CN116356064A - Identification method, primer pair and kit for cryptococcus - Google Patents
Identification method, primer pair and kit for cryptococcus Download PDFInfo
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Abstract
The application relates to an identification method, a primer pair and a kit of cryptococcus, wherein the method comprises the following steps: obtaining genome DNA of a sample to be tested; amplifying the URA5 gene and/or SOD1 gene of the genome DNA by polymerase chain reaction PCR to obtain an amplified product; performing sanger sequencing on the amplification product; determining the type of the cryptococcus based on the sequencing result of the URA5 gene and/or the SOD1 gene. The cryptococcus identification method can determine the secondary type of the cryptococcus based on the primary type of the cryptococcus, and accurately judge the subtype of the cryptococcus, thereby helping to promote early intervention and treatment of diseases caused by the cryptococcus.
Description
Technical Field
The invention belongs to the technical field of fungal etiology detection, and particularly relates to an identification method, a primer pair and a kit of cryptococcus.
Background
Cryptococcus is a fungus that can cause cryptococcosis and belongs to the family Cryptococcus of the phylum Deuteromycotina, class of Bacillus in the mycotaxonomy. Cryptococcosis is a pulmonary or disseminated infectious disease caused by cryptococcus, mainly causing pneumonia, meningitis, skin or visceral infections. Cryptococcus is of a wide variety of genus, with the main species responsible for human infection being Cryptococcus neoformans and Cryptococcus glaucocalycis. There are significant differences in epidemiological and clinical pathogenic processes among different types of cryptococcus, for example, cryptococcus neoformans primarily infect immunosuppressed populations, while cryptococcus gartertiaryana primarily infects immunocompetent populations and predominates in pulmonary infections.
Since the pathogenicity and lethality of different types of cryptococcus are different, it is judged that the disease caused by that cryptococcus is critical for diagnosis and treatment of cryptococcosis. Therefore, how to accurately molecular type cryptococcus is a prime problem to be solved.
Disclosure of Invention
The invention provides a cryptococcus identification method, a primer pair and a kit, wherein the method can identify the subtype of cryptococcus on the basis of the primary type of cryptococcus, and can more accurately judge the type of pathogenic cryptococcus so as to help promote early intervention and treatment of diseases caused by cryptococcus.
In a first aspect, the present application provides a method of identifying cryptococcus comprising: obtaining genome DNA of a sample to be tested; amplifying the URA5 gene and/or SOD1 gene of the genome DNA by polymerase chain reaction PCR to obtain an amplified product; performing sanger sequencing on the amplification product; determining the type of the cryptococcus based on the sequencing result of the URA5 gene and/or the SOD1 gene.
According to the method, the multiple-site sequence typing is carried out on the sanger sequencing result of the URA5 gene and the SOD1 gene of the cryptococcus, so that the molecular typing of the cryptococcus can be accurately judged, more specifically, which of VGI types the cryptococcus belongs to can be judged, the secondary typing and detection of the cryptococcus are realized, and the type of the cryptococcus causing infection can be judged in clinical diagnosis. In addition, the cryptococcus typing method can be realized without using complex and expensive instruments and equipment, so that the detection cost of the cryptococcus can be saved, and the application scene of clinical detection can be expanded.
In one possible implementation, the types of cryptococcus include: cryptococcus VGIc, cryptococcus VGId and Cryptococcus wildtype.
It is understood that the wild-type cryptococcus described herein is other types of cryptococcus besides Cryptococcus of VGic type and Cryptococcus of VGId type.
The method can identify the subtype of the cryptococcus to obtain more accurate cryptococcus types.
In one possible implementation, the sequencing result of the URA5 gene and/or the SOD1 gene comprises: sequences of different sites of the URA5 gene and/or the SOD1 gene.
The method for classifying the cryptococcus by using the multi-site sequence classification method has the advantages that the mutation of the strain is analyzed based on the sanger sequencing result, the operation is simple, and the result can be obtained quickly and efficiently.
In one possible implementation, the sites include: 353 site of the URA5 gene, 54 site of the SOD1 gene or 457 site of the SOD1 gene.
In one possible implementation, the determining the type of the cryptococcus includes: in the case where the 353 site of the URA5 gene is guanine G, the cryptococcus is determined to be a VGic type cryptococcus.
In one possible implementation, the determining the type of the cryptococcus includes: in the case where the 54 th site of the SOD1 gene is thymine T and the 457 th site of the SOD1 gene is cytosine C, the cryptococcus is determined to be a VGId type cryptococcus.
In one possible implementation, the determining the type of the cryptococcus includes: in the case where the 353 site of the URA5 gene is adenine a, cytosine C or thymine T; or in the case where the 54 th site of the SOD1 gene is adenine A, cytosine C or guanine G; or in the case where the 457 site of the SOD1 gene is adenine A, thymine T or guanine G, the cryptococcus is determined to be a wild-type cryptococcus.
In one possible implementation, the PCR reaction comprises: a URA5 forward primer and a URA5 reverse primer, and/or a SOD1 forward primer and a SOD1 reverse primer; the nucleotide sequence of the URA5 forward primer is shown as a Seq ID No.1, the nucleotide sequence of the URA5 reverse primer is shown as a Seq ID No.2, the nucleotide sequence of the SOD1 forward primer is shown as a Seq ID No.3, and the nucleotide sequence of the SOD1 reverse primer is shown as a Seq ID No. 4.
In one possible implementation, after obtaining the amplification product, the method further comprises: confirming that the URA5 gene and/or the SOD1 gene was successfully amplified.
In one possible implementation, the success of amplification of the URA5 gene and/or the SOD1 gene is confirmed by gel electrophoresis.
In the embodiment of the application, after the amplification product is obtained and before sanger sequencing is carried out, whether amplification is successful or not is confirmed by using a gel electrophoresis method, so that the waste of time and cost caused by unsuccessful amplification, namely sanger sequencing, can be avoided, and the identification efficiency of cryptococcus is improved.
In one possible implementation manner, the obtaining the genomic DNA of the sample to be tested includes: grinding the sample to be tested by a liquid nitrogen grinding method; the genomic DNA was extracted by standard phenol chloroform method.
In one possible implementation, the sample to be tested comprises blood and/or body fluid.
In a second aspect, there is provided a primer pair for identifying cryptococcus, the primer pair comprising a first forward primer and a first reverse primer, and a second primer pair comprising a second forward primer and a second reverse primer, the nucleotide sequence of the first forward primer being as set forth in Seq ID No.1, the nucleotide sequence of the first reverse primer being as set forth in Seq ID No.2, the nucleotide sequence of the second forward primer being as set forth in Seq ID No.3, and the nucleotide sequence of the second reverse primer being as set forth in Seq ID No. 4.
In one possible implementation, the first primer pair is used to recognize 353 sites of the URA5 gene sequence of the cryptococcus, and the second primer pair is used to recognize 54 sites and 457 sites of the SOD1 gene sequence of the cryptococcus.
In a third aspect, there is provided a kit for identifying cryptococcus, wherein the kit comprises a primer pair according to any one of the embodiments of the second aspect.
In some embodiments, the kit further comprises: buffer solution, template DNA, deoxynucleoside triphosphate dNTPs and DNA polymerase.
In a fourth aspect, there is provided the use of a primer pair as in any one of the embodiments of the second aspect for identifying cryptococcus VGIa.
The cryptococcus identification method can accurately judge the type of the cryptococcus by amplifying the URA5 gene and/or SOD1 gene of the sample to be detected and detecting the sequences of a plurality of specific sites by sanger sequencing so as to identify the type of the cryptococcus, more particularly, can judge the subtype of the cryptococcus, thereby realizing the secondary type detection of the cryptococcus, helping to carry out rapid and accurate clinical diagnosis and reducing drug abuse.
Drawings
FIG. 1 is a schematic flow chart of an identification method of Cryptococcus in an embodiment of the present application.
FIG. 2 is another schematic flow chart of a method for determining cryptococcus in accordance with an embodiment of the present application.
FIG. 3 is a gel electrophoresis pattern of an amplification product according to the embodiment of the present application.
FIG. 4 is a gel electrophoresis spectrum of another amplification product according to the embodiment of the present application.
FIG. 5 is a graph showing the NGS sequencing results of wild-type cryptococcus and VGic cryptococcus in the examples of the present application.
FIG. 6 is a graph showing the peak sequencing of the wild-type cryptococcus and the VGId-type cryptococcus of the examples of the present application.
FIG. 7 is a diagram showing another gene sequencing peaks of wild-type cryptococcus and VGId-type cryptococcus of the embodiment of the present application.
Detailed Description
The technical scheme of the application is further described below in connection with specific embodiments.
The "range" disclosed herein is defined in terms of lower and upper limits, with a given range being defined by the selection of a lower and an upper limit, the selected lower and upper limits defining the boundaries of the particular range. Ranges that are defined in this way can be inclusive or exclusive of the endpoints, and any combination can be made, i.e., any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60-120 and 80-110 are listed for a particular parameter, it is understood that ranges of 60-110 and 80-120 are also contemplated. Furthermore, if the minimum range values 1 and 2 are listed, and if the maximum range values 3,4 and 5 are listed, the following ranges are all contemplated: 1-3, 1-4, 1-5, 2-3, 2-4 and 2-5. In this application, unless otherwise indicated, the numerical range "a-b" represents a shorthand representation of any combination of real numbers between a and b, where a and b are both real numbers. For example, the numerical range "0-5" means that all real numbers between "0-5" have been listed throughout, and "0-5" is simply a shorthand representation of a combination of these values. When a certain parameter is expressed as an integer of 2 or more, it is disclosed that the parameter is, for example, an integer of 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12 or the like.
In the description of the present application, the terms "first," "second," "third," and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.
Reference herein to "comprising" and "including" means open ended, as well as closed ended, unless otherwise noted. For example, the terms "comprising" and "comprises" may mean that other components not listed may be included or included, or that only listed components may be included or included.
The term "or" is inclusive in this application, unless otherwise specified. For example, the phrase "a or B" means "a, B, or both a and B. More specifically, either of the following conditions satisfies the condition "a or B": a is true (or present) and B is false (or absent); a is false (or absent) and B is true (or present); or both A and B are true (or present).
All embodiments and alternative embodiments of the present application may be combined with each other to form new solutions, unless specifically stated otherwise.
First, some terms are briefly introduced:
next generation sequencing (Next Generation Sequencing, NGS) is a high throughput sequencing technology that can rapidly and comprehensively detect DNA mutations in the genome, commonly used for circulating tumor DNA (Circulating Tumor DNA, ctDNA), copy number variation (Copy Number Variations, CNVs) and gene fusion (using RNA sequencing panel). NGS may detect a variety of different types of samples, such as blood, tumor tissue, and bone marrow samples.
Sanger sequencing (Sanger Sequencing) is a method of obtaining a base sequence of a visible DNA, specifically, starting at a certain fixed point according to nucleotides, randomly terminating at a certain specific base, and fluorescence labeling after each base, producing four sets of nucleotides of different lengths ending with Adenine (A), thymine (T), cytosine (C), guanine (G), and then performing polyacrylamide gel electrophoresis (Polyacrylamide Gel Eectrophoresis, PAGE), thereby obtaining a base sequence of a visible DNA. The ability to analyze long and continuous nucleic acid sequences to detect DNA strands to identify mutations by sanger sequencing is one of the important means to achieve molecular typing.
The polymerase chain reaction (Polymerase Chain Reaction, PCR) is a molecular biological technique for amplifying specific DNA fragments, which can be regarded as specific DNA replication in vitro.
Multi-site sequence typing (Multilocus Sequence Typing, MLST) is a bacterial typing method based on nucleic acid sequence determination. This method analyzes the variation of the strain by amplifying a plurality of housekeeping gene internal fragments by PCR and determining the sequence thereof. MLST has the advantages of simple operation, capability of rapidly obtaining results and the like.
The Real-time fluorescence quantitative nucleic acid amplification detection system (Real-time Quantitative PCR Detecting System, QPCR) is a method for adding a fluorescent group into a PCR reaction system, monitoring the whole PCR process in Real time by utilizing fluorescent signal accumulation, and finally quantitatively analyzing an unknown template through a standard curve.
URA5 gene: the nucleotide sequence of the phosphoribosyl transferase gene of cryptococcus is shown in Seq ID No. 5.
SOD1 gene: the superoxide dismutase gene of cryptococcus has the nucleotide sequence shown in Seq ID No. 6.
The embodiments of the present application are described below.
Cryptococcus is a fungus that mainly invades the central system of the human body, causing cryptococcosis. Cryptococcus comprises 17 and 8 variants of neutralization, of which novel Cryptococcus is the main pathogenic strain, hereinafter referred to as Cryptococcus. Different cryptococcus pathogenic rates, infected people have obvious differences, and therefore, it is critical for clinical diagnosis to identify which cryptococcus is responsible for the infection. With the improvement of gene detection means, cryptococcus has 8 molecular types: VNI, VNII, VNIII, VNIV, VGI, VGII, VGIII, VGIV. The inventors of the present application have found through NGS in combination with clinical epidemiological analysis that cryptococcus can be further classified into type VGIa, VGIb, VGIc and type VGId cryptococcus, i.e., type two, in addition to the above 8 types of primary classification; in addition, in the infection caused by cryptococcus, the pathogenicity rate, the lethality rate and the clinical manifestation of the cryptococcus of different subtypes are different, so that the detection of the cryptococcus subtype is extremely important in early clinical diagnosis of the infection caused by cryptococcus.
The currently used typing method can only conduct one-stage typing on cryptococcus, namely, can identify which of the molecular types in 8 belongs to cryptococcus, and cannot conduct two-stage typing. Means for realizing secondary typing such as QPCR are limited by expensive and complex instruments and equipment, and for hospitals in less developed areas, corresponding instruments and equipment and technicians cannot be equipped, so that secondary typing and accurate identification of cryptococcus cannot be performed.
In view of the above, the present application provides a method for identifying cryptococcus, which can be implemented without expensive instruments and complicated detection techniques, and can accurately implement secondary typing detection of cryptococcus, thereby helping to promote early diagnosis and treatment of cryptococcus.
Referring to FIG. 1, a schematic flow chart of a method 100 for identifying cryptococcus in accordance with an embodiment of the present application is shown. The method 100 comprises the following steps:
s101, obtaining genome DNA of a sample to be detected;
s102, amplifying URA5 genes and SOD1 genes of the genome DNA by PCR to obtain amplified products;
s103, performing sanger sequencing on the amplified product;
s104, determining the type of the cryptococcus according to the sequencing result of the URA5 gene and/or the SOD1 gene.
Specifically, the present inventors found that secondary typing of cryptococcus can be judged by detecting whether mutation occurs in URA5 gene and/or SOD1 gene of cryptococcus by combining the previous results with computational biology method. For example, cryptococcus VGIa, cryptococcus VGIb or Cryptococcus VGIc can be identified by whether mutation occurs at different sites of the URA5 gene. The VGId type cryptococcus can be identified by whether mutation occurs at different sites of the SOD1 gene. Specifically, the URA5 gene sequence of the wild-type cryptococcus or the VGId-type cryptococcus is shown as Seq ID No.5, and the URA5 gene sequence of the VGic-type cryptococcus is shown as Seq ID No. 7. As another example, the SOD1 gene sequence of wild-type cryptococcus or VGic-type cryptococcus is shown as Seq ID No.6, and the gene sequence of VGId-type cryptococcus is shown as Seq ID No. 8. It is understood that the wild-type cryptococcus described herein is other types of cryptococcus besides Cryptococcus of VGic type and Cryptococcus of VGId type.
Thus, the typing method 100 of the present application can achieve a secondary typing of cryptococcus by detecting mutation of URA5 gene and/or SOD1 gene. In addition, mutation of URA5 gene and/or SOD1 gene can be detected only by combining sanger sequencing with MLST or other common molecular typing methods, and expensive testing instruments and complex testing techniques are not needed, so that medical staff in underdeveloped areas can accurately determine secondary molecular typing of cryptococcus, and the detection cost of cryptococcus is effectively saved.
Alternatively, the PCR reaction in S102 includes: URA5 forward primer and URA5 reverse primer, and/or SOD1 forward primer and SOD1 reverse primer.
Wherein the nucleotide sequence of the URA5 forward primer is shown as Seq ID No.1, namely the nucleotide sequence of the ATGTCCTCCCAAGCCCTCGAC, URA reverse primer is shown as Seq ID No.2, namely the nucleotide sequence of the TTAAGACCTCTGAACACCGTACTC, SOD forward primer is shown as Seq ID No.3, namely the nucleotide sequence of the GATCCTCACGCCATTACG, SOD reverse primer is shown as Seq ID No.4, namely GAATGATGCGCTTAGTTGGA.
Optionally, the typing of cryptococcus comprises: cryptococcus VGIc, cryptococcus VGId and Cryptococcus wildtype. In other words, the typing method 100 of the present application is used for the secondary typing of cryptococcus.
In the examples herein, by accurately identifying whether an infection is caused by a more lethal cryptococcus subtype, the treatment regimen and medication can be adjusted early in the clinical diagnosis, thereby helping to reduce drug abuse.
Alternatively, the gene sequencing result in S104 includes: sequences of URA5 gene and/or SOD1 gene locus. Optionally, the site comprises at least one of 353 site of URA5 gene, 54 site of SOD1 gene or 457 site of SOD1 gene.
Specifically, the embodiment of the application detects mutation by detecting sequences of a plurality of specific sites of URA5 gene and/or SOD1 gene, thereby judging molecular typing of cryptococcus, and the method can be realized through MLST. On one hand, the subtype of cryptococcus can be judged by detecting and analyzing mutation of specific sites without detecting and analyzing complete gene sequences, so that the detection and analysis workload is saved; on the other hand, compared with other molecular typing modes, the MLST can simply and rapidly obtain results, and the efficiency of subtype identification of cryptococcus is improved.
FIG. 2 is another schematic flow chart of a method 100 for identifying cryptococcus. Optionally, after obtaining the amplification product, the method 100 further comprises:
s105 confirms that URA5 gene and/or SOD1 gene was successfully amplified.
Alternatively, in S105, success of the URA5 gene and/or SOD1 gene amplification was confirmed by gel electrophoresis.
It is to be understood that the gel electrophoresis described herein includes, but is not limited to, agarose gel electrophoresis. In some other embodiments, the success of the URA5 gene and/or SOD1 gene amplification can also be confirmed by polyacrylamide gel electrophoresis.
Specifically, the cryptococcus typing method can carry out gel electrophoresis on an amplified product after PCR amplification, so as to judge whether the amplification of the target genes URA5 and/or SOD1 is successful. If the amplification is successful, step S103 is executed, namely, the amplified product is subjected to sanger sequencing; if the amplification is unsuccessful, steps S101 and S102 are re-executed. By judging whether the target gene is amplified successfully, the reliability of the subsequent sanger sequencing can be improved, and the waste of sequencing time and sequencing cost caused by unsuccessful amplification of the target gene is avoided, so that the identification efficiency of the cryptococcus subtype is improved.
Optionally, in S104, determining the type of cryptococcus comprises:
under the condition that 353 site of URA5 gene is G, determining cryptococcus as VGic cryptococcus;
under the condition that 54 sites of the SOD1 gene are T and 457 sites of the SOD1 gene are C, determining that the cryptococcus is VGId type cryptococcus;
determining cryptococcus as wild-type cryptococcus in the case that 353 of URA5 gene is A, C or T;
under the condition that 54 site of SOD1 gene is A, C or G, determining cryptococcus as wild cryptococcus;
in the case where the 457 site of the SOD1 gene is A, T or G, cryptococcus is determined to be wild-type cryptococcus.
Specifically, by early experiments and clinical pathology analysis, the secondary molecular typing of cryptococcus can be judged by detecting the above sites of URA5 gene and SOD1 gene, thereby determining whether the infection is caused by cryptococcus of VGic type or cryptococcus of VGId type.
In some embodiments, obtaining genomic DNA of a test sample comprises:
grinding a sample to be detected by a liquid nitrogen grinding method; genomic DNA was extracted by standard phenol chloroform method.
The present application also contemplates a primer pair for identifying cryptococcus comprising a first primer pair and/or a second primer pair, wherein the first primer pair comprises a first forward primer and a first reverse primer and the second primer pair comprises a second forward primer and a second reverse primer.
Wherein the nucleotide sequence of the first forward primer is shown as Seq ID No.1, the nucleotide sequence of the first reverse primer is shown as Seq ID No.2, the nucleotide sequence of the second forward primer is shown as Seq ID No.3, and the nucleotide sequence of the second reverse primer is shown as Seq ID No. 4.
Alternatively, the first primer pair is used to recognize 353 and the second primer pair is used to recognize 54 and 457 positions of the URA5 gene sequence of cryptococcus. Specifically, the first primer pair is capable of specifically recognizing the URA5 gene sequence of cryptococcus so that the 353 site of the URA5 gene sequence is detected, and the second primer pair is capable of specifically recognizing the SOD1 gene sequence of cryptococcus so that the 54 site and the 353 site of the SOD1 gene sequence are detected.
The application also provides a kit for identifying cryptococcus, which comprises the primer pair for identifying cryptococcus.
Optionally, the kit further comprises at least one of a buffer, a template DNA, deoxynucleoside triphosphate dntps, and a DNA polymerase.
In addition, the use of a first primer pair and/or a second primer pair in the identification of cryptococcus.
Next, specific steps of the parting method 100 provided in the present application are further described. It is to be understood that the following examples are merely illustrative of embodiments of the typing methods of the present application, wherein the specific reagent amounts or use of the apparatus may be adjusted as desired, and are not to be construed as limiting the examples of the present application.
Example 1: extraction of genomic DNA
1. Test materials: sample to be measured
The sample to be tested described herein includes, but is not limited to, blood, body fluids, or lung tissue.
2. Main reagent and instrument
Reagents include, but are not limited to: liquid nitrogen, 100% alcohol, 75% alcohol, 50% alcohol, ultrapure water, DNA lysate, protease K, tris saturated phenol, chloroform, isoamyl alcohol.
Instruments include, but are not limited to: mortar, pipette, centrifuge, freeze dryer.
3. Experimental method
Get greater than 1 x 10 7 After liquid nitrogen milling in a mortar, genomic DNA was extracted using standard phenol chloroform DNA extraction procedure for each test bacterial sample.
The standard phenol chloroform extraction step may include the steps of:
1) And (3) placing the ground sample to be tested into a centrifuge tube, and respectively carrying out gradient dealcoholization by using 75% alcohol, 50% alcohol and ultrapure water. The dehydration time for each gradient was 5-10min.
2) The sample to be tested after step 1) is placed in a mortar, and an appropriate amount of DNA lysate, for example 300. Mu.L, is added.
3) The ground sample to be tested is transferred into a first centrifuge tube by a pipette, and an appropriate amount of proteinase K, for example 10. Mu.L, is added into the first centrifuge tube, and the first centrifuge tube is sealed and placed into a shaker (56 ℃ C., 5 h).
4) An equal volume of Tris saturated phenol was added to the first centrifuge tube and shaken well (10 min).
5) Putting the first centrifugal tube into a centrifugal machine for centrifugation, wherein the centrifugation conditions are as follows: 12000R,7min,4 ℃. After centrifugation, the mixture is divided into an upper layer, a middle layer and a lower layer, wherein the upper layer is DNA, the middle layer is protein, and the lower layer is organic matter.
6) The supernatant was pipetted and transferred into a second centrifuge tube, to which was added 450. Mu.L of a mixture of Tris-saturated phenol, chloroform and isoamyl alcohol and shaken for 10min. Wherein, the volume ratio of each component in the mixed solution is Tris saturated phenol: chloroform: isoamyl alcohol=25: 24:1.
7) Putting the second centrifugal tube into a centrifugal machine for centrifugation, wherein the centrifugation conditions are as follows: 12000R,7min,4 ℃.
8) Sucking the supernatant into a third centrifuge tube by using a pipette, and adding 400 mu L of a mixed solution of chloroform and isoamyl alcohol in equal volume, wherein the volume ratio of each component in the mixed solution is chloroform: isoamyl alcohol=24: 1.
9) Putting the third centrifugal tube into a centrifugal machine for centrifugation, wherein the centrifugation conditions are as follows: 12000R,7min,4 ℃.
10 Pipette the supernatant into a fourth centrifuge tube, add 2.5 volumes of 100% alcohol frozen at-20 ℃. Overnight at-20 ℃.
11 Placing the fourth centrifugal tube into a centrifugal machine for centrifugation, wherein the centrifugation conditions are as follows: 12000R,7min,4 ℃.
12 A white precipitate (DNA) at the bottom of the fourth centrifuge tube was left, 400. Mu.L of 75% alcohol frozen at-20℃was added thereto, and the mixture was repeatedly blown and dissolved.
13 Repeating step 12) twice.
14 Extracting genome DNA of the sample to be detected.
Example 2: PCR amplification reaction
1. Test materials: 10 ng/. Mu.L of genomic DNA, 10. Mu.L of URA5 forward primer, 2. Mu.L of URA5 reverse primer, 0. Mu.M, 2. Mu.L of SOD1 forward primer, and 2. Mu.L of SOD1 reverse primer, respectively.
2. Main reagent and instrument
Reagents include, but are not limited to: ultrapure water, dNTPs, a buffer solution and DNA polymerase.
3. Experimental method
The PCR amplification reaction was performed using a PCR amplification kit of Prime STAR 2X Mix from TAKARA company, and the PCR amplification process can be seen in the product instructions of the kit. In this example, the nucleotide sequence of the URA5 forward primer is shown as Seq ID No.1 and the nucleotide sequence of the URA5 reverse primer is shown as Seq ID No. 2. The nucleotide sequence of the SOD1 forward primer is shown as SEQ ID No.3, and the nucleotide sequence of the SOD reverse primer is shown as SEQ ID No. 4.
Example 3: agarose gel electrophoresis
The amplification products of example 2 were subjected to agarose gel electrophoresis, and the electropherograms were measured as shown in FIGS. 3 and 4. The single band can be known by electrophoresis pattern, and when the single band is the electrophoresis band with 638bp size, the next step of sequencing is performed.
Example 4: sanger sequencing and site analysis
1. Test materials: amplification products.
2. Main reagent and instrument
Reagents include, but are not limited to: ultrapure water, ddNTP, DNA polymerase.
Instruments include, but are not limited to: sequencer, centrifuge.
3. Experimental method
1) Purifying the amplified product;
2) The purified amplified product is subjected to a program reaction: pre-denaturation at 96 ℃ for 2min, followed by 25 cycles of the following steps: 96 ℃ for 10s;55 ℃ for 5s; and at 60℃for 90s. Finally cooling to 4 ℃ or 15 ℃.
3) After denaturation, the sample was sent to a sequencer for sequencing.
4. Analysis and judgment of amplification result
After the sequencing result was obtained, the sites shown in Table 1 were analyzed, and cryptococcus was typed according to the sequencing result of the sites.
TABLE 1
In other words, when the 353 site of the URA5 gene is mutated to guanine G, cryptococcus is determined to be cryptococcus of VGic type;
when the 54 site of SOD1 is mutated to thymine T and the 457 site of SOD1 is mutated to cytosine C, determining the cryptococcus as VGId type cryptococcus;
when there is no mutation at both 353 and 54 and 457 sites of the URA5 gene and the SOD1 gene, cryptococcus is determined to be wild-type cryptococcus.
FIG. 5 shows partial NGS sequencing results of wild-type cryptococcus and VGic cryptococcus of different genomes, with wild-type cryptococcus having VGIa type cryptococcus and VGIb type cryptococcus. The position indicated by the square in FIG. 5 is the 353 position of the URA5 gene. As can be seen from FIG. 5, the Cryptococcus VGIc is clearly distinguished from the wild-type Cryptococcus at 353, and the Cryptococcus VGIc is mutated to G at 353. It was demonstrated that cryptococcus VGIc can be effectively identified by 353 site of URA5 gene.
The gene sequencing peak diagrams of SOD1 genes of wild-type cryptococcus and VGId cryptococcus at 54 and 457 sites are shown in FIG. 6 and FIG. 7. As can be seen from fig. 6 and 7, the VGId type cryptococcus is clearly distinguished from other types of cryptococcus at 54 and 457 sites, in which the 54 site is mutated to T and the 457 site is mutated to C. The 54 site and 457 site of SOD1 gene can identify VGId cryptococcus effectively.
In summary, the cryptococcus typing method of the present application can accurately determine the type of cryptococcus by performing multi-site sequence typing on the sanger sequencing result of the URA5 gene and/or the SOD1 gene to identify the type of cryptococcus, more specifically, which of VGI types the cryptococcus belongs to, to realize secondary typing detection of the cryptococcus, and to determine the type of the cryptococcus; the method is applied to clinic, is favorable for rapidly and accurately identifying and diagnosing the infection caused by cryptococcus, thereby improving the treatment scheme and reducing the abuse of drugs; the technical scheme of the application is convenient to operate and easy to popularize, and has wide application prospect.
In the description of the present specification, the descriptions of the terms "one embodiment," "one implementation," "some embodiments," "an exemplary embodiment," "an example," "a particular example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present application. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present application, and not for limiting the same; although the present application has been described in detail with reference to the foregoing embodiments, it should be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present application.
Claims (17)
1. A method of identifying cryptococcus, said method comprising:
obtaining genome DNA of a sample to be tested;
amplifying the URA5 gene and/or SOD1 gene of the genome DNA by polymerase chain reaction PCR to obtain an amplified product;
performing sanger sequencing on the amplification product;
determining the type of the cryptococcus based on the sequencing result of the URA5 gene and/or the SOD1 gene.
2. The method of claim 1, wherein the type of cryptococcus comprises cryptococcus of VGIc type, cryptococcus of VGId type, and cryptococcus of wild type.
3. The method according to claim 1 or 2, wherein the sequencing of the URA5 gene and/or the SOD1 gene comprises:
the sequence of the URA5 gene and/or the SOD1 gene locus.
4. The method of claim 3, wherein the site comprises at least one of a 353 site of the URA5 gene, a 54 site of the SOD1 gene, or a 457 site of the SOD1 gene.
5. The method of any one of claims 1-4, wherein said determining the type of cryptococcus comprises:
in the case where the 353 site of the URA5 gene is guanine G, the cryptococcus is determined to be a VGic type cryptococcus.
6. The method of any one of claims 1-4, wherein said determining the type of cryptococcus comprises:
in the case where the 54 th site of the SOD1 gene is thymine T and the 457 th site of the SOD1 gene is cytosine C, the cryptococcus is determined to be a VGId type cryptococcus.
7. The method of any one of claims 1-4, wherein said determining the type of cryptococcus comprises:
in the case where the 353 site of the URA5 gene is adenine a, cytosine C or thymine T; or (b)
In the case where the 54 th site of the SOD1 gene is adenine a, cytosine C or guanine G; or (b)
In the case where the 457 site of the SOD1 gene is adenine A, thymine T or guanine G, the cryptococcus is determined to be a wild-type cryptococcus.
8. The method of any one of claims 1-7, wherein the PCR reaction comprises: a URA5 forward primer and a URA5 reverse primer, and/or a SOD1 forward primer and a SOD1 reverse primer; the nucleotide sequence of the URA5 forward primer is shown as a Seq ID No.1, the nucleotide sequence of the URA5 reverse primer is shown as a Seq ID No.2, the nucleotide sequence of the SOD1 forward primer is shown as a Seq ID No.3, and the nucleotide sequence of the SOD1 reverse primer is shown as a Seq ID No. 4.
9. The method of any one of claims 1-8, wherein after obtaining the amplification product, the method further comprises:
confirming that the URA5 gene and/or the SOD1 gene was successfully amplified.
10. The method according to claim 9, wherein successful amplification of the URA5 gene and/or the SOD1 gene is confirmed by gel electrophoresis.
11. The method of any one of claims 1-10, wherein the obtaining genomic DNA of the test sample comprises:
grinding the sample to be tested by a liquid nitrogen grinding method;
the genomic DNA was extracted by standard phenol chloroform method.
12. The method according to any one of claims 1-11, wherein the sample to be tested comprises blood and/or body fluid.
13. A primer pair for identifying cryptococcus, wherein the primer pair comprises a first primer pair and/or a second primer pair, wherein the first primer pair comprises a first forward primer and a first reverse primer, the second primer pair comprises a second forward primer and a second reverse primer, the nucleotide sequence of the first forward primer is shown as Seq ID No.1, the nucleotide sequence of the first reverse primer is shown as Seq ID No.2, the nucleotide sequence of the second forward primer is shown as Seq ID No.3, and the nucleotide sequence of the second reverse primer is shown as Seq ID No. 4.
14. The primer pair of claim 13, wherein the first primer pair is used to recognize 353 and the second primer pair is used to recognize 54 and 457 sites of the URA5 gene sequence of cryptococcus.
15. A kit for identifying cryptococcus, comprising the primer pair of claim 13 or 14.
16. The kit of claim 15, further comprising: buffer solution, template DNA, deoxynucleoside triphosphate dNTPs and DNA polymerase.
17. Use of a primer pair according to claim 13 or 14 for identifying cryptococcus.
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