionized water; the concentration of the forward primer is 5-20 μM, and the concentration of the reverse primer is 5-20 μM; wherein the 5' end of the forward primer is labeled with FAM and / or HEX by a fluorescent dye.
[0010] Optionally, the reaction program of the PCR amplification reaction includes: 94°C 5 min, 94°C 30 s, 60°C 45 s, 72°C 50 s, 35 cycles; 72°C 5 min.
[0011] Optionally, the polymorphism detection includes: detecting fluorescently labeled DNA fragments in PCR amplification products by capillary electrophoresis, and determining the positions and relative molecular weights of allelic fragments of the PCR amplification products by molecular weight internal standards.
[0012] The third aspect of the present disclosure provides a primer set for amplifying the Yarrowia lipolytica microsatellite locus described in the first aspect, the primer set comprising forward primers with nucleotide sequences as shown in SEQ ID NOs. 5-8 and reverse primers with nucleotide sequences as shown in SEQ ID NOs. 9-12.
[0013] A fourth aspect of the present disclosure provides a kit for detecting the Yarrowia lipolytica microsatellite locus described in the first aspect, wherein the kit comprises the primer set for amplifying the Yarrowia lipolytica microsatellite locus described in the fourth aspect.
[0014] The fifth aspect of the present disclosure provides the application of the Yarrowia lipolytica microsatellite locus described in the first aspect or the detection method described in the third aspect in the molecular typing, molecular tracing, hospital infection monitoring, population genetics and phylogenetic analysis of Yarrowia lipolytica.
[0015] A sixth aspect of the present disclosure provides a method for detecting Yarrowia lipolytica for non-diagnostic purposes, comprising the following steps: S1. Extracting total DNA of the sample to be tested; S2, using the total DNA as a template and using a fluorescently labeled primer set to perform a PCR amplification reaction in a PCR reaction system; S3, analyzing PCR amplification products; The primer set comprises forward primers with nucleotide sequences as shown in SEQ ID NOs. 5-8 and reverse primers with nucleotide sequences as shown in SEQ ID NOs. 9-12.
[0016] Through the above technical scheme, the present invention provides for the first time a Yarrowia lipolytica microsatellite locus, a detection method for the Yarrowia lipolytica microsatellite locus and a specific primer set. The detection of Yarrowia lipolytica using the above Yarrowia lipolytica microsatellite locus, the detection method and the specific primer set has the advantages of high polymorphism, good specificity, high throughput and low cost, and is of great significance in the molecular epidemiology and population genetics research of Yarrowia lipolytica.
[0017] Other features and advantages of the present disclosure will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] The accompanying drawings are used to provide a further understanding of the present disclosure and constitute a part of the specification. Together with the following specific embodiments, they are used to explain the present disclosure but do not constitute a limitation of the present disclosure. In the accompanying drawings: Figure 1 It is the peak diagram of the YL01 site of the sample detected by capillary electrophoresis.
[0019] Figure 2 This is the peak diagram of the YL06 site of the capillary electrophoresis test sample.
[0020] Figure 3 This is the peak diagram of the YL85 site of the sample detected by capillary electrophoresis.
[0021] Figure 4 It is the peak diagram of the YL88 site of the sample detected by capillary electrophoresis. DETAILED DESCRIPTION
[0022] The specific implementation of the present disclosure is described in detail below in conjunction with the accompanying drawings. It should be understood that the specific implementation described herein is only used to illustrate and explain the present disclosure, and is not used to limit the present disclosure.
[0023] The present disclosure provides a first aspect of a Yarrowia lipolytica microsatellite locus, wherein the Yarrowia lipolytica microsatellite locus is any one of the nucleotide sequences shown in SEQ ID NOs. 1-4.
[0024] In one embodiment of the present disclosure, the Yarrowia lipolytica microsatellite locus is a combination of any two, three or four of the nucleotide sequences shown in SEQ ID NOs. 1-4. Preferably, the Yarrowia lipolytica microsatellite locus is a combination of four of the nucleotide sequences shown in SEQ ID NOs. 1-4.
[0025] The type of the Yarrowia lipolytica microsatellite locus disclosed in the present invention is a simple SSR composed of a sequence consisting of a single repeating unit. The specific information of the Yarrowia lipolytica microsatellite locus is shown in Table 1.
[0026] Table 1
[0027] A second aspect of the present disclosure provides a method for detecting a microsatellite locus of Yarrowia lipolytica, the detection method comprising the following steps: S1, extracting total DNA of Yarrowia lipolytica; S2, using the total DNA as a template and using a fluorescently labeled primer set to perform a PCR amplification reaction in a PCR reaction system; S3, performing polymorphism detection on the fluorescently labeled DNA fragments in the PCR amplification products; The Yarrowia lipolytica microsatellite locus is the Yarrowia lipolytica microsatellite locus described in the first aspect; The primer set comprises forward primers with nucleotide sequences as shown in SEQ ID NOs. 5-8 and reverse primers with nucleotide sequences as shown in SEQ ID NOs. 9-12.
[0028] According to the present disclosure, each 20μL PCR reaction system contains 2µL amplification buffer, 1.6-4µl dNTP mixture, 0.5-1µL Taq DNA polymerase, 1-3µL gDNA, 0.2-0.4µL forward primer, 0.2-0.4µL reverse primer and the remainder of deionized water; the concentration of the forward primer is 5-20μM, preferably 20μM; the concentration of the reverse primer is 5-20μM, preferably 20μM; wherein the 5' end of the forward primer is labeled with FAM and / or HEX by a fluorescent dye. In the present disclosure, the amplification buffer is a 10× amplification buffer, and the pH at room temperature is 8.3; "10×" means that the amplification buffer needs to be diluted ten times when used.
[0029] According to the present disclosure, the reaction procedure of the PCR amplification reaction includes: 94°C 5 min, 94°C 30 s, 60°C 45 s, 72°C 50 s, 35 cycles; 72°C 5 min.
[0030] According to the present disclosure, the polymorphism detection includes: using capillary electrophoresis to detect fluorescently labeled DNA fragments in PCR amplification products, and determining the position and relative molecular weight of the allelic fragments of the PCR amplification products by molecular weight internal standards.
[0031] In a specific embodiment, the molecular weight internal standard and formamide are mixed at a volume ratio of 1: (50-100) (preferably 1: 100), 15 μL of the mixture is added to the sample plate, and then 1 μL of the PCR product diluted 10 times is added. Capillary electrophoresis is performed using an ABI3730XL sequencer. The raw data obtained by the sequencer is analyzed using the Fragment (Plant) fragment analysis software in Genemarker, and the positions of the molecular weight internal standard and the peaks of each PCR amplification product in each lane are compared and analyzed to obtain the fragment size.
[0032] The third aspect of the present disclosure provides a primer set for amplifying the Yarrowia lipolytica microsatellite locus described in the first aspect, the primer set comprising forward primers with nucleotide sequences as shown in SEQ ID NOs. 5-8 and reverse primers with nucleotide sequences as shown in SEQ ID NOs. 9-12.
[0033] A fourth aspect of the present disclosure provides a kit for detecting the Yarrowia lipolytica microsatellite locus described in the first aspect, wherein the kit comprises the primer set for amplifying the Yarrowia lipolytica microsatellite locus described in the fourth aspect.
[0034] The fifth aspect of the present disclosure provides the application of the Yarrowia lipolytica microsatellite locus described in the first aspect or the detection method described in the third aspect in the molecular typing, molecular tracing, hospital infection monitoring, population genetics and phylogenetic analysis of Yarrowia lipolytica.
[0035] A sixth aspect of the present disclosure provides a method for detecting Yarrowia lipolytica for non-diagnostic purposes, comprising the following steps: S1. Extracting total DNA of the sample to be tested; S2, using the total DNA as a template and using a fluorescently labeled primer set to perform a PCR amplification reaction in a PCR reaction system; S3, analyzing PCR amplification products; The primer set comprises forward primers with nucleotide sequences as shown in SEQ ID NOs. 5-8 and reverse primers with nucleotide sequences as shown in SEQ ID NOs. 9-12.
[0036] The present invention is further described below by way of examples, but the present invention is not limited thereto. The raw materials, reagents, instruments and equipment involved in the examples of the present invention can be purchased unless otherwise specified.
[0037] Example 1 This example is used to illustrate the detection of genetic diversity of Yarrowia lipolytica populations.
[0038] Total DNA extraction from Yarrowia lipolytica: The QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) was used to extract total DNA from 75 strains of Yarrowia lipolytica. The operation procedure was carried out according to the instructions of the kit. Yarrowia lipolytica was provided by the Laboratory Department of Peking Union Medical College Hospital.
[0039] 2. PCR amplification of microsatellite loci of Yarrowia lipolytica: The total DNA of the 75 strains of Yarrowia lipolytica extracted above was amplified using the specific primer set disclosed in the present invention. The specific primer set includes forward primers with nucleotide sequences as shown in SEQ ID NOs. 5-8 and reverse primers with nucleotide sequences as shown in SEQ ID NOs. 9-12, as shown in Table 2.
[0040] Table 2
[0041] The 5' end of the forward primer was labeled with a fluorescent dye, FAM.
[0042] The PCR reaction system was 20 μL, which contained 7.4 μL of ddH2O, 10 μL of DNA polymerase, 0.3 μL of forward primer (20 uM), 0.3 μL of reverse primer (20 μM), and 2 μL of DNA template; SSR PCR amplification program: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 60°C for 45 s, and extension at 72°C for 50 s, for a total of 35 cycles; and final extension at 72°C for 5 min.
[0043] 3. Capillary electrophoresis method for polymorphism detection: After formamide and molecular weight internal standard are mixed at a volume ratio of 100:1, 15 μL is taken and added to the sample plate, and then 1 μL of PCR product diluted 10 times is added. Then, the ABI 3730XL sequencer is used for capillary electrophoresis, and the raw data obtained by the sequencer are analyzed using the Fragment (Plant) fragment analysis software in Genemarker. The position of the molecular weight internal standard in each lane is compared with the position of each sample peak to obtain the fragment size. The example result is as follows Figure 1-4 As shown, four STR loci were determined to be real polymorphic loci.
[0044] DNA sequence identification: The measured sequence was compared with the NCBI genome sequence, and the sequencing results were consistent with the STR locus sequence, confirming that the four STR loci were real polymorphic loci.
[0045] 4. Test results The selected four microsatellite loci of Yarrowia lipolytica were used to type 75 strains of Yarrowia lipolytica and common clinical pathogenic fungi (Candida albicans ATCC 90028; Candida albicans SC 5314; Candida glabrata ATCC 2001; Candida glabrata CBS 138; Candida parapsilosis ATCC 22019; Candida tropicalis ATCC 750; Candida krusei ATCC 6258; Cryptococcus neoformans H 99; Trichosporon asahii ATCC 2479; Candida guilliermonensis ATCC 6260) collected from 2010 to 2020 in the Chinese Hospital Invasive Fungal Surveillance (CHIF-NET) project. The 75 strains were successfully divided into 34 types, and the results are shown in Table 3. At the same time, the microsatellite locus primer set has good specificity, and specific bands cannot be amplified for other common clinical pathogenic fungi, and there is no crossover phenomenon.
[0046] Table 3
[0047] Using this typing method, outbreaks were detected in two hospitals, namely, an outbreak of MT08 type infection in H01 hospital, 11 strains were detected, involving 10 patients in the same ward; an outbreak of MT29 type infection in H02 hospital, 12 strains were detected, involving 8 patients in the same ward. The test results show that the four microsatellite loci of the present invention are authentic and effective, can be used for molecular typing of Yarrowia lipolytica, and obtain good typing results, which is of great significance for the clinical epidemiology and hospital infection monitoring of Yarrowia lipolytica.
[0048] The preferred embodiments of the present disclosure are described in detail above in conjunction with the accompanying drawings; however, the present disclosure is not limited to the specific details in the above embodiments. Within the technical concept of the present disclosure, a variety of simple modifications can be made to the technical solution of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.
[0049] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present disclosure will not further describe various possible combinations.
[0050] In addition, various embodiments of the present disclosure may be arbitrarily combined, and as long as they do not violate the concept of the present disclosure, they should also be regarded as the contents disclosed by the present disclosure.
Claims
1. A Yarrowia lipolytica microsatellite locus, characterized in that The Yarrowia lipolytica microsatellite locus is any one of the nucleotide sequences shown in SEQ ID NO. 1-4.
2. The Yarrowia lipolytica microsatellite locus according to claim 1, wherein The Yarrowia lipolytica microsatellite locus is a combination of any two, three or four of the nucleotide sequences shown in SEQ ID NO. 1-4.
3. A method for detecting microsatellite loci of Yarrowia lipolytica, characterized in that: The detection method comprises the following steps: S1. Extract total DNA of Yarrowia lipolytica; S2, using the total DNA as a template and using a fluorescently labeled primer set to perform a PCR amplification reaction in a PCR reaction system; S3, performing polymorphism detection on the fluorescently labeled DNA fragments in the PCR amplification products; The Yarrowia lipolytica microsatellite locus is the Yarrowia lipolytica microsatellite locus according to claim 1 or 2; The primer set comprises forward primers with nucleotide sequences as shown in SEQ ID NOs. 5-8 and reverse primers with nucleotide sequences as shown in SEQ ID NOs. 9-12.
4. The detection method according to claim 3, wherein: Each 20 μL PCR reaction system contains 2 μL amplification buffer, 1.6-4 μL dNTP mixture, 0.5-1 μL Taq DNA polymerase, 1-3 μL gDNA, 0.2-0.4 μL forward primer, 0.2-0.4 μL reverse primer and the balance deionized water; The concentration of the forward primer is 5-20 μM, and the concentration of the reverse primer is 5-20 μM; Wherein, the 5' end of the forward primer is labeled with FAM and / or HEX by a fluorescent dye.
5. The detection method according to claim 3, wherein: The reaction procedure of the PCR amplification reaction includes: 94°C for 5 min, 94°C for 30 s, 60°C for 45 s, 72°C for 50 s, 35 cycles; 72°C for 5 min.
6. The detection method according to claim 3, wherein: The polymorphism detection comprises: using capillary electrophoresis to detect fluorescently labeled DNA fragments in PCR amplification products, and determining the position and relative molecular weight of the allelic fragments of the PCR amplification products by molecular weight internal standards.
7. A primer set for amplifying the microsatellite locus of Yarrowia lipolytica according to claim 1, characterized in that: The primer set includes forward primers with nucleotide sequences as shown in SEQ ID NOs. 5-8 and reverse primers with nucleotide sequences as shown in SEQ ID NOs. 9-12.
8. A kit for detecting the microsatellite loci of Yarrowia lipolytica according to claim 1, characterized in that: The kit comprises the primer set for amplifying the Yarrowia lipolytica microsatellite locus according to claim 7.
9. Use of the Yarrowia lipolytica microsatellite locus according to any one of claims 1 to 2 or the detection method according to any one of claims 3 to 6 in molecular typing, molecular tracing, hospital infection monitoring, population genetics and phylogenetic analysis of Yarrowia lipolytica.
10. A method for detecting Yarrowia lipolytica for non-diagnostic purposes, characterized in that: The steps include: S1. Extracting total DNA of the sample to be tested; S2, using the total DNA as a template and using a fluorescently labeled primer set to perform a PCR amplification reaction in a PCR reaction system; S3, analyzing PCR amplification products; The primer set comprises forward primers with nucleotide sequences as shown in SEQ ID NOs. 5-8 and reverse primers with nucleotide sequences as shown in SEQ ID NOs. 9-12.