CN110055347A - A method of identifying five kinds of dermatophytes using high-resolution fusion curve - Google Patents
A method of identifying five kinds of dermatophytes using high-resolution fusion curve Download PDFInfo
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- CN110055347A CN110055347A CN201910298852.6A CN201910298852A CN110055347A CN 110055347 A CN110055347 A CN 110055347A CN 201910298852 A CN201910298852 A CN 201910298852A CN 110055347 A CN110055347 A CN 110055347A
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Abstract
The present invention provides a kind of methods for identifying five kinds of dermatophytes using high-resolution fusion curve, belong to high-resolution fusion curve analysis technical field.The method of the present invention uses 1 pair of specific primer, identifies Trichophyton rubrum, trichophyton interdigitale, acrothesium floccosum, microsporum canis and Microsporum incurvatum based on real-time fluorescence PCR (Real-time PCR) melting curve.The sequence of 1 pair of specific primer is as shown in SEQ ID NO.1-2.High sensitivity of the present invention, specificity are good, it is fast to detect speed, it can be used for the identification of clinical diagnosis, environmental monitoring, food safety etc. to dermatophyte, so that the monitoring for the treatment of infection of dermatophyte, environmental sanitation and food safety provides infallible foundation.
Description
Technical field
The present invention relates to the applications of high-resolution fusion curve analysis method, and in particular to a kind of to utilize high-resolution fusion curve
Identify the method for five kinds of dermatophytes, five kinds of dermatophytes are Trichophyton rubrum (T.rubrum), hair tinea between toe respectively
Bacterium (T.interdigitale), acrothesium floccosum (E.floccosum), microsporum canis (M.canis) and
Microsporum incurvatum。
Background technique
Superficial mycoses refers to by pathomycete or opportunistic fungus infection application on human skin, refers to the superficials such as (toe) first or hair
Disease caused by organizing, is the common disease in Dermatology Outpatient Department, frequently-occurring disease.It is reported that illness rate of the disease in crowd can be high
Up to 20%-25%, and disease incidence is still in rise year by year trend.And dermatophyte is the most common cause of disease in mycotic infection of superficial part
Bacterium is the pathogenic epiphyte of a kind of thermophilic cutin, parasitic or saprophytic in the keratin tissue at these positions, causes ringworm of the body, jock itch
With the dermatophytosis such as tinea of feet and hands, it is detrimental to health, influences its normal life.Therefore, it is necessary to fast and accurately identify skin
The strain of skin tinea bacterium provides foundation for clinical precisely medication.
Dermatophyte is generally divided into 3 categories: trichophyton, Epidermophyton and Microsporon.Traditional skin tinea
The dientification of bacteria depends on colonial morphology, Arthroscopic characteristic (the mainly morphological feature of macroconidium), and physiology, biochemical isophenous are special
Sign.But Morphological Identification time and effort consuming, and it is more demanding to staff's operating technology.With the rapid hair of molecular biology
Exhibition, more and more molecular diagnostics technologies are applied to the quick diagnosis of dermatophyte.Chitin is a kind of polysaccharide of threadiness,
Constitute the cell wall of many fungies including dermatophyte.Chitin synthetase (CHS) gene is in a variety of fungies (such as white
Candida albicans, aspergillus nidulans etc.) in expressed, by compare sequence find the gene region have it is well-conserved, be suitable for
Phylogenetic Analysis in the Molecular Identification and category of fungal species between species or between the obvious flora of intraspecies variation.Therefore,
By using being sequenced after primer pair amplifies CHS gene, basic Local Alignment is carried out in NCBI, bacterial strain identification can be carried out,
But this method is difficult to carry out all bacterial strains precise Identification, and exists without amplification phenomenon.In addition, common technology also has polymerization
Enzyme chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP), i.e., after carrying out PCR amplification for a certain genetic fragment,
Digestion is carried out to PCR product with suitable restriction enzyme, according to the length polymorphism of digestion products, to carry out strain mirror
It is fixed, but the experimentation of this method is complicated, is unfavorable for promoting；Or Analysis of random amplified polymorphic DNA (RAPD) is used, although
Have the advantages that required amount of DNA is small, sensibility is high, easy to operate rapid, low etc. convenient for large-scale use and expense, but its repeatability
It is often restricted by reaction condition with specificity, the change of reaction condition often influences the accuracy of testing result.
High-resolution fusion curve (High resolution melting, HRM) analytical technology, principle is by real-time
The combination situation of temperature-rise period double center chain DNA fluorescent dye and PCR product is monitored, the difference of the single base of DNA fragmentation can all make
Melting temperature (hereinafter referred to as Tm) changes, so that DNA fragmentation is made to form different melting curves in heating dehybridization procedure,
And then distinguish different strain.HRM is not limited to by mutating alkali yl site and type, is not necessarily to sequence-specific probes, with other heredity
Typing method is compared, and has many advantages, such as easy to operate, high sensitivity, good, at low cost, quick, the high-throughput detection of specificity, and tie
Fruit is accurate, realizes real stopped pipe operation.
Summary of the invention
The purpose of the invention is to make up the deficiencies in the prior art, provide a kind of based on RT-PCR melting curve analysis
Identify the method for five kinds of dermatophytes, it is especially a kind of to identify Trichophyton rubrum using high-resolution fusion curve analytical technology
(T.rubrum), trichophyton interdigitale (T.interdigitale), acrothesium floccosum (E.floccosum), microsporum canis
(M.canis) and the method for Microsporum incurvatum.
Trichophyton rubrum can be identified simultaneously by being suitable for high-resolution fusion curve another object of the present invention is to provide one kind
(T.rubrum), trichophyton interdigitale (T.interdigitale), acrothesium floccosum (E.floccosum), microsporum canis
(M.canis) and a specific primer pair of Microsporum incurvatum.
Present invention application high-resolution fusion curve analytical technology.It is contaminated by real-time monitoring temperature-rise period double center chain DNA fluorescence
The combination situation of material and PCR product, CHS gene order difference can make the Tm value of double-stranded DNA change, so that double-stranded DNA exists
Successive unwinding in temperature-rise period forms different melting curve shapes.Fluorescent dye is discharged from the DNA molecular of local unwinding,
Discrepant CHS genetic fragment, and the difference of CHS gene order can be judged whether there is from fluorescence intensity and time graph
It will affect the peak shape of melting curve, can effectively distinguish the CHS gene of different strain.
In order to achieve the object of the present invention, the present invention has carefully studied Trichophyton rubrum (T.rubrum), trichophyton interdigitale
(T.interdigitale), acrothesium floccosum (E.floccosum), microsporum canis (M.canis) and Microsporum
The genome sequence of this five kinds of dermatophytes of incurvatum is found otherness target sequence in CHS gene order, is determined above-mentioned
The otherness target sequence of five kinds of dermatophytes is respectively as shown in SEQ ID NO.3-7.By well-designed and screening, design is a pair of
Specific primer can specifically identify above-mentioned five kinds of dermatophytes by HRM technology to primer using this.
Present invention firstly provides the specificity that can identify above-mentioned five kinds of dermatophytes suitable for high-resolution fusion curve to draw
Object pair, nucleotide sequence is as shown in SEQ ID NO.1-2.
The present invention provides the kits or detection reagent that contain above-mentioned specific primer pair.
The present invention provides the specific primers in identification Trichophyton rubrum (T.rubrum), trichophyton interdigitale
(T.interdigitale), acrothesium floccosum (E.floccosum), microsporum canis (M.canis) and Microsporum
Application in incurvatum.
The present invention provides the specific primers to identify Trichophyton rubrum (T.rubrum) in preparation, trichophyton interdigitale
(T.interdigitale), acrothesium floccosum (E.floccosum), microsporum canis (M.canis) and Microsporum
The kit of incurvatum or the application in detection reagent.
The present invention provides above-mentioned specific primer ensure food safety or environmental health monitoring and surveilliance in application.
The present invention provides a kind of identification Trichophyton rubrum (T.rubrum), trichophyton interdigitale (T.interdigitale), wadding
Shape Epidermophyton (E.floccosum), the method for microsporum canis (M.canis) and Microsporum incurvatum are
Using the above-mentioned specific primer that designs of the present invention, using the DNA of sample to be tested as template, using Real-time PCR method into
Row detection determines result according to the peak shape for melting melting curve in peak value figure.
30 μ L PCR reaction system in above-mentioned Real-time PCR method are as follows: 2 × Mix Taqman PCR Master 15
Each 0.9 μ L of μ L, upstream and downstream primer, 0.3 μ L of Rox Reference Dye II (100 ×), Evagreen, 20 × in Water
1.5 μ L, 2 μ L of DNA profiling to be measured supply system to 30 μ L with sterilizing pure water.
Real-time PCR reaction condition are as follows: initial denaturation: 95 DEG C of 10min；95 DEG C of 15s, 65 DEG C of 45s, 35 circulations, rise
1.6 DEG C/s of cooling rate.
The melting curve manufacturing conditions of the above method of the present invention are as follows: 95 DEG C of 10s, 60 DEG C of 1min, (0.025 DEG C/s is warming up to)
95 DEG C of 15s, 60 DEG C of 15s, with 1.6 DEG C/s rate heating and cooling, while continuous fluorescence intensity；With fluorescence signal to the one of temperature
The negative derivative of rank is ordinate, and temperature is abscissa, obtains melting peak value figure.
Specifically, the judgment principle of the method for the present invention identification result is the melting peakss of each specific amplification products Tm value
A kind of specific dermatophyte is represented,
Wherein, there are melting peakss within the scope of 82-85 DEG C for Trichophyton rubrum (T.rubrum) detection in Tm value；
There are melting peakss within the scope of 83-86 DEG C for trichophyton interdigitale (T.interdigitale) detection in Tm value；
There are melting peakss within the scope of 84-87 DEG C for acrothesium floccosum (E.floccosum) detection in Tm value；
There are melting peakss within the scope of 88-90 DEG C for microsporum canis (M.canis) detection in Tm value；
There are melting peakss within the scope of 86-88 DEG C for Microsporum incurvatum detection in Tm value.
The present invention also provides the target sequence combination for identifying five kinds of dermatophytes, five kinds of dermatophytes are respectively
Trichophyton rubrum (T.rubrum), trichophyton interdigitale (T.interdigitale), acrothesium floccosum (E.floccosum), dog
Sporidiole bacteria (M.canis) and Microsporum incurvatum, for identifying the target sequence of Trichophyton rubrum (T.rubrum)
Column contain the nucleotide sequence as shown in SEQ ID NO.3；
For identifying that the target sequence of trichophyton interdigitale (T.interdigitale) contains the core as shown in SEQ ID NO.4
Nucleotide sequence；
For identifying that the target sequence of acrothesium floccosum (E.floccosum) contains the nucleosides as shown in SEQ ID NO.5
Acid sequence；
For identifying that the target sequence of microsporum canis (M.canis) contains the nucleotide sequence as shown in SEQ ID NO.6；
Target sequence for identification of M icrosporum incurvatum contains the nucleotides sequence as shown in SEQ ID NO.7
Column.
Specifically, the present invention provides for identifying the target sequence of Trichophyton rubrum (T.rubrum), contain such as SEQ
Nucleotide sequence shown in ID NO.3.
The present invention provides for identifying the target sequence of trichophyton interdigitale (T.interdigitale), contain such as SEQ
Nucleotide sequence shown in ID NO.4；
The present invention provides for identifying the target sequence of acrothesium floccosum (E.floccosum), contain such as SEQ ID
Nucleotide sequence shown in NO.5；
The present invention provides for identifying the target sequence of microsporum canis (M.canis), contain such as SEQ ID NO.6 institute
The nucleotide sequence shown；
The present invention provides the target sequence for identification of M icrosporum incurvatum, contain such as SEQ ID
Nucleotide sequence shown in NO.7.
The present invention provides application of the above method in environmental health monitoring and surveilliance, food safety monitoring.
Real-time PCR melting curve analysis method of the invention is detected using totally-enclosed reaction tube and interpretation of result,
Without carrying out subsequent PCR product electrophoresis detection and analysis, streamline operation reduces and tests testing cost, raising detection efficiency,
Avoid the cross contamination of sample and environment.Method of the invention simultaneously has good specificity, guarantees that this method can be special
The detection Trichophyton rubrum (T.rubrum) of property, trichophyton interdigitale (T.interdigitale), acrothesium floccosum
(E.floccosum), microsporum canis (M.canis) and Microsporum incurvatum, avoid false positive.
HRM analysis method of the invention sensitivity also with higher is 20- to above-mentioned five kinds of dermatophyte Monitoring lower-cuts
100 copies, have very highly sensitive, and standard peak shape is distinguished obvious, can effectively distinguish the CHS sequence of aforementioned different strain.This
Invention is not necessarily to design specific probe using HRM analysis method, easy to operate compared with other identify the method for strains, has
The advantages that high sensitivity, specific good, at low cost, quick, high-throughput detection, realizes stopped pipe without carrying out electrophoresis after PCR amplification
Operation, prevents cross contamination.The method of the present invention can be used for clinical diagnosis, environmental health monitoring and surveilliance, food safety etc. to above-mentioned
The identification of five kinds of fungies, thus for the treatment of infection of dermatophyte, environmental sanitation and food safety monitoring provide it is infallible
Foundation.
Detailed description of the invention
Fig. 1 is the high-resolution melting peak value map that the present invention identifies five kinds of dermatophytes.
Fig. 2 is that the method for the present invention limits result figure to the detection of five kinds of dermatophyte DNA profilings.Curve is by left-to-right point in figure
Not Dui Ying, 5 × 106Copy, 5 × 105Copy, 5 × 104Copy, 5 × 103Copy, 5 × 102Copy, 5 × 10 copies.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Without departing substantially from spirit of that invention
In the case where essence, to modifications or substitutions made by the method for the present invention, step or condition, all belong to the scope of the present invention.
Unless otherwise specified, chemical reagent used in embodiment is conventional commercial reagent, skill used in embodiment
The conventional means that art means are well known to those skilled in the art.
Embodiment 1 identifies the foundation and specificity, sensitivity verifying of five kinds of dermatophyte methods
The present invention has carefully studied Trichophyton rubrum (T.rubrum), trichophyton interdigitale (T.interdigitale), cotton-shaped
This five kinds of skin tineas of Epidermophyton (E.floccosum), microsporum canis (M.canis) and Microsporum incurvatum
The genome sequence of bacterium finds otherness target sequence in CHS sequence, by well-designed and screening, designs a pair of of specificity
Primer, on nucleotide sequence can specifically be identified primer by HRM technology as shown in SEQ ID NO.1-2, using this
State five kinds of dermatophytes.The design of the primer can not be automatically derived by computer software, needs to choose according to experience abundant
Target sequence segment and length calculate Tm value according to following formula while considering sequence G/C content.It is carried out after synthetic primer anti-
Retrial is tested, and a pair of best primer that can distinguish above-mentioned five kinds of dermatophytes is obtained.
Identify five kinds of dermatophytes using high-resolution fusion curve analytical technology, its step are as follows:
1. extract the genomic DNA of fungi sample to be measured using thick formulation, running gel imaging method is to the concentration of DNA and pure
Degree detects.
2. comparing five kinds of dermatophytes (Trichophyton rubrum (T.rubrum), the trichophyton interdigitale identified
(T.interdigitale), acrothesium floccosum (E.floccosum), microsporum canis (M.canis) and Microsporum
Incurvatum) the sequence (sequence is shown in SEQ ID NO.3-7 respectively) in the area CHS, using primer shown in SEQ ID NO.1-2 into
Row PCR amplification, PCR product length about 200bp, primer information are as follows:
F 5 '-TCGCGCACCAGCAGCAAGAC-3 ', (SEQ ID NO.1)
R 5’-CCTTGACCTCCATGCCTATCT-3’(SEQ ID NO.2)
3. preparing reaction system.2 × Mix Taqman PCR is added according to following table in each PCR reacting hole
Master 15ul, 0.9 μ l of primers F, 0.9 μ l of primer R (primer concentration is 10 μM), Rox Reference Dye II (100
×) 0.3ul, Evagreen, 20 × in Water1.5 μ l, 2 μ l of DNA profiling to be measured supply system to 30 μ l with sterilizing pure water.
4. being reacted on ABI QuantStudio 6flex, PCR response procedures such as table 1:
Table 1
5. carrying out interpretation of result using ABI software, curve offset is based on by standard curve and curve shape variation shows
The CHS difference of different skin tinea strain.Its standard peak shape is distinguished obviously, as shown in Figure 1.Tm value occurs within the scope of 82-85 DEG C
Melting peakss are Trichophyton rubrum (T.rubrum) detection；It is trichophyton interdigitale that melting peakss occurs within the scope of 83-86 DEG C in Tm value
(T.interdigitale) it detects；It is acrothesium floccosum that melting peakss occurs within the scope of 84-87 DEG C in Tm value
(E.floccosum) it detects；There are melting peakss within the scope of 88-90 DEG C for microsporum canis (M.canis) detection in Tm value；Tm value
There are melting peakss within the scope of 86-88 DEG C for Microsporum incurvatum detection.
6.HRM sensitivity verifying
(Trichophyton rubrum, trichophyton interdigitale, acrothesium floccosum, dog are small for five kinds of dermatophytes being serially diluted with 10 times
Pityrosporion ovale and Microsporum incurvatum) standard DNA be template carry out HRM amplification, analyze its standard peak shape.As a result
Show that this method copies the Monitoring lower-cut 20-100 of five kinds of dermatophyte DNA profilings, there is very high sensitivity, see Fig. 2.
7.HRM specificity verification
Using the present invention simultaneously to a variety of fungies (Trichophyton rubrum, Trichophyton
Interdigitale, Epidermophyton floccosum, Microsporum canis, Microsporum
Incurvatum, Talaromyces funiculosus, Curvularia hominis, Aspergillus sydowii,
Meyerozyma caribbica, Kurtzmaniella cleridarum, Pichia kluyveri, Wickerhamomyces
Anomalus, Trichosporon asahii, Cladosporium sphaerospermum, Microascus murinus,
Hortaea werneckii, Fusarium oxysporum, Candida albicans) it is identified.
Use high-resolution fusion curve analysis method and PCR sequencing PCR simultaneously, to the bacterial strain from the 10 different provinces and cities in the whole nation into
Row identification.By strain culturing, DNA extract and etc. after, with high-resolution fusion curve analytical technology referring to the present embodiment it is aforementioned
Method is identified, while using PCR sequencing PCR, uses Claudia Cafarchia (Molecular characterization
of selected dermatophytes and their identification by electrophoretic
Mutation scanning) region used primer pair targeted fungal bacterial strain CHS accurately expands, surveyed after obtaining PCR product
Sequencing result is compared in NCBI, obtains its strain by sequence.The qualification result of two methods is compared.As a result it shows
Show, utilizes sequencing side after the method and conventional PCR amplification of high-resolution fusion curve five kinds of dermatophytes of identification of the invention established
Five kinds of dermatophyte bacterium that method identifies can correspond；Rather than dermatophyte expands not successfully.Show the method for the present invention
It can differentiation identification that is special, being efficiently used for five kinds of dermatophytes.
The application of 2 the method for the present invention of embodiment
1. patient's sample of the medical dermatophytosis of 100 hospital dermatology departments in the 10 different provinces and cities in the whole nation is chosen,
Using the potato dextrose agar base (PDA) for containing 0.5% chloramphenicol, it is separately cultured under 28 DEG C of constant temperatures, to training
The sample of establishing the yang function property is identified.
2. extracting the genomic DNA of fungi sample to be measured using thick formulation, operation of the subsequent identification method referring to embodiment 1
Method carries out.Interpretation of result is carried out using ABI software, curve offset is based on by standard curve and curve shape variation shows not
With the CHS difference of dermatophyte.Qualification result such as the following table 2.
Table 2
The sample of corresponding dermatophyte is identified for use 1 method of embodiment, then uses PCR same as Example 1
Sequencing approach detects the sample respectively after amplification, determines that two methods testing result is consistent.It can be seen that the method for the present invention has
Good accuracy.
The sample not expanded is shown for testing result in table 2, is distinguished using PCR sequencing approach same as Example 1
The sample is detected, result is not Trichophyton rubrum (T.rubrum), trichophyton interdigitale (T.interdigitale), cotton-shaped table
This five kinds of dermatophytes of dermatophytes (E.floccosum), microsporum canis (M.canis) and Microsporum incurvatum
Any one of, it is seen that the method specificity that the present invention identifies five kinds of dermatophytes is high, no false negative or non-false positive.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>Jinhua central hospital
<120>a kind of method for identifying five kinds of dermatophytes using high-resolution fusion curve
<130> KHP191111398.4
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
tcgcgcacca gcagcaagac 20
<210> 2
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ccttgacctc catgcctatc t 21
<210> 3
<211> 414
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
gggtccagat ggggcaggag gcatggagag attgtcgtct gcatcgtctc agacggccgt 60
ggaaagatca atccacgcac cagagctgtc ctggctggtc tcggtgtcta tcaggacggc 120
attgccaaac agcaggtcaa cggcaaagac gtcactgccc acatctacga gtataccacc 180
cagataggca tggaggtcaa ggggacccaa gtcctcctca agccccggcc agggatgcca 240
gtccagctcc tcttctgtct caaggagaag aaccagaaga agatcaactc ccacagatgg 300
ttcttccagg cctttggccg tgtcctcgac cccaacatct gcgtactcct cgacgccgga 360
acaaaaccag gcaggcagag catataccag ctctggcgtg ccttttgacc tcga 414
<210> 4
<211> 419
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
atgacgctag actggggcag gaggcctgga gagatcgtcg tctgcatcgt ctccgacggc 60
cgtgcaaaaa tcaatccgcg cacgagagcc gtgctggccg ggctgggggt ctaccaggac 120
ggcattgcca aacagcaggt caacggcaag gacgtcaccg cccacatcta cgagtacacc 180
acccagatcg gcatggaggt caagggcacc caggtcatcc tcaagcctcg gccgggcatg 240
cccgtccagc tcctcttctg cctcaaggag aagaaccaga agaagatcaa ctcgcaccgg 300
tggttcttcc aggccttcgg ccgcatcctc gacccaaaca tctgcgtcct cctcgacgcc 360
gggacaaagc cggggaagca gagcatatac cagctctggc gtgcctttgg acctcgatg 419
<210> 5
<211> 420
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
gatgctacga ctgcggcagg agcctggaag aagattgtcg tctgtatcgt ctcagacggc 60
cgtgcaaaga tcaatccacg cacgagagct gtccttgccg gtctgggtgt ctatcaagat 120
ggtattgcca aacagcaggt caacggtaaa gacgtcaccg ctcacatcta cgagtacacc 180
acccagatag gcatggaggt taagggcacc caggtcgttc tcaagccccg gccgggaatg 240
ccagtacaac tccttttctg tctcaaggag aagaaccaga agaagatcaa ctctcacaga 300
tggttcttcc aggccttcgg ccgtgtcctc gaccccaaca tctgtgtcct cctcgacgcc 360
ggaacaaagc caggcaggca gagtatatac caactctggc gtgccttttt aacctcgaat 420
<210> 6
<211> 411
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<213>artificial sequence (Artificial Sequence)
<400> 6
gcagactggg gtagagcctg gaagaagatt gtcgtttgta tcgtctcaga cggtcgtgca 60
aagataaatc cgcgtacgag agctgtcctt gccggtctag gcgtttacca agatggcatt 120
gccaaacagc aggtcaacgg taaagacgtc actgcgcaca tctacgaata taccacccag 180
ataggcatgg aggtcaaggg cacccaggtt attctcaagc cgcggccggg aatgccggtc 240
cagctcctct tctgtcttaa agagaagaac cagaagaaga tcaactctca cagatggttc 300
ttccaagcct ttggtcgcgt cctcgacccc aatatctgtg ttctcctcga cgcgggaaca 360
aaaccaggcg gccgaagtat ataccaactc tggcgtgcct tttgacctcg a 411
<210> 7
<211> 412
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
gagtctagac tggggtagag cctggagagt tgtcgtttgt attgtctcag acggtcgtgc 60
aaagataaac ccacgtacaa gagctgtcct tgccggtcta ggtgtttacc aagatggcat 120
tgctaagcag caggttaacg gtaaagacgt cactgctcac atctacgaat ataccaccca 180
gataggcatg gaggtcaagg gcacccaggt tatcctcaag ccgcggccag gaatgccagt 240
ccagcttctc ttctgtctta aagagaagaa tcagaaaaag atcaactctc acagatggtt 300
cttccaagcc tttggccgtg tcctcgaccc caatatctgt gttctcctcg atgctggaac 360
aaaaccaggc gggcgaagta tataccagct ctggcgtgcc ttttgacctc ga 412
Claims (10)
1. being suitable for the specific primer pair that high-resolution fusion curve identifies five kinds of dermatophytes, five kinds of dermatophytes difference
For Trichophyton rubrum (T.rubrum), trichophyton interdigitale (T.interdigitale), acrothesium floccosum (E.floccosum),
Microsporum canis (M.canis) and Microsporum incurvatum, which is characterized in that the nucleotide of the specific primer
Sequence contains the sequence as shown in SEQ ID NO.1-2 or its specific sequence.
2. kit or detection reagent containing specific primer pair described in claim 1.
3. specific primer described in claim 1 identifies Trichophyton rubrum (T.rubrum) in preparation, trichophyton interdigitale
(T.interdigitale), acrothesium floccosum (E.floccosum), microsporum canis (M.canis) and Microsporum
The kit of incurvatum or the application in detection reagent.
4. specific primer described in claim 1 to ensure food safety or environmental health monitoring and surveilliance in application.
5. a kind of identification Trichophyton rubrum (T.rubrum), trichophyton interdigitale (T.interdigitale), acrothesium floccosum
(E.floccosum), the method for microsporum canis (M.canis) and Microsporum incurvatum, which is characterized in that answer
It is carried out using the DNA of sample to be tested as template using Real-time PCR method with specific primer pair described in claim 1
Detection determines result according to the peak shape for melting melting curve in peak value figure.
6. method as claimed in claim 5, which is characterized in that 30 μ L PCR reaction system in Real-time PCR method are as follows:
2 × Mix Taqman PCR Master, 15 μ L, each 0.9 μ L of upstream and downstream primer, 100 × Rox Reference Dye II 0.3
μ L, Evagreen, 20 × in Water1.5 μ L, 2 μ L of DNA profiling to be measured supply system to 30 μ L with sterilizing pure water.
7. method as claimed in claim 5, which is characterized in that Real-time PCR reaction condition are as follows: initial denaturation: 95 DEG C
10min；95 DEG C of 15s, 65 DEG C of 45s, 35 circulations, 1.6 DEG C/s of warming and cooling rate.
8. method as claimed in claim 5, which is characterized in that melting curve manufacturing conditions are as follows: 95 DEG C of 10s, 60 DEG C of 1min,
0.025 DEG C/s is warming up to 95 DEG C of 15s, 60 DEG C of 15s, with 1.6 DEG C/s rate heating and cooling, while continuous fluorescence intensity；With glimmering
Optical signal is ordinate to the negative derivative of the single order of temperature, and temperature is abscissa, obtains melting peak value figure.
9. the method as described in claim 5-8 is any, which is characterized in that the melting peakss of each specific amplification products Tm value
A kind of specific dermatophyte is represented,
Wherein, there are melting peakss within the scope of 82-85 DEG C for Trichophyton rubrum (T.rubrum) detection in Tm value；
There are melting peakss within the scope of 83-85 DEG C for trichophyton interdigitale (T.interdigitale) detection in Tm value；
There are melting peakss within the scope of 84-87 DEG C for acrothesium floccosum (E.floccosum) detection in Tm value；
There are melting peakss within the scope of 88-90 DEG C for microsporum canis (M.canis) detection in Tm value；
There are melting peakss within the scope of 86-88 DEG C for Microsporum incurvatum detection in Tm value.
10. the target sequence combination for identifying five kinds of dermatophytes, five kinds of dermatophytes are respectively Trichophyton rubrum
(T.rubrum), trichophyton interdigitale (T.interdigitale), acrothesium floccosum (E.floccosum), microsporum canis
(M.canis) and Microsporum incurvatum, which is characterized in that
For identifying that the target sequence of Trichophyton rubrum (T.rubrum) contains the nucleotide sequence as shown in SEQ ID NO.3；
For identifying that the target sequence of trichophyton interdigitale (T.interdigitale) contains the nucleotide as shown in SEQ ID NO.4
Sequence；
For identifying that the target sequence of acrothesium floccosum (E.floccosum) contains the nucleotides sequence as shown in SEQ ID NO.5
Column；
For identifying that the target sequence of microsporum canis (M.canis) contains the nucleotide sequence as shown in SEQ ID NO.6；
Target sequence for identification of M icrosporum incurvatum contains the nucleotide sequence as shown in SEQ ID NO.7.
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| CN110484641A (en) * | 2019-08-07 | 2019-11-22 | 中国医学科学院北京协和医院 | A kind of method of high-resolution melting curve identification Candida glabrata |
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| CN111206114A (en) * | 2020-03-03 | 2020-05-29 | 杭州缔蓝生物技术有限公司 | Primer and kit for fluorescence PCR (polymerase chain reaction) detection of nine dermatophytes |
| CN111961745A (en) * | 2020-09-02 | 2020-11-20 | 上海捷诺生物科技有限公司 | Method and kit for detecting multiple dermatophytes at one time |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110408720A (en) * | 2019-08-07 | 2019-11-05 | 中国医学科学院北京协和医院 | A kind of method of high-resolution melting curve identification ear candida albicans |
| CN110484641A (en) * | 2019-08-07 | 2019-11-22 | 中国医学科学院北京协和医院 | A kind of method of high-resolution melting curve identification Candida glabrata |
| CN110512013A (en) * | 2019-09-04 | 2019-11-29 | 中国疾病预防控制中心传染病预防控制所 | A method of identifying three kinds of corynebacterias using high-resolution melting curve method |
| CN110512013B (en) * | 2019-09-04 | 2022-07-08 | 中国疾病预防控制中心传染病预防控制所 | Method for identifying three corynebacteria by using high-resolution melting curve method |
| CN111206114A (en) * | 2020-03-03 | 2020-05-29 | 杭州缔蓝生物技术有限公司 | Primer and kit for fluorescence PCR (polymerase chain reaction) detection of nine dermatophytes |
| CN111206114B (en) * | 2020-03-03 | 2023-10-03 | 杭州缔蓝生物技术有限公司 | Nine primers and kit for fluorescence PCR detection of dermatophytes |
| CN111961745A (en) * | 2020-09-02 | 2020-11-20 | 上海捷诺生物科技有限公司 | Method and kit for detecting multiple dermatophytes at one time |
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