CN117089648A - Primer probe combination, method and kit for detecting VGIa type cryptococcus - Google Patents
Primer probe combination, method and kit for detecting VGIa type cryptococcus Download PDFInfo
- Publication number
- CN117089648A CN117089648A CN202311312481.5A CN202311312481A CN117089648A CN 117089648 A CN117089648 A CN 117089648A CN 202311312481 A CN202311312481 A CN 202311312481A CN 117089648 A CN117089648 A CN 117089648A
- Authority
- CN
- China
- Prior art keywords
- cryptococcus
- vgia
- primer
- fluorescence
- probe combination
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 241001337994 Cryptococcus <scale insect> Species 0.000 title claims abstract description 108
- 239000000523 sample Substances 0.000 title claims abstract description 75
- 238000000034 method Methods 0.000 title claims abstract description 20
- 239000002773 nucleotide Substances 0.000 claims abstract description 21
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 21
- 230000003321 amplification Effects 0.000 claims description 26
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 26
- 238000001514 detection method Methods 0.000 claims description 23
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 4
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 4
- 238000003752 polymerase chain reaction Methods 0.000 claims description 4
- 238000010791 quenching Methods 0.000 claims description 4
- 230000000171 quenching effect Effects 0.000 claims description 4
- 239000000872 buffer Substances 0.000 claims description 3
- 239000000376 reactant Substances 0.000 claims description 3
- 238000012360 testing method Methods 0.000 claims description 3
- 239000001226 triphosphate Substances 0.000 claims description 3
- 235000011178 triphosphate Nutrition 0.000 claims description 3
- 125000002264 triphosphate group Chemical class [H]OP(=O)(O[H])OP(=O)(O[H])OP(=O)(O[H])O* 0.000 claims description 3
- 238000011282 treatment Methods 0.000 abstract description 4
- 108020004414 DNA Proteins 0.000 description 11
- 238000006243 chemical reaction Methods 0.000 description 6
- 201000007336 Cryptococcosis Diseases 0.000 description 5
- 241000221204 Cryptococcus neoformans Species 0.000 description 4
- 238000012408 PCR amplification Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 230000007918 pathogenicity Effects 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000003759 clinical diagnosis Methods 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 125000006853 reporter group Chemical group 0.000 description 3
- 241000894007 species Species 0.000 description 3
- COCMHKNAGZHBDZ-UHFFFAOYSA-N 4-carboxy-3-[3-(dimethylamino)-6-dimethylazaniumylidenexanthen-9-yl]benzoate Chemical compound C=12C=CC(=[N+](C)C)C=C2OC2=CC(N(C)C)=CC=C2C=1C1=CC(C([O-])=O)=CC=C1C(O)=O COCMHKNAGZHBDZ-UHFFFAOYSA-N 0.000 description 2
- 238000007400 DNA extraction Methods 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000001124 body fluid Anatomy 0.000 description 2
- 239000010839 body fluid Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 238000013399 early diagnosis Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 description 2
- UDGUGZTYGWUUSG-UHFFFAOYSA-N 4-[4-[[2,5-dimethoxy-4-[(4-nitrophenyl)diazenyl]phenyl]diazenyl]-n-methylanilino]butanoic acid Chemical compound COC=1C=C(N=NC=2C=CC(=CC=2)N(C)CCCC(O)=O)C(OC)=CC=1N=NC1=CC=C([N+]([O-])=O)C=C1 UDGUGZTYGWUUSG-UHFFFAOYSA-N 0.000 description 1
- WCKQPPQRFNHPRJ-UHFFFAOYSA-N 4-[[4-(dimethylamino)phenyl]diazenyl]benzoic acid Chemical compound C1=CC(N(C)C)=CC=C1N=NC1=CC=C(C(O)=O)C=C1 WCKQPPQRFNHPRJ-UHFFFAOYSA-N 0.000 description 1
- BZTDTCNHAFUJOG-UHFFFAOYSA-N 6-carboxyfluorescein Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C11OC(=O)C2=CC=C(C(=O)O)C=C21 BZTDTCNHAFUJOG-UHFFFAOYSA-N 0.000 description 1
- 239000005711 Benzoic acid Substances 0.000 description 1
- 206010013654 Drug abuse Diseases 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 229930002877 anthocyanin Natural products 0.000 description 1
- 235000010208 anthocyanin Nutrition 0.000 description 1
- 239000004410 anthocyanin Substances 0.000 description 1
- 150000004636 anthocyanins Chemical class 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 235000010233 benzoic acid Nutrition 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 231100000676 disease causative agent Toxicity 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 244000053095 fungal pathogen Species 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 208000011117 substance-related disease Diseases 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Botany (AREA)
- Mycology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The application relates to a primer probe combination, a method and a kit for detecting Cryptococcus VGIa. Wherein the primer comprises a forward primer and a reverse primer, the nucleotide sequence of the forward primer is shown as a Seq ID No.1, the nucleotide sequence of the reverse primer is shown as a Seq ID No.2, and the nucleotide sequence of the probe is shown as a Seq ID No. 3. The primer probe combination provided by the application can accurately and rapidly detect the cryptococcus and identify the cryptococcus as VGIa type cryptococcus, and determine the subtype of the cryptococcus when detecting the cryptococcus, thereby helping to distinguish the cryptococcus of different subtypes in early clinical stage and making a proper treatment scheme.
Description
Technical Field
The application belongs to the technical field of fungal etiology detection, and particularly relates to a primer probe combination, a method and a kit for detecting Cryptococcus VGIa.
Background
Cryptococcus is a kind of pathogenic fungi of various kinds in genus, and after being infected, the cryptococcus can invade central nerve of human body or cause infection of other viscera along with blood flow. From epidemiological observations, there are significant differences in epidemics, pathogenicity, and mortality among cryptococcus of different species, and the applicable therapies are different. Thus, in early diagnosis of cryptococcus, detection and identification of the type of cryptococcus is of great importance for the assignment of treatment regimens.
Cryptococcus is generally classified into 8 types by molecular typing, i.e., one-stage typing. It has been found that there is a significant difference in clinical performance of cryptococcus of different subspecies under the same type-one subtype, but there is still no research on cryptococcus subtype and no corresponding detection and identification method. Therefore, how to accurately detect cryptococcus in the secondary typing is a technical problem to be solved.
Disclosure of Invention
The application provides a primer probe combination, a method and a kit for detecting VGIa type cryptococcus and application of the primer probe combination in detecting VGIa type cryptococcus, and the specific primer probe combination is designed aiming at specific gene fragments of the VGIa type cryptococcus, so that the VGIa type cryptococcus can be accurately detected, and early clinical diagnosis of cryptococcosis is facilitated.
In a first aspect, the present application provides a primer probe combination for detecting cryptococcus of VGIa type, the primer comprising a forward primer and a reverse primer, the nucleotide sequence of the forward primer being shown as Seq ID No.1, the nucleotide sequence of the reverse primer being shown as Seq ID No. 2; the nucleotide sequence of the probe is shown as Seq ID No. 3.
In some embodiments, the primer is used to recognize a nucleotide sequence as set forth in Seq ID No.4 in Cryptococcus VGIa.
In some embodiments, the 5 'end of the probe is attached to a fluorescent reporter group and the 3' end of the probe is attached to a fluorescence quenching group.
In a second aspect, there is provided a method of detecting cryptococcus VGIa, the method comprising: obtaining template DNA of a sample to be detected; amplifying the template DNA by polymerase chain reaction, PCR, to obtain an amplification result, wherein the reactants of the PCR comprise a primer probe combination as in any of the embodiments of the first aspect; and judging whether the detection result of the Cryptococcus VGIa in the sample to be detected is positive or negative according to the amplification result.
In some embodiments, the determining that the detection result of the cryptococcus VGIa in the sample to be detected is positive or negative according to the amplification result comprises: judging that the detection result of the Cryptococcus VGIa in the sample to be detected is positive when the fluorescence report group has fluorescence logarithmic growth in the amplification result and the cycle threshold Ct is smaller than or equal to 30.0; and judging that the detection result of the Cryptococcus VGIa in the sample to be detected is negative under the condition that the fluorescence report group does not increase in fluorescence or the fluorescence report group increases in fluorescence logarithm and the cycle threshold Ct is less than or equal to 30.0 in the amplification result.
In a third aspect, there is provided a kit for detecting cryptococcus VGIa, the kit comprising the primer probe combination of any one of the embodiments of the first aspect.
In some embodiments, the kit further comprises: buffers, template DNA, DNA polymerase and/or deoxynucleotide triphosphates dntps.
In a fourth aspect, there is provided the use of a primer probe combination as in any of the embodiments of the first aspect for detecting cryptococcus VGIa.
Drawings
FIG. 1 is a schematic flow chart of a detection method of Cryptococcus VGIa according to an embodiment of the present application.
FIG. 2 is a graph showing the specific amplification of Cryptococcus VGIa of the present application.
Detailed Description
The technical scheme of the application is further described below with reference to specific embodiments.
The "range" disclosed herein is defined in terms of lower and upper limits, with the 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 the present 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.
The terms "comprising" and "including" as used herein mean open ended or 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 of the application and alternative embodiments may be combined with each other to form new solutions, unless otherwise specified.
Cryptococcus is a fungus widely existing in nature and belongs to the phylum of the subdivision Deuteromycotina, class of the genus Cryptococcus and the family Cryptococcus. Cryptococcus comprises 17 species and 8 varieties, of which the causative agent is mainly Cryptococcus neoformans, hereinafter referred to as Cryptococcus. There are significant differences in the pathogenicity of cryptococcus of different species among infected individuals. For example, cryptococcus neoformans primarily infect immunosuppressed people, while Cryptococcus glatirami primarily infects immunocompetent people.
With the improvement of gene detection means, cryptococcus is classified into 8 types on the primary typing, which includes: cryptococcus VNI, cryptococcus VNII, cryptococcus VNIII, cryptococcus VNIV, cryptococcus VGI, cryptococcus VGII, cryptococcus VGIII, and cryptococcus VGIV. Furthermore, the inventors have found that under the above 8 types of cryptococcus, cryptococcus can be further subdivided, for example, cryptococcus of VGI type can be further divided into a plurality of subtypes of cryptococcus of VGIa type, cryptococcus of VGIb type, cryptococcus of VGIc type, etc. There are also significant differences in the popularity, pathogenicity and mortality of cryptococcus of different subtypes. Therefore, how to detect and identify the subtype of cryptococcus has important significance for clinical diagnosis.
In view of the above, the present application provides a primer probe combination for detecting cryptococcus VGIa, and polymerase chain reaction PCR based on the primer probe combination can accurately and rapidly detect and identify cryptococcus VGIa.
Firstly, the embodiment of the application provides a primer probe combination for detecting the Cryptococcus VGIa.
Wherein the primer comprises a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown as a Seq ID No.1, and the nucleotide sequence of the reverse primer is shown as a Seq ID No. 2. The nucleotide sequence of the probe is shown in Seq ID No. 3.
Specifically, the inventors have designed primers having nucleotide sequences shown in Seq ID No.1 and Seq ID No.2 and probes having nucleotide sequences shown in Seq ID No.3 by comparing the entire genome of cryptococcus of various subtypes by bioinformatics, not limited to a single coding gene, but searching for a differential site on the whole genome level. The primer probe combination can be used for accurately detecting and identifying the Cryptococcus VGIa, thereby helping early diagnosis of diseases caused by the Cryptococcus VGIa.
The nucleotide sequence of the forward primer is specifically as follows:
TACAGTTACTGCACGGCATG。
the nucleotide sequence of the reverse primer is specifically as follows:
TACGAAGGATGAGCAATGCC。
the primer is used for identifying a nucleotide sequence shown as a Seq ID No.4 in the cryptococcus VGIa. In other words, the primer designed for cryptococcus VGIa is capable of specifically recognizing the nucleotide sequence shown in Seq ID No.4, which is present only in cryptococcus VGIa.
Optionally, a fluorescent reporter group is attached to the 5 'end of the probe, and a fluorescence quenching group is attached to the 3' end of the probe.
Among them, fluorescent reporter groups include, but are not limited to, 6-carboxyfluorescein (FAM), tetra-chloro-6-carboxyfluorescein (TET), 2, 7-dimethyl-4, 5-dichloro-6-carboxyfluorescein (JOE), hexachloro-6-methylfluorescein (HEX), anthocyanin fluorescent dye CY3, 6-carboxy-tetramethyl rhodamine (TAMRA). Fluorescence quenching groups include, but are not limited to, 6-carboxy-tetramethyl rhodamine (TAMRA), 4- (4-oxaminophenylazo) benzoic acid (DABCYL), or black hole quenchers BHQ1, BHQ2, BHQ3.
For example, the 5 'end of the probe is connected with a FAM group, and the 3' end of the probe is connected with a BHQ1 group. Namely, the probe is as follows:
FAM-TGGGAGCATCCACGCCAGTTTCCCGAGTA-BHQ1。
the primer probe combination has the obvious advantages of high sensitivity and strong specificity when detecting and identifying the VGIa type cryptococcus.
The embodiment of the application also provides a method 100 for detecting the Cryptococcus VGIa.
FIG. 1 is a schematic flow chart of a method 100 for detecting Cryptococcus VGIa according to an embodiment of the present application. As shown in fig. 1, the method 100 includes:
s101, obtaining template DNA of a sample to be detected.
S102, amplifying template DNA of the sample to be detected by polymerase chain reaction PCR to obtain an amplification result. Wherein, the reactants of the PCR comprise the primer probe combination in any embodiment of the application.
S103, judging whether the detection result of the Cryptococcus VGIa in the sample to be detected is positive or negative according to the amplification result.
Specifically, the primer probe combination provided by the application can effectively detect the VGIa type cryptococcus in a sample to be detected through fluorescent quantitative PCR.
Optionally, the sample to be tested comprises blood, body fluid, and/or lung tissue.
Optionally, in S103, determining that the detection result of the cryptococcus VGIa in the sample to be detected is positive or negative according to the amplification result includes:
in the amplification result, under the condition that the fluorescence logarithm of the fluorescence reporting group is increased and the cycle threshold Ct is smaller than or equal to 30.0, the detection result of the VGIa type cryptococcus in the sample to be detected is judged to be positive.
In the amplification result, under the condition that the fluorescence report group does not have fluorescence logarithmic growth or the fluorescence report group has fluorescence logarithmic growth but the cycle threshold Ct is more than 30.0, judging that the detection result of the VGIa type cryptococcus in the sample to be detected is negative.
The embodiment of the application also provides a kit for detecting the Cryptococcus VGIa, which comprises the primer probe combination in any embodiment of the application.
Optionally, the kit further comprises: buffers, template DNA, DNA polymerase and/or deoxynucleotide triphosphates dntps.
Optionally, the kit further comprises MgCl 2 。
The kit provided by the embodiment of the application can accurately and rapidly detect the Cryptococcus VGIa. The method has the advantages of simple operation, short detection time and strong adaptability, and can identify the subtype of pathogenic bacteria, thereby carrying out rapid and accurate clinical diagnosis and winning precious time for disease treatment.
In addition, the embodiment of the application also provides application of the primer probe combination in any embodiment in detecting the VGIa type cryptococcus.
Next, specific steps of the detection method 100 provided in the present application will be further described. It is to be understood that the following examples are merely illustrative of embodiments of the identification method 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 template DNA
1. Test materials: sample to be measured
The sample to be tested according to the present application includes, but is not limited to, blood, body fluids or lung tissue.
2. Main reagent and instrument
DNA extraction adopts a yeast gene extraction kit of Tiangen company; the synthesis of primers and probes was done by bioengineering limited; DNA polymerase was purchased from Takara; fluorescent quantitative PCR instrument was purchased from Roche company.
3. Experimental method
DNA was extracted according to the DNA extraction kit instructions, and the content of DNA in the sample was measured using a spectrophotometer to determine the OD260/OD280 ratio. The sample to be tested provided in this example contains Cryptococcus neoformans, cryptococcus VGIa and Cryptococcus VGIb.
Example 2: PCR amplification reaction
2.1 PCR detection of Cryptococcus VGIa type
1. Test materials
Template DNA. The specific PCR reaction system is shown in Table 1.
2. Main reagent and instrument
Table 1: real-time fluorescence PCR reaction system (25 mu L)
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 forward primer in the reaction system is shown as Seq ID No.1, the nucleotide sequence of the reverse primer is shown as Seq ID No.2, and the nucleotide sequence of the probe is shown as Seq ID No. 3.
In this example, the amplification conditions for the PCR reaction were: 95 o C, pre-denaturation for 5min;95 o C 10s，68 o C60 s,45 cycles, amplification results were obtained.
4. Analysis and judgment of amplification result
FIG. 2 shows amplification curves of Cryptococcus VGIa obtained by the above procedure in the examples of the present application. In the amplification curve of FIG. 2, the ordinate indicates the relative fluorescence units (Relative fluorescence unit, RFU) and the abscissa indicates the number of cycles (cycles) of amplification.
As shown in FIG. 2, in the detection result of the sample to be detected, only the Cryptococcus VGIa is specifically amplified, the amplification curve is corresponding, the fluorescence of FAM has logarithmic increase of fluorescence, and Ct is less than or equal to 30.0. In parallel experiments, the VGIb type cryptococcus and the neonatal cryptococcus have no specific amplification curve. The primer probe combination has good specificity to the VGIa type cryptococcus, and can accurately identify the VGIa type cryptococcus in a sample to be detected. In addition, 3 amplification curves of the Cryptococcus VGIa have almost consistent fluorescence logarithmic growth, which shows that the method for detecting the Cryptococcus VGIa by the primer probe combination has good repeatability. In addition, the template DNA concentration in FIG. 2 is 0.1 ng/. Mu.L, and the fluorescence logarithmic response can be still good after fluorescence PCR, which shows that the method for detecting the VGIa type cryptococcus by the primer probe combination has excellent sensitivity.
Therefore, the primer probe combination provided by the application can accurately and efficiently detect the VGIa type cryptococcus.
Through the detection of the VGIa type cryptococcus, the VGIb type cryptococcus and the neoformans, the primer probe combination provided by the application can carry out specific identification on the VGIa type cryptococcus, and the results of other strains in parallel experiments are negative without specific amplification curves, so that the primer probe combination has good specificity. In addition, when the primer probe combination provided by the application is used for detecting the VGIa type cryptococcus, not only can the cryptococcus be detected, but also the subtype of the cryptococcus can be directly determined, so that the primer probe combination is beneficial to early intervention and treatment of cryptococcus subspecies with higher pathogenicity or mortality in clinic. The detection kit provided by the application is used for detecting the cryptococcus subtype, has simple operation and reliable and accurate result, can help doctors to rapidly diagnose cryptococcus infection diseases, is beneficial to formulating a proper treatment method as early as possible, and reduces drug abuse.
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 application has been described in detail with reference to the foregoing embodiments, it will 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 application.
Claims (8)
1. A primer probe combination for detecting cryptococcus of VGIa type, which is characterized in that the primer comprises a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown as a Seq ID No.1, and the nucleotide sequence of the reverse primer is shown as a Seq ID No. 2; the nucleotide sequence of the probe is shown as Seq ID No. 3.
2. The primer probe combination according to claim 1, wherein the primer is used for identifying a nucleotide sequence as shown in Seq ID No.4 in Cryptococcus VGIa.
3. The primer probe combination according to claim 1 or 2, wherein the 5 'end of the probe is connected with a fluorescence reporting group, and the 3' end of the probe is connected with a fluorescence quenching group.
4. A method of detecting cryptococcus of VGIa type, comprising:
obtaining template DNA of a sample to be detected;
amplifying the template DNA by polymerase chain reaction PCR to obtain an amplification result, wherein the reactants of the PCR comprise the primer probe combination of any one of claims 1-3;
and judging whether the detection result of the Cryptococcus VGIa in the sample to be detected is positive or negative according to the amplification result.
5. The method of claim 4, wherein the determining that the detection result of the cryptococcus VGIa in the test sample is positive or negative according to the amplification result comprises:
judging that the detection result of the Cryptococcus VGIa in the sample to be detected is positive when the fluorescence report group has fluorescence logarithmic growth in the amplification result and the cycle threshold Ct is smaller than or equal to 30.0;
and judging that the detection result of the Cryptococcus VGIa in the sample to be detected is negative under the condition that the fluorescence report group does not increase in fluorescence or the fluorescence report group increases in fluorescence logarithm and the cycle threshold Ct is less than or equal to 30.0 in the amplification result.
6. A kit for detecting cryptococcus of VGIa type, comprising the primer probe combination according to any one of claims 1 to 3.
7. The kit of claim 6, further comprising: buffers, template DNA, DNA polymerase and/or deoxynucleoside triphosphates dntps.
8. Use of a primer probe combination according to any one of claims 1-3 for detecting cryptococcus VGIa.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311312481.5A CN117089648B (en) | 2023-10-11 | 2023-10-11 | Primer probe combination, method and kit for detecting VGIa cryptococcus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311312481.5A CN117089648B (en) | 2023-10-11 | 2023-10-11 | Primer probe combination, method and kit for detecting VGIa cryptococcus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117089648A true CN117089648A (en) | 2023-11-21 |
| CN117089648B CN117089648B (en) | 2024-07-19 |
Family
ID=88783562
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311312481.5A Active CN117089648B (en) | 2023-10-11 | 2023-10-11 | Primer probe combination, method and kit for detecting VGIa cryptococcus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117089648B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20180274011A1 (en) * | 2014-08-06 | 2018-09-27 | The Translational Genomics Research Institute | Methods and kits to detect and genotype cryptococcus species |
| CN116287388A (en) * | 2023-02-17 | 2023-06-23 | 首都医科大学附属北京世纪坛医院 | Identification method, primer pair and kit for cryptococcus |
-
2023
- 2023-10-11 CN CN202311312481.5A patent/CN117089648B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20180274011A1 (en) * | 2014-08-06 | 2018-09-27 | The Translational Genomics Research Institute | Methods and kits to detect and genotype cryptococcus species |
| CN116287388A (en) * | 2023-02-17 | 2023-06-23 | 首都医科大学附属北京世纪坛医院 | Identification method, primer pair and kit for cryptococcus |
Non-Patent Citations (1)
| Title |
|---|
| 冯晓博;凌波;付小花;王磊;廖万清;姚志荣;: "格特隐球菌核基因、交配型位点和线粒体基因的多位点序列分型及重组分析", 微生物学杂志, no. 06, 15 December 2013 (2013-12-15), pages 26 - 31 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117089648B (en) | 2024-07-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111004862B (en) | Primer and probe for rapidly detecting and identifying cryptococcus and application thereof | |
| CN103725798B (en) | For detecting primer, test kit, the detection method of hemorrhagic fever with renal syndrome virus with RT-LAMP method | |
| CN113652505B (en) | Method and kit for detecting novel coronavirus and VOC-202012/01 mutant strain thereof | |
| CN113999933A (en) | Method for accurately detecting cryptococcus neoformans | |
| CN117144049A (en) | Primer probe combination, method and kit for detecting cryptococcus subtype | |
| CN110055347A (en) | A method of identifying five kinds of dermatophytes using high-resolution fusion curve | |
| CN113862393A (en) | Method for rapidly detecting cryptococcus gatherensis | |
| CN112831597A (en) | Real-time fluorescent PCR amplification primer pair and probe primer for gene identification and detection of African swine fever virus and prepared kit | |
| CN113913543B (en) | EHP real-time fluorescence quantitative PCR detection primer probe combination, kit and method | |
| KR102453357B1 (en) | Primer set for multiplex loop-mediated isothermal amplification reaction for detection and identification of Candida albicans, Candida auris and Candida glabrata using fluorescent probe | |
| CN117089648B (en) | Primer probe combination, method and kit for detecting VGIa cryptococcus | |
| CN111926110B (en) | African swine fever virus real-time fluorescent PCR amplification primer pair, probe primer and prepared kit | |
| CN117248068B (en) | Primer probe combination, method and kit for detecting VGIb cryptococcus | |
| CN117265162B (en) | Primer probe combination, method and kit for detecting cryptococcus garvieae | |
| CN105296668B (en) | Primer, probe and kit for specifically detecting type 3 ungulate bocavirus parvovirus | |
| CN117106966B (en) | Primer pair, method and kit for detecting cryptococcus garvieae | |
| CN117165714A (en) | Primer probe combination, method and kit for detecting cryptococcus neoformans | |
| CN110257544B (en) | Ergota germ fluorescent quantitative PCR detection reagent, detection kit and application | |
| CN117051168A (en) | Primer probe combination, method and kit for detecting cryptococcus | |
| CN105567838B (en) | MiR-331-5p is as the application in acute HIV infection detection marker | |
| KR102653475B1 (en) | Composition for detecting coronavirus simultaneously and method for detecting coronavirus simultaneously comprising the same | |
| CN117904341B (en) | Primer probe combination, kit and method for detecting mycoplasma gallisepticum by real-time fluorescence quantitative PCR | |
| CN117757991B (en) | POCT detection method for DNA and mRNA of HPV16/18 virus gene in cervical exfoliated cells | |
| CN114908084B (en) | Primer probe of cercospora spinosa, aspergillus terreus and aspergillus nidulans and application thereof | |
| KR102692361B1 (en) | Primer set for multiplex loop-mediated isothermal amplification reaction for detection of Candida pan and Candida albicans |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |