CN114075564B - Malassezia detection composition, kit and detection method thereof - Google Patents
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Abstract
The invention discloses a detection composition, a kit and a detection method of malassezia. The Malassezia detection primer group and the kit provided by the invention combine the advantages of specific targeting of the RPA high-efficiency amplification and CRISPR/Cas detection technology, have the characteristics of high amplification efficiency, strong specificity, simple operation and the like, and have no cross reaction with other common pathogenic fungi such as candida, mould, sporothrix and the like and human genome DNA. All detection steps of the kit provided by the invention do not depend on temperature change, and an expensive PCR instrument or complex temperature change conditions are not needed, so that the reaction speed is higher, and the cost is lower. The kit provided by the invention can be directly used for detecting clinical pathogenic fungi samples without culture, meets the requirement of quick and accurate diagnosis in early clinical period, and is helpful for quickly identifying strains which are difficult to identify by conventional morphology or histopathology.
Description
Technical Field
The invention relates to the technical field of gene detection, in particular to a detection composition, a kit and a detection method of malassezia.
Background
Malassezia related Diseases (The Malassezia Genus in Skin and Systemic Diseases) are a group of Diseases caused by common fungal infections, such as: pityriasis versicolor, malassezia folliculitis (pityriasis versicolor) ((R))Malassezia folliculolitis), seborrheic dermatitis (seborreic dermatitis), Atopic dermatitis (Atopic dermatitis), Psoriasis (Psoriasis) and the like are closely related to malassezia. The above diseases not only harm the physical and mental health of people in all age groups, but also are easy to misdiagnose in hospitals at all levels, and bring huge economic burden to patients, society and national medical insurance. Taking atopic dermatitis and psoriasis as examples, according to the data published by the national center for disease control in the United states, the average natural population prevalence of atopic dermatitis is 10%, and the conservative estimate of the diagnosis and treatment cost of atopic dermatitis in the United states is $ 52.97 billion in 2015 alone. Psoriasis is a common chronic skin disease with a natural population prevalence of about 3% and about 740 million adults with psoriasis, and the annual treatment cost of the disease is estimated to be $ 352 million. Therefore, the prevention and control of malassezia related diseases are related to the public health safety of the country, and the rapid and accurate identification of malassezia at an early stage is the key for timely diagnosing and treating malassezia infected diseases.
The current diagnosis of fungal infectious diseases relies mainly on laboratory tests for the pathogenic pathogens, and traditional laboratory diagnostic methods are: microscopic examination of fungi (KOH method, fluorescent staining method), isolated culture of fungi, and histopathological examination. The fungus isolation culture and the histopathological examination are used as gold standards for diagnosing fungal infectious diseases and are also suitable for malassezia infection, but the fungus isolation culture has the defects of long identification period, low positive rate, high morphological identification difficulty and the like; therefore, how to realize more rapid, simple and accurate detection is the future development direction of pathogenic pathogen detection.
Disclosure of Invention
The invention aims to provide a detection composition, a detection kit and a detection method of malassezia, so as to solve one or more technical problems in the prior art and provide at least one beneficial choice or creation condition.
In a first aspect of the invention, crRNA is provided. The crRNA comprises an anchor sequence segment near the 5 'end and a guide sequence segment near the 3' end, wherein the nucleotide sequence of the guide sequence segment is shown in Table 1:
TABLE 1 guide sequences for crRNA
The guide sequence fragment is specifically combined with the ITS sequence of the Malassezia, so that the identification and identification of five different types of common Malassezia bacteria, namely Malassezia furfur (Malassezia furfur), Malassezia blunt (Malassezia obtusa), Malassezia globosa (Malassezia globosa), Malassezia symptomata (Malassezia sympodialis) and Malassezia pachydermatis (Malassezia pachydermata) can be accurately realized.
Further, the nucleotide sequence of the anchor sequence fragment is 5'-UAAUUUCUACUAAGUGUAGAU-3' (SEQ ID NO. 11). That is, the sequence of the specific crRNA is shown in Table 2:
TABLE 2 sequence of crRNA
The design of the crRNA fully considers the reaction characteristics of a CRISPR (Clustered regularly Interspaced Short Palindromic Repeats) system, namely, the Cas protein can specifically recognize a target sequence under the guidance of the crRNA so as to start the 'additional cutting' activity, and the PAM (PAM) (protospacer adjacent motif) sequence at the downstream of the target plays an important role in the specific recognition process of the Cas protein.
A second aspect of the invention provides a kit. The kit comprises a CRISPR/Cas detection system, wherein the CRISPR/Cas detection system comprises at least one crRNA with a nucleotide sequence shown in SEQ ID NO.12 to SEQ ID NO. 21. When a CRISPR/Cas detection system has a plurality of crRNAs, detection and identification of different malassezia in a sample can be realized.
Further, the CRISPR/Cas detection system also comprises a Cas protein and a signal report probe. The Cas protein is Cas12a protein, and the anchor sequence segment shown as SEQ ID No.11 is capable of specifically binding to Cas12a protein. The sequence of the signal reporting probe is as follows: 5'-TTTTTATTTTT-3' (SEQ ID NO. 34). The 5 'end of the signal report probe is marked with a fluorescence report group, and the 3' end of the signal report probe is marked with a fluorescence quenching group.
Further, the fluorescent reporter group is selected from one of FAM, VIC, HEX, ROX, JOE, Cy3 and Cy 5; the fluorescence quenching group is selected from one of TAMRA, BHQ-1, BHQ-2 and BHQ-3.
Further, the CRISPR/Cas detection system also includes other required reagent components, such as NEB Buffer.
Further, the kit also comprises an RPA (Recombinase Polymerase Amplification) Amplification system, which comprises an upstream primer and a downstream primer, wherein the upstream primer: 5'-GCAGAATTCCGTGAATCATCGAATCTTTGAAC-3' (SEQ ID NO. 27); a downstream primer: 5'-ACACACAGCAAATGACGTATCATGCCATGC-3' (SEQ ID NO. 28). The primer pair is used for specifically amplifying the ITS sequence of the malassezia. The introduction of the RPA amplification system can complete the amplification of the sample at normal temperature without special equipment, so that the sensitivity of the CRISPR/Cas detection system is greatly improved.
Further, the RPA amplification system may further comprise other desired reagent components, such as RPA enzyme premix, activator, ddH2O, etc. toThe RPA enzyme premix comprises phosphocreatine, creatine kinase, dNTPs, ATP, DTT, potassium acetate, recombinase UvsX, recombinase UvsY, single-chain binding protein and Bsu polymerase.
Further, the reaction temperature of the RPA amplification system is 37-42 ℃. And (3) enriching the ITS sequences of the malassezia in the sample by RPA amplification for 15-20min, and then directly adding the ITS sequences into each component of the CRISPR/Cas detection system for detection reaction, namely reading a detection signal by using a fluorescence detection device. The fluorescence detection device can be a real-time fluorescence quantitative PCR instrument or a microplate reader.
The specific kit using method comprises the following steps:
(1) amplification: taking DNA extracted and purified from a sample to be detected as a template to carry out RPA amplification, wherein an RPA amplification system comprises the following components: 20. mu.L of RPA enzyme premix, 2.5. mu.L each of forward primer (10. mu.M) and reverse primer (10. mu.M), 1. mu.L of sample to be tested, 2. mu.L of activator, and ddH2O22. mu.L. The reaction temperature was 37 ℃ and the reaction time was 20 min.
(2) And (3) detection: and taking the amplification product, adding corresponding crRNA, a signal report probe and Cas protein to perform CRISPR reaction detection, and reading a detection signal by using fluorescence detection equipment.
(3) And (4) interpretation of results: the fluorescent signal can be read by a real-time fluorescent PCR instrument or a microplate reader. When a real-time fluorescence PCR instrument is used for fluorescence detection, if the reaction is positive, the fluorescence signal is obviously enhanced and exceeds a threshold line; if the reaction is negative, the fluorescence signal is not obviously enhanced and is lower than the threshold line. When the enzyme-linked immunosorbent assay is used for fluorescence detection, the excitation wavelength is 485nm, the emission wavelength is 520nm, and if the fluorescence signal value is 2 times higher than the negative signal value, the fluorescence signal value is determined to be positive.
The crRNA, the primer pair or the kit can be applied to identification of malassezia species for non-disease diagnosis.
The invention has the following beneficial effects:
the Malassezia detection primer group and the kit provided by the invention combine the advantages of specific targeting of the RPA high-efficiency amplification and CRISPR/Cas detection technology, have the characteristics of high amplification efficiency, strong specificity, simple operation and the like, and have no cross reaction with other common pathogenic fungi such as candida, mould, sporothrix and the like and human genome DNA. All detection steps of the kit provided by the invention do not depend on temperature change, and an expensive PCR instrument or complex temperature change conditions are not needed, so that the reaction speed is higher, and the cost is lower. The kit provided by the invention can be directly used for detecting clinical pathogenic fungi samples without culture, meets the requirement of quick and accurate diagnosis in early clinical period, and is helpful for quickly identifying strains which are difficult to identify by conventional morphology or histopathology.
Drawings
FIG. 1 is an electrophoretogram of the efficiency of the RPA amplification primers tested in example 1;
FIG. 2 is a bar graph of fluorescence signals for testing the efficiency of crRNA detection in example 2;
FIG. 3 is a graph of fluorescence signals for identifying different species of Malassezia in example 3;
FIG. 4 is a graph of the fluorescence signal of the specificity test in example 4;
FIG. 5 is a graph showing the fluorescence signal curves of clinical examination in example 6, in which reference numeral 1 is the fluorescence signal curve of the sample to be examined and reference numeral 2 is the fluorescence signal curve of the negative control;
FIG. 6 is a sample culture microscopy image of the clinical assay of example 6.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to specific embodiments, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 primer design and testing of RPA amplification System
Molecular marker sequences such as an ITS sequence, a beta-tubulin gene sequence, a Translation Elongation Factor (TEF), an actin gene and a mitochondrial cytochrome B gene of the malassezia are collected from public databases such as EMBL-EBI, NCBI and DDBJ, and an ITS region sequence with good species specificity is finally screened out to be used as a detection target sequence of the malassezia with the species specificity.
And designing an amplification primer for RPA aiming at the malassezia detection target sequence. Because the prior art lacks effective RPA amplification primer design software and the amplification effect of a primer pair is difficult to predict in advance, a plurality of upstream primers and a plurality of downstream primers need to be designed respectively to carry out pairing screening of different combinations, and candidate primers are shown in Table 3:
TABLE 3 RPA primer sequences
Specifically, the standard template to be detected adopts a plasmid carried by escherichia coli engineering bacteria and inserted with malassezia furfur ITS gene sequence, the plasmid is synthesized by biological engineering (Shanghai) GmbH, and the concentration of the plasmid is 10 cobies/mu L. The amplification process of the RPA amplification system is carried out according to the operation instruction of the RPA basic isothermal amplification kit.
The RPA amplification system is 50 μ L: wherein the RPA enzyme premix is 20 mu L, the upstream primer with the concentration of 10 mu M and the downstream primer with the concentration of 10 mu M are respectively 2.5 mu L, the standard template to be detected is 1 mu L, the magnesium acetate activator is 2 mu L and ddH2O22. mu.L. The RPA enzyme premix contains: creatine phosphate, creatine kinase, dNTPs, ATP, DTT, potassium acetate, recombinase UvsX, UvsY, single-chain binding protein and Bsu polymerase. Adding the RPA amplification system into a PCR tube, centrifuging briefly, quickly putting into a constant-temperature incubation system for constant-temperature amplification reaction, and setting reaction conditions as follows: 15-20min at 37-42 ℃.
After the reaction is finished, 50 mu L of phenol/chloroform mixed solution with the volume ratio of 1:1 is added into each reaction tube, the mixture is fully and uniformly shaken, centrifuged at 12000rpm for 1min, and 10 mu L of supernatant is absorbed for agarose gel electrophoresis detection.
Experimental tests prove that a considerable part of 36 groups of RPA constant-temperature amplification primer pairs have poor effects and cannot obtain amplification products, and 6 pairs of primer combinations have good effects and can obtain the amplification products, specifically F1-R3, F2-R2, F2-R3, F4-R6, F6-R1 and F6-R2. Further, the amplification efficiencies of the preliminarily screened 6 primer sets are compared in parallel, and the result is shown in fig. 1, wherein the product band obtained by amplification of the primer set combination F6-R1 is brightest, which indicates that the amplification efficiency is higher, and it is determined that F6 (SEQ ID No. 27) is selected as the upstream primer and R1 (SEQ ID No. 28) is selected as the downstream primer.
Example 2 establishment of CRISPR/Cas detection System
Selecting interspecific polytropic region from ITS region of Malasse, using region containing Cas12a protein recognition sequence as target, designing single-stranded crRNA with length range of 20-24bp and homology with target, wherein the crRNA is shown in SEQ ID NO. 12-SEQ ID NO. 21.
The crRNA and Cas12a protein were incubated in a2 × NEB Buffer at a 2:3 molar ratio for 10 minutes at 25 ℃ to facilitate binding of Cas12a to the crRNA to form a complex. And adding the RPA amplification product, the signal report probe and other reagents of the detection system into the detection system, reacting for 30min at 37 ℃, and reading signals in real time by a real-time fluorescence PCR instrument.
The signal reporting probe in this example was synthesized by Biotechnology engineering (Shanghai) Inc., and the sequence is shown in SEQ ID NO. 34: 5 '-6-FAM-TTTTTATTTTT-BHQ 1-3'; after the Cas12a protein recognizes double-stranded DNA with a targeting sequence under the guidance of crRNA, reverse cleavage activity is activated, and then the DNA with a signal reporter probe is degraded, so that a fluorescent signal group is separated from a quenching group, and a fluorescent signal is released.
The results of the crRNA effect test are shown in FIG. 2, in which the ordinate represents the intensity of fluorescence signal, and finally, crRNA 1-Malasszia furfur, crRNA 1-Malasszia obtusa, crRNA 2-Malasszia globosa, crRNA 1-Malasszia sympodialis, crRNA 2-Malasszia pachyderma are selected as the preferred 5 crRNAs.
Example 3 assay for identifying Malassezia species
Since the crRNA has a precise specific target-targeting effect, and can guide the Cas12a protein to recognize a corresponding target sequence and further activate the nonspecific cleavage activity thereof, 5 crrnas preferably obtained in example 2 are used to establish detection systems for precisely recognizing Malassezia furfur (Malassezia furfur), Malassezia blunted (Malassezia oblisa), Malassezia globosa (Malassezia globosa), Malassezia symptomata (Malassezia sympodialis), and Malassezia pachydermatis (Malassezia pachyderma), respectively.
In this example, the detection system established by the present invention was used to perform the identification and verification experiments of different malassezia species. Specifically, standard genomic DNAs of Malassezia furfur (Malassezia furfur), Malassezia blume obtusia (Malassezia obtusia), Malassezia globosa (Malassezia globosa), Malassezia symptomata (Malassezia sympodialis) and Malassezia pachydermata (Malassezia pachydermatis) are respectively extracted by using an adsorption column method genome extraction kit, CRISPR/Cas detection is carried out, and real-time fluorescent signals are read by using a real-time fluorescent quantitative PCR instrument.
The results of the experiment are shown in FIG. 3. In the differential verification experiment in which the Malassezia furfur sample is dripped, only the reaction system of the crRNA1-Malassezia furfur has a fluorescence signal curve, and the other reaction systems do not detect positive signals, so that the detection system provided by the embodiment 1 can identify and identify the Malassezia furfur and simultaneously can not have cross reaction on other types of Malassezia furfur. In the identification and verification experiment of the dropwise addition of the Malassezia blunt sample, only the reaction system of crRNA1-Malassezia obtusa shows a fluorescence signal curve, and the other reaction systems do not detect positive signals, so that the detection system provided by the embodiment 1 can identify and identify the Malassezia blunt and can not generate cross reaction on other types of Malassezia. In the identification and verification experiment of the added Malassezia globosa sample, only the reaction system of crRNA2-Malassezia globosa has a fluorescence signal curve, and the other reaction systems do not detect positive signals, so that the detection system provided by the example 1 can identify and identify the Malassezia globosa and simultaneously can not generate cross reaction on other types of Malassezia globosa. In the identification and verification experiment added with the Malassezia julibrissin sample, only the reaction system of crRNA1-Malassezia sympodialis shows a fluorescent signal curve, and the other reaction systems do not detect positive signals, so that the detection system provided by the embodiment 1 can identify and identify the Malassezia julibrissin and simultaneously can not generate cross reaction on other types of Malassezia julibrissin. In the identification and verification experiment of the added Malassezia pachydermatis sample, only the reaction system of crRNA2-Malassezia pachydermatis has a fluorescence signal curve, and the other reaction systems do not detect positive signals, so that the detection system provided by the embodiment 1 can identify and identify the Malassezia pachydermatis and simultaneously can not generate cross reaction on other types of Malassezia pachydermatis.
Example 4 specificity test
Specificity testing was performed using the CRISPR/Cas detection system provided in example 2. The genomic DNA templates to be detected are respectively from Candida albicans, Candida tropicalis, Aspergillus flavus, Aspergillus niger, Trichophyton mentagrophytes, Trichophyton rubrum and Sporothrix coccorum standard strains. The genomic DNA of a standard fungus strain is extracted and purified, the obtained products are respectively diluted to 50 ng/mu L, and 2 mu L is taken as a sample after mixing according to the equal volume.
The standard genomic DNA mixture of Candida albicans, Candida tropicalis, Aspergillus flavus, Aspergillus niger, Trichophyton mentagrophytes, Trichophyton rubrum and Sporothrix cocci was subjected to amplification detection according to the detection method provided in example 3, and the specificity of the Malassezia detection kit was tested using the corresponding Malassezia DNA as a positive control.
In this example, the QPCR instrument was used to perform real-time fluorescence signal collection and detection on the reaction system. The experimental result is shown in fig. 4, only the corresponding malassezia positive control DNA can be specifically detected, and other fungi DNA mixtures have no obvious signal, which indicates that the malassezia detection kit constructed by the invention has strong specificity.
Example 5 Malassezia detection kit and detection method
1) Composition of the kit
RPA amplification system: the nucleotide sequences of the upstream primer and the downstream primer are respectively shown as SEQ ID NO.27 and SEQ ID NO. 28; RPA enzyme premix (containing creatine phosphate, creatine kinase, dNTPs, ATP, DTT, potassium acetate, recombinase UvsX, UvsY, single-strand binding protein, Bsu polymerase, etc.); an activator;
CRISPR/Cas detection system: crRNA (nucleotide sequences are shown as SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.17, SEQ ID NO.18 and SEQ ID NO. 21) aiming at five common malassezia;
the signal reporting probe (the nucleotide sequence is shown as SEQ ID NO.34, the 5 'end of the signal reporting probe is marked with 6-FAM, the 3' end of the signal reporting probe is marked with BHQ 1)
Cas12a protein;
2×NEB Buffer；
the positive control is Malassezia furfur, Malassezia blume, Malassezia globosa, and Malassezia pachydermatis standard DNA;
2) kit using method
(1) Extracting DNA of a sample to be detected, and adding 0.01-1ng of the DNA as a template into an RPA amplification system, wherein the RPA amplification system comprises the following components: 20. mu.L of RPA enzyme premix, 2.5. mu.L each of the forward primer at a concentration of 10. mu.M and the reverse primer at a concentration of 10. mu.M, 1. mu.L of template, 2. mu.L of activator, and ddH2O22 mu L, the reaction temperature of the RPA amplification system is 37 ℃, and the amplification time is 20 min.
(2) The obtained RPA amplification product, the crRNA corresponding to a single species of malassezia, the Cas12a protein, the 2 XNEB Buffer, the signal report probe and the like are used for preparing a CRISPR/Cas detection system according to the table 4.
TABLE 4
(3) The CRISPR/Cas detection system is placed in a real-time fluorescence quantitative PCR instrument, the reaction program is set to be 37 ℃, 30s (fluorescence collection), and 30-40 cycles.
(4) If the reaction is positive, the fluorescence signal is obviously enhanced and exceeds a threshold line; if the reaction is negative, the fluorescence signal is not obviously enhanced and is lower than the threshold line.
Example 6 clinical sample testing
Micro-dandruff samples of 20 clinical subjects such as pityriasis versicolor, seborrheic dermatitis and the like are collected, and the samples are from dermatology department of a third hospital affiliated to Zhongshan university. The subject has signed an informed consent.
The DNA of the sample to be tested is extracted by adopting a phenol/chloroform method, the kit provided by the embodiment 5 is used for detection, and the specific experimental method is carried out according to the description of the embodiment 5. The detection results were compared with the dideoxy chain termination method (Sanger) sequencing results of the cultures to be tested, and the rate of agreement was 100% (as shown in Table 5).
TABLE 5 comparison of coincidence rates of the detection method established in the invention and the sequencing results
The results of 1 of the clinical samples were used for the detailed description. The detection result of the clinical sample of the example obtained by the method of the present invention is shown in fig. 5, and the fluorescence reaction is detected in the reaction well of malassezia collina, and the result of the identification is malassezia collina. The microscopic examination of the sample cultures in this example is shown in FIG. 6, which is consistent with the morphological characterization of Malassezia. Meanwhile, the Sanger sequencing result of the sample is shown in SEQ ID NO.35, and the sample is analyzed and determined to be malassezia axyridis. The microscopic examination result and the sequencing result are both consistent with the detection result of the kit, which shows that the malassezia detection kit has higher accuracy.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
SEQUENCE LISTING
<110> Guangdong Huamei Zhongyuan Biotech Co., Ltd, Zhongshan university affiliated third Hospital
<120> detection composition, kit and detection method for malassezia
<130> 2021
<160> 35
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> RNA
<213> Malassezia furfur
<400> 1
gcaagaaaca ggcucgcccg 20
<210> 2
<211> 23
<212> RNA
<213> Malassezia furfur
<400> 2
gcaagaaaca ggcucgcccg aaa 23
<210> 3
<211> 22
<212> RNA
<213> Malassezia obtusa
<400> 3
uugcuaaaca ggcucgcccg aa 22
<210> 4
<211> 21
<212> RNA
<213> Malassezia obtusa
<400> 4
uugcuaaaca ggcucgcccg a 21
<210> 5
<211> 22
<212> RNA
<213> Malassezia globosa
<400> 5
uucucuagaa aaagcucguc cg 22
<210> 6
<211> 23
<212> RNA
<213> Malassezia globosa
<400> 6
agugccguga auucucccau ccc 23
<210> 7
<211> 23
<212> RNA
<213> Malassezia sympodialis
<400> 7
agugccgcga auucucccuc ccc 23
<210> 8
<211> 22
<212> RNA
<213> Malassezia sympodialis
<400> 8
agugccgcga auucucccuc cc 22
<210> 9
<211> 22
<212> RNA
<213> Malassezia pachydermatis
<400> 9
<210> 10
<211> 24
<212> RNA
<213> Malassezia pachydermatis
<400> 10
agugccgcga auucucccac ccca 24
<210> 11
<211> 21
<212> RNA
<213> Artificial sequence
<400> 11
<210> 12
<211> 41
<212> RNA
<213> Malassezia furfur
<400> 12
uaauuucuac uaaguguaga ugcaagaaac aggcucgccc g 41
<210> 13
<211> 44
<212> RNA
<213> Malassezia furfur
<400> 13
uaauuucuac uaaguguaga ugcaagaaac aggcucgccc gaaa 44
<210> 14
<211> 43
<212> RNA
<213> Malassezia obtusa
<400> 14
uaauuucuac uaaguguaga uuugcuaaac aggcucgccc gaa 43
<210> 15
<211> 42
<212> RNA
<213> Malassezia obtusa
<400> 15
uaauuucuac uaaguguaga uuugcuaaac aggcucgccc ga 42
<210> 16
<211> 43
<212> RNA
<213> Malassezia globosa
<400> 16
uaauuucuac uaaguguaga uuucucuaga aaaagcucgu ccg 43
<210> 17
<211> 44
<212> RNA
<213> Malassezia globosa
<400> 17
uaauuucuac uaaguguaga uagugccgug aauucuccca uccc 44
<210> 18
<211> 44
<212> RNA
<213> Malassezia sympodialis
<400> 18
uaauuucuac uaaguguaga uagugccgcg aauucucccu cccc 44
<210> 19
<211> 43
<212> RNA
<213> Malassezia sympodialis
<400> 19
uaauuucuac uaaguguaga uagugccgcg aauucucccu ccc 43
<210> 20
<211> 43
<212> RNA
<213> Malassezia pachydermatis
<400> 20
uaauuucuac uaaguguaga uagugccgcg aauucuccca ccc 43
<210> 21
<211> 45
<212> RNA
<213> Malassezia pachydermatis
<400> 21
uaauuucuac uaaguguaga uagugccgcg aauucuccca cccca 45
<210> 22
<211> 31
<212> DNA
<213> Artificial sequence
<400> 22
cgtgaatcat cgaatctttg aacgcacctt g 31
<210> 23
<211> 32
<212> DNA
<213> Artificial sequence
<400> 23
ccgtgaatca tcgaatcttt gaacgcacct tg 32
<210> 24
<211> 32
<212> DNA
<213> Artificial sequence
<400> 24
aattccgtga atcatcgaat ctttgaacgc ac 32
<210> 25
<211> 30
<212> DNA
<213> Artificial sequence
<400> 25
tccgtgaatc atcgaatctt tgaacgcacc 30
<210> 26
<211> 32
<212> DNA
<213> Artificial sequence
<400> 26
attccgtgaa tcatcgaatc tttgaacgca cc 32
<210> 27
<211> 32
<212> DNA
<213> Artificial sequence
<400> 27
gcagaattcc gtgaatcatc gaatctttga ac 32
<210> 28
<211> 30
<212> DNA
<213> Artificial sequence
<400> 28
acacacagca aatgacgtat catgccatgc 30
<210> 29
<211> 31
<212> DNA
<213> Artificial sequence
<400> 29
cacacatagc aaatgacgta tcatgccatg c 31
<210> 30
<211> 30
<212> DNA
<213> Artificial sequence
<400> 30
acacatagca aatgacgtat catgccatgc 30
<210> 31
<211> 30
<212> DNA
<213> Artificial sequence
<400> 31
cacactcagc aaatgacgta tcatgccatg 30
<210> 32
<211> 30
<212> DNA
<213> Artificial sequence
<400> 32
acacatagca aatgacgtat catgccatgc 30
<210> 33
<211> 30
<212> DNA
<213> Artificial sequence
<400> 33
acacatagca aatgacgtat catgccatgc 30
<210> 34
<211> 11
<212> DNA
<213> Artificial sequence
<400> 34
tttttatttt t 11
<210> 35
<211> 642
<212> DNA
<213> Malassezia sympodialis
<400> 35
gaaaggcagg ggactgcaga ggatcattag tgaagtttcg ggcctgccat acggacgcaa 60
acacgtctct ggcgcccatc actatatcca taccaacccc tgtgcactgt gatgacgaat 120
gtcatcgaac aaaaaaaact cgtatggttg aatgtacgtg aaattgtagg tatagcctac 180
gaactataca caactttcga caacggatct cttggttctc ccatcgatga agaacgcagc 240
gaaacgcgat aggtaatgtg aattgcagaa ttccgtgaat catcgaatct ttgaacgcac 300
cttgcgctcc atggtattcc gtggagcatg cctgtttgag tgccgcgaat tctccctccc 360
cttacggtgg ccgaaaggcc gaagtagggc ggacggggta ggatgggtgt tgctgcctgg 420
ggattgtacc aggctcgccc gaaatgcata agcgccagga ccctcgctac cgctctctag 480
ggaagagtgg ctaagcgacc gctgagcatg gcatgatacg tcatttgctg tgtgtgggcg 540
gccggttgga gaggtgtctg ctttaccagc ccttttttaa ttctggtctc aaatcaggta 600
ggatcacccg ctgaacttaa gcatatcaaa agccgggagg aa 642
Claims (7)
1. A kit is characterized by comprising a CRISPR/Cas detection system and an RPA amplification system, wherein the CRISPR/Cas detection system comprises a crRNA, a Cas protein and a signal report probe, the crRNA comprises an anchor sequence fragment and a guide sequence fragment, the nucleotide sequence of the guide sequence fragment is shown as at least one of SEQ ID NO.01, SEQ ID NO.03, SEQ ID NO.06, SEQ ID NO.07 and SEQ ID NO.10, and the nucleotide sequence of the anchor sequence fragment is shown as SEQ ID NO. 11; the anchor sequence segment specifically binds to the Cas protein; the sequence of the signal report probe is shown in SEQ ID NO.34, a fluorescent report group is marked at the 5 'end, and a fluorescent quenching group is marked at the 3' end; the RPA amplification system comprises an upstream primer segment and a downstream primer segment, wherein the nucleotide sequence of the upstream primer segment is shown as SEQ ID NO.27, and the nucleotide sequence of the downstream primer segment is shown as SEQ ID NO. 28.
2. The kit of claim 1, wherein the fluorescent reporter group is selected from one of FAM, VIC, HEX, ROX, JOE, Cy3, and Cy 5.
3. The kit according to claim 1, wherein the fluorescence quenching group is one selected from TAMRA, BHQ-1, BHQ-2 and BHQ-3.
4. The kit of claim 1, wherein the CRISPR/Cas detection system further comprises at least one of NEB Buffer, magnesium acetate activator, creatine phosphate, creatine kinase, dNTPs, ATP, DTT, potassium acetate, recombinase UvsX, recombinase UvsY, single-strand binding protein, or Bsu polymerase.
5. The kit of claim 1, wherein the RPA amplification system further comprises at least one of an RPA enzyme premix or an activator.
6. The kit according to claim 1, wherein the reaction temperature of the RPA amplification system is 37-42 ℃.
7. Use of a kit according to any one of claims 1 to 6 for the identification of malassezia species for non-disease diagnostic purposes.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105483278A (en) * | 2016-02-04 | 2016-04-13 | 中国人民解放军第二军医大学 | Histoplasma capsulatum infectious molecular diagnosis reagent kit based on recombinase and polymerase amplification technological principle and application thereof |
| CN109295054A (en) * | 2017-07-25 | 2019-02-01 | 广州普世利华科技有限公司 | The detection method of gRNA for pathogen targeting gene RNA and the pathogen gene based on C2c2, detection kit |
| CN111363842A (en) * | 2020-04-13 | 2020-07-03 | 广州医科大学附属第一医院 | Sequence, kit, method and application for rapidly detecting aspergillus fumigatus |
| CN111394490A (en) * | 2020-05-15 | 2020-07-10 | 中国人民解放军军事科学院军事医学研究院 | CRISPR-Cas12a detection primer group for eupolyphaga and application thereof |
| CN113122648A (en) * | 2021-03-30 | 2021-07-16 | 中国检验检疫科学研究院 | Rapid detection kit for pathogenic bacteria of rape black leg disease and use method thereof |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016138337A1 (en) * | 2015-02-27 | 2016-09-01 | Nawana Namal | Microbiome diagnostics |
| EP3728613A4 (en) * | 2017-12-22 | 2022-01-12 | The Broad Institute, Inc. | Crispr effector system based multiplex diagnostics |
| CN112522429B (en) * | 2020-12-16 | 2021-07-16 | 中国人民解放军军事科学院军事医学研究院 | Method and reagent set for detecting bacillus anthracis by RPA (reverse transcriptase polymerase chain reaction) combined CRISPR (clustered regularly interspaced short palindromic repeats) technology |
| CN112522434B (en) * | 2020-12-24 | 2023-02-03 | 中山大学附属第三医院 | Primer group and kit for simultaneously detecting multiple pathogenic fungi |
-
2022
- 2022-01-17 CN CN202210046149.8A patent/CN114075564B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105483278A (en) * | 2016-02-04 | 2016-04-13 | 中国人民解放军第二军医大学 | Histoplasma capsulatum infectious molecular diagnosis reagent kit based on recombinase and polymerase amplification technological principle and application thereof |
| CN109295054A (en) * | 2017-07-25 | 2019-02-01 | 广州普世利华科技有限公司 | The detection method of gRNA for pathogen targeting gene RNA and the pathogen gene based on C2c2, detection kit |
| CN111363842A (en) * | 2020-04-13 | 2020-07-03 | 广州医科大学附属第一医院 | Sequence, kit, method and application for rapidly detecting aspergillus fumigatus |
| CN111394490A (en) * | 2020-05-15 | 2020-07-10 | 中国人民解放军军事科学院军事医学研究院 | CRISPR-Cas12a detection primer group for eupolyphaga and application thereof |
| CN113122648A (en) * | 2021-03-30 | 2021-07-16 | 中国检验检疫科学研究院 | Rapid detection kit for pathogenic bacteria of rape black leg disease and use method thereof |
Non-Patent Citations (1)
| Title |
|---|
| CRISPR/Cas9技术在微生物研究中的应用进展;张昆等;《微生物学通报》;20170808(第02期);第451-464页 * |
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