CN101555523A - Kit used for detecting candida krusei in intestinal tract by fluorescence quantitative PCR method - Google Patents
Kit used for detecting candida krusei in intestinal tract by fluorescence quantitative PCR method Download PDFInfo
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- CN101555523A CN101555523A CNA2009100985568A CN200910098556A CN101555523A CN 101555523 A CN101555523 A CN 101555523A CN A2009100985568 A CNA2009100985568 A CN A2009100985568A CN 200910098556 A CN200910098556 A CN 200910098556A CN 101555523 A CN101555523 A CN 101555523A
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- candida krusei
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Abstract
The invention relates to a kit, and aims at providing a kit used for detecting candida krusei in the intestinal tract by a fluorescence quantitative PCR method. The kit comprises a standard positive template and two primers of a specificity amplification candida krusei ITS zone used in the PCR amplification process, wherein the gene of a coded amplification product has the nucleotide sequence shown by SEQ ID NO: 3. The operation is simple and convenient and fast; the specificity is good, and the sensitivity is high; the detection based on nucleotide is not limited by culture conditions; the quantitative detection can truly reflect the situations of candida krusei permanent planting and infection in the intestinal tract; and high-flux sample detection can be implemented simultaneously. The kit can carry out fast and quantitative detection to the candida krusei in the intestinal tract, can replace the traditional diagnostic method of isolated culture which is used for a long time, and is suitable for being widely popularized and applied in clinical laboratory.
Description
Technical field
The present invention relates to a kind of test kit, be specifically related to a kind of test kit that fluorescence quantitative PCR method detects the enteron aisle candida krusei that is used for.
Background technology
In the normal human subject enteron aisle, there is abundant, a various fungal colonization.People adopt traditional isolated culture the human intestine fungi to be carried out many researchs (Khatib R, Riederer KM, Ramanathan J, et al.Faecalfungal flora in healthy volunteers and inpatients.Mycoses, 2001,44:151-6.), this method is by diagnosing the fungi phenotype, observe colony growth and breeding situation on the solid medium, according to colony shape, size, color, viscosity and other constitutional featuress are identified fungi, in conjunction with different physiology, biochemical and thermal test etc. carries out the morphology diagnosis.The utilization isolated culture can be diagnosed some pathogenic fungus accurately, but, at some in particular cases, colonial morphology such as separation and Culture is not true to type, special culture medium can't obtain, incubation time is long, phenotype result is non-special or the like, then can't accurately diagnose, need to rely on non-cultivation diagnostic techniques to carry out the fungi evaluation fungal pathogens.
The real-time fluorescence quantitative PCR technology has been widely used in every field (the Espy MJ of microorganism, Uhl JR, et al.Real-time PCR in clinical microbiology:applications for routine laboratorytesting.Clin Microbiol, 2006, Rev 19 (1): 165-256.).This technology not only can be from increasing the sensitivity of pathogen detection in essence, in conjunction with species-specific primer and the analysis of amplified production melt curve analysis can be special detection and identify certain pathogenic agent, testing process need not separation and Culture, and is easy and simple to handle, quick, shortened Diagnostic Time greatly; And can be used as the monitoring means of pathogenic infection patient treatment curative effect.But, because the little synusium complex structure of human intestine exists about 10
14Individual different microbial organisms, and the fungi proportion is lower, less than 1%; The growth of some fungi strain also needs complicated culture condition, and isolated culture can only detect the microorganism that the sub-fraction condition is fit to, and detection sensitivity is low and interval between diagnosis is long.And the real-time fluorescence quantitative PCR technology is based on the detection to pathogenic agent Nucleotide, is not subjected to culture condition restriction and detection sensitivity height, helps more accurately, comprehensively understanding the effect of fungal colonization in the enteron aisle pathophysiological process.Utilize the real-time fluorescence quantitative PCR technology can detect and identify fungal pathogens accurately and rapidly, this is for timely treatment and effectively the control intestinal canal fungus infection is all most important.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, a kind of test kit that fluorescence quantitative PCR method detects the enteron aisle candida krusei that is used for is provided.
In order to solve the problems of the technologies described above, the present invention is achieved by the following technical solutions:
A kind of test kit that fluorescence quantitative PCR method detects the enteron aisle candida krusei that is used for is provided, and this test kit comprises:
10 * Taqman PCR buffer of reaction cumulative volume 1/10;
The upstream and downstream primer of each 0.15 μ mol/L～1 μ mol/L;
DATP, dTTP, dGTP, the dCTP of each 200 μ mol/L;
2.0mmol/L the MgCl of～3.5mmol/L
2
0.05U/ μ l HOTSTART Taq archaeal dna polymerase;
0.02 * SYBR green I fluorescence dye;
The standard positive masterplate, its concentration is by the requirement serial dilution of quantitative criterion curve preparation;
Surplus is an aseptic double-distilled water;
Described two primers are the primer in the specific amplification candida krusei ITS district that uses in the pcr amplification process, wherein:
The upstream primer sequence is: 5 '-AAAAGTCTAGTACGCTCGG-3 ' (SEQ ID NO:1);
The downstream primer sequence is: 5 '-GCATCCATGAAGAACGCAGC-3 ' (SEQ ID NO:2);
Described standard positive masterplate be candida krusei reference culture DNA through aforementioned primer amplification, amplified production is connected the standard plasmid DNA that the back obtains with the pGEMT-TEasy carrier; The gene of described amplified production of encoding has the nucleotide sequence shown in the SEQ ID NO:3.
Compared with prior art, the invention has the beneficial effects as follows:
Easy and simple to handle, quick; Specificity is good, and is highly sensitive; Based on detection, not limited by culture condition to Nucleotide; Detection by quantitative, the situation that can truly reflect the field planting of enteron aisle candida krusei and take place to infect; Can carry out high-throughout pattern detection simultaneously.Test kit provided by the invention can carry out fast quantification to the enteron aisle candida krusei and detect, and can substitute the traditional diagnosis method of the separation and Culture of always continuing to use, and be suitable for the wide popularization and application in clinical labororatory.
Description of drawings
Fig. 1 is with behind the test kit standard positive masterplate gradient dilution of the present invention, carries out the dynamic curve figure of fluorescent quantitative PCR gained, and X-coordinate is represented cycle number, and ordinate zou is represented relative intensity of fluorescence, and the straight line that is parallel to X-coordinate among the figure is represented fluorescence threshold.
Fig. 2 is with behind the test kit standard positive masterplate gradient dilution of the present invention, carries out the canonical plotting of fluorescent quantitative PCR gained, and X-coordinate is represented fluorescence threshold, and ordinate zou is represented the concentration of different extent of dilution standard positive masterplates.
Fig. 3 is the melt curve analysis figure of fluorescent quantitative PCR product described in the specific embodiment, and X-coordinate is represented melting temp, and ordinate zou is represented relative intensity of fluorescence.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention in further detail.
The test kit that is used for fluorescence quantitative PCR method detection enteron aisle candida krusei of the present invention comprises primer, Taqman PCRbuffer, dNTPs, MgCl
2, Taq archaeal dna polymerase, SYBR green I fluorescence dye, standard positive masterplate and aseptic double-distilled water.Wherein:
(1) primer: the primer that is specific amplification candida krusei ITS district;
The upstream primer sequence is: 5 '-AAAAGTCTAGTACGCTCGG-3 ' (SEQ ID NO:1)
The downstream primer sequence is: 5 '-GCATCCATGAAGAACGCAGC-3 ' (SEQ ID NO:2)
(2) standard positive masterplate:
Candida krusei reference culture DNA is through aforementioned primer amplification, and amplified production is connected with pGEMT-T Easy carrier and is the standard positive masterplate; The gene of described amplified production of encoding has the nucleotide sequence shown in the SEQ ID NO:3; Amplified production with can in bacillus coli DH 5 alpha, breed after pGEMT-T Easy carrier is connected.Storing concentration is 10
10Copy/ul carries out serial dilution before the use.
This test kit is stored in-20 ℃, reduces multigelation as far as possible.
Candida krusei ATCC 6258 bacterial strains that the reference culture of candida krusei described in the present invention is produced available from Biomerieux SA, pGEMT-T Easy carrier is a Promega company product, PCR Buffer, MgCl
2, dNTPs, HOTSTART TaqDNA polysaccharase, SYBR green I fluorescence dye be available from the precious biotech company in Dalian.
Specific embodiment 1:
Test kit in the present embodiment comprises:
10 * Taqman PCR buffer of reaction cumulative volume 1/10;
The upstream and downstream primer of each 0.15 μ mol/L;
DATP, dTTP, dGTP, the dCTP of each 200 μ mol/L;
2.0mmol/L MgCl
2
0.05U/ μ l HOTSTART Taq archaeal dna polymerase;
0.02 * SYBR green I fluorescence dye;
The standard positive masterplate, its concentration is by the requirement serial dilution of quantitative criterion curve preparation;
Surplus is an aseptic double-distilled water.
Specific embodiment 2:
The test kit composition is as follows:
10 * Taqman PCR buffer of reaction cumulative volume 1/10;
The upstream and downstream primer of each 1.0 μ mol/L;
DATP, dTTP, dGTP, the dCTP of each 200 μ mol/L;
3.5mmol/L MgCl
2
0.05U/ μ l HOTSTART Taq archaeal dna polymerase;
0.02 * SYBR green I fluorescence dye;
The standard positive masterplate, its concentration is by the requirement serial dilution of quantitative criterion curve preparation;
Surplus is an aseptic double-distilled water.
Specific embodiment 3:
The test kit composition is as follows:
10 * Taqman PCR buffer of reaction cumulative volume 1/10;
The upstream and downstream primer of each 0.5 μ mol/L;
DATP, dTTP, dGTP, the dCTP of each 200 μ mol/L;
3.0mmol/L MgCl
2
0.05U/ μ l HOTSTART Taq archaeal dna polymerase;
0.02 * SYBR green I fluorescence dye;
The standard positive masterplate, its concentration is by the requirement serial dilution of quantitative criterion curve preparation;
Surplus is an aseptic double-distilled water.
The principle of work introduction of test kit of the present invention:
In the PCR reaction system, add excessive SYBR fluorescence dye, after the SYBR fluorescence dye mixes the dna double chain specifically, the emitting fluorescence signal, and the SYBR dye molecule that does not mix in the chain can not launched any fluorescent signal, thereby the increase of the increase of assurance fluorescent signal and PCR product is synchronous fully.Reaction comprises following step: (1) PCR denaturation process produces single stranded DNA, and the SYBR fluorescence dye is unbound state, and fluorescent signal is very low; (2) in the primer annealing process, double-stranded DNA partly forms, and the SYBR fluorescence dye mixes the dna double chain simultaneously specifically, and fluorescent signal increases; (3) in the PCR extension process, double-stranded DNA forms gradually, and the SYBR fluorescence dye constantly mixes in the dna double chain, and fluorescent signal constantly increases; (4) after the end of the stage of extension at last, all DNA form two strands, and the fluorescence dye that mixes in the dna double chain reaches maximum value, and fluorescent signal also reaches maximum value.
Test kit using method of the present invention is given an example:
(1) reaction system: 10 * Taqman PCR buffer, 4 μ l, the concentration of dATP, dTTP, dGTP, dCTP is respectively 200 μ mol/L, and the concentration of upstream and downstream primer is 0.5 μ mol/L, MgCl
2Concentration be 3.0mmol/L, 0.02 * SYBR green I fluorescence dye, 2U HOTSTART Taq archaeal dna polymerase, masterplate DNA 2 μ l, aseptic double-distilled water is supplied volume to 40ul.
(2) response procedures: 95 ℃ of sex change 5min, 94 ℃ of pre-sex change 30s subsequently, 60 ℃ of annealing 30s, 72 ℃ are extended 45s, circulates 35 times, last 72 ℃ of extension 5min.
(3) quantitative criterion curve preparation: the standard positive masterplate is carried out continuous 10 times of dilutions (as 10
9, 10
8, 10
7, 10
6), obtain the standard plasmid DNA of serial copy number gradient.According to above-mentioned reaction system and response procedures, each concentration triplicate increases.After reaction finishes, according to the logarithmic value and C (T) the value drawing standard curve Y=aX+b of initial masterplate copy number, R
2
(4) sample detection: get just 200mg of the fresh tail of experimenter, extract DNA, get 2ul DNA, increase according to above-mentioned reaction system and response procedures as masterplate according to ordinary method.
(5) result calculates: C (T) value according to typical curve and gained is calculated initial masterplate copy number.
Application example introduction of the present invention:
Get the fresh tail of 40 routine healthy volunteers just, adopt husky Bao Shi substratum separation and Culture and fluorescent quantificationally PCR detecting kit of the present invention to detect simultaneously.
1. the preparation of masterplate DAN:
(1) reagent: adopt
DNA stool mini kit is in conjunction with granulated glass sphere driving extracting experimenter stool sample DNA,
DNA stool mini kit test kit is available from Beijing new experimental technique of east KingMax company limited, and granulated glass sphere is available from Shanghai bio-engineering corporation.
(2) extraction of stool sample DNA: take by weighing just 200mg/ part of the fresh tail of experimenter with electronic balance, add ASL damping fluid and 0.3g granulated glass sphere, insert the quick nucleic acid extraction instrument of Fast Prep FP120,6.5m/s * 45s carries out broken wall, then according to
DNA stool mini kit specification sheets is operated, last AE eluted dna.The DNA that all extractings obtain all places-80 ℃ of preservations standby.
2. quantitative fluorescent PCR reaction
(1) reaction system: 10 * Taqman PCR buffer, 4 μ l, the concentration of dATP, dTTP, dGTP, dCTP is respectively 200 μ mol/L, and the concentration of upstream and downstream primer is 0.5 μ mol/L, MgCl
2Concentration be 3.0mmol/L, 0.02 * SYBR green I fluorescence dye, 2U HOTSTART Taq archaeal dna polymerase, masterplate DNA 2 μ l, aseptic double-distilled water is supplied volume to 40ul.
(2) response procedures: 95 ℃ of sex change 5min, 94 ℃ of pre-sex change 30s subsequently, 60 ℃ of annealing 30s, 72 ℃ are extended 45s, circulates 35 times, last 72 ℃ of extension 5min.
(3) quantitative criterion curve preparation: the standard positive masterplate is carried out continuous 10 times of dilutions (as 10
9, 10
8, 10
7, 10
6), obtain the standard plasmid DNA of serial copy number gradient.According to above-mentioned reaction system and response procedures, each concentration triplicate increases.Reaction is drawn out typical curve Y=-0.2867X+10.19, R according to the logarithmic value and C (T) value of initial masterplate copy number after finishing
2=0.994 (Fig. 1,2).
(4) sample detection: get 40 routine healthy volunteer's stool sample DNA as masterplate, carry out fluorescent quantitative PCR according to above-mentioned reaction system and response procedures.By the PCR product is carried out the specificity that the melting temp tracing analysis decides product, promptly from 75 ℃ to 95 ℃, 0.5 ℃ of every intensification continues to gather after 2 seconds the fluorescent signal of reaction solution in the reaction tubes when loop ends.The standard positive masterplate that each quantitative fluorescent PCR reaction all is provided with doubling dilution makes up typical curve, obtains different strain gene copy number in the sample with this.Various fungi quantity represent that with the logarithmic value of every gram ight soil gene copy number data are with mean (x) ± standard deviation (s) expression.
The result shows that the relation conefficient that test kit of the present invention detects candida krusei gained typical curve is 0.99, and minimum linear detectability is 10
2Copy/ul (Fig. 1,2).The melting temp of amplified production is 91.0 ℃, and melt curve analysis shows amplified reaction specificity better (Fig. 3).The detection positive rate (60.0%) of test kit of the present invention is significantly higher than isolated culture (5.0%) (P＜0.001), and the fluorescence quantitative PCR detection result of 40 routine healthy volunteer's enteron aisle candida kruseis is 6.521 ± 0.318 (log
10Copy/g).Experimental result shows that test kit of the present invention and traditional isolated culture compare, and it is good to have specificity, highly sensitive, detects characteristics such as quick.
At last, it is also to be noted that what more than enumerate only is specific embodiments of the invention.Obviously, the invention is not restricted to above examples of implementation, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Sequence table
SEQ?ID?NO：1
aaaagtctag?tacgctcgg?19
SEQ?ID?NO：2
gcatccatga?agaacgcagc?20
SEQ?ID?NO：3
aaaagtctag?tacgctcggc?cagcttcgct?ccctttcagg?cgagtcgcag?ctccgacgct?60
ctttacacgt?cgtccgctcc?gctcccccaa?ctctgcgcac?gcgcaagatg?gaaacgacgc?120
tcaaacaggc?atgccccccg?gaatgccgag?gggcgcaatg?tgcgttcaag?aactcgatga?180
ttcacgatgg?ctgcaattca?cactaggtat?cgcatttcgc?tgcgttcttc?atggatgc 238
Claims (1)
1, a kind of test kit that is used for fluorescence quantitative PCR method detection enteron aisle candida krusei is characterized in that this test kit comprises:
10 * Taqman PCR buffer of reaction cumulative volume 1/10;
The upstream and downstream primer of each 0.15 μ mol/L～1 μ mol/L;
DATP, dTTP, dGTP, the dCTP of each 200 μ mol/L;
2.0mmol/L the MgCl of～3.5mmol/L
2
0.05U/ μ l HOTSTART Taq archaeal dna polymerase;
0.02 * SYBR green I fluorescence dye;
The standard positive masterplate, its concentration is by the requirement serial dilution of quantitative criterion curve preparation;
Surplus is an aseptic double-distilled water;
Described two primers are the primer in the specific amplification candida krusei ITS district that uses in the pcr amplification process, wherein:
The upstream primer sequence is: 5 '-AAAAGTCTAGTACGCTCGG-3 ';
The downstream primer sequence is: 5 '-GCATCCATGAAGAACGCAGC-3 ';
Described standard positive masterplate be candida krusei reference culture DNA through aforementioned primer amplification, amplified production is connected the standard plasmid DNA that the back obtains with pGEMT-T Easy carrier; The gene of described amplified production of encoding has the nucleotide sequence shown in the SEQ ID NO:3.
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| CN2009100985568A CN101555523B (en) | 2009-05-14 | 2009-05-14 | Kit used for detecting candida krusei in intestinal tract by fluorescence quantitative PCR method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009100985568A CN101555523B (en) | 2009-05-14 | 2009-05-14 | Kit used for detecting candida krusei in intestinal tract by fluorescence quantitative PCR method |
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| CN101555523A true CN101555523A (en) | 2009-10-14 |
| CN101555523B CN101555523B (en) | 2011-07-20 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105950740A (en) * | 2016-05-30 | 2016-09-21 | 中国人民解放军第二军医大学 | LAMP (loop-mediated isothermal amplification) kit for early diagnosis of candida krusei and special primer |
| CN106048051A (en) * | 2016-07-25 | 2016-10-26 | 泰普生物科学（中国）有限公司 | Candida krusei fluorescence PCR detection kit |
| CN112695128A (en) * | 2021-02-05 | 2021-04-23 | 杭州医学院 | Rapid analysis method for yeast fungi based on membrane culture |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1286989C (en) * | 2005-04-22 | 2006-11-29 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Biochip for detecting pathogenesis fungus |
-
2009
- 2009-05-14 CN CN2009100985568A patent/CN101555523B/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105950740A (en) * | 2016-05-30 | 2016-09-21 | 中国人民解放军第二军医大学 | LAMP (loop-mediated isothermal amplification) kit for early diagnosis of candida krusei and special primer |
| CN105950740B (en) * | 2016-05-30 | 2019-12-10 | 中国人民解放军第二军医大学 | LAMP kit for early diagnosis of candida krusei and special primer thereof |
| CN106048051A (en) * | 2016-07-25 | 2016-10-26 | 泰普生物科学（中国）有限公司 | Candida krusei fluorescence PCR detection kit |
| CN106048051B (en) * | 2016-07-25 | 2019-11-29 | 泰普生物科学（中国）有限公司 | A kind of candida krusei fluorescence PCR detection reagent kit |
| CN112695128A (en) * | 2021-02-05 | 2021-04-23 | 杭州医学院 | Rapid analysis method for yeast fungi based on membrane culture |
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| CN101555523B (en) | 2011-07-20 |
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