CN101974640A - Rapid Fungus detection kit and detection method using same - Google Patents
Rapid Fungus detection kit and detection method using same Download PDFInfo
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Abstract
The invention relates to the field of clinical microorganism identification, and provides a rapid fungus detection kit and a detection method using the same. The kit comprises two universal primers for amplifying an internal transcribed spacer (ITS1) of a fungus 18S, 5.8S, a sequencing primer for determining a sequence of a reverse section of the ITS1, a magnetic bead marked by an avidin, and a sequencing result and database comparison and judgment species. By using the kit, the fungus in a clinical sample can be distinguished by one-time sequencing, so the kit can be used for clinical diagnosis. The kit not only gains precious rescue time for clinical diagnosis and treatment, but also has the advantages that: the cost is low, the kit is convenient to operate and the specificity is high.
Description
Technical field
The invention belongs to the clinical microorganism identification technical field, be specifically related to test kit and the detection method of a kind of Rapid identification clinical sample fungi.
Background technology
Along with the widespread use of radiotherapy, chemotherapy, Broad spectrum antibiotics and the immunological reagent of tumour, systematicness, opportunistic fungal infection increase day by day, particularly in blood system malignant disease, tumour and bone marrow transplantation and organ transplantation.For example infectivity fungi infestation takes place in 18%～50% patient after bone marrow transplantation; The morbidity of fungi infestation is up to 20%～40% in organ transplant recipients and the malignant tumor patient.Detect net analysis according to China's hospital infection, hospital's fungi infestation rises to 17.1%～24.4% of the latter stage nineties from 13.9% of the early stage nineties, and along with fungi infestation is increasing, the use of antifungal drug causes the fungi resistance also to increase gradually.The infectivity mycosis mostly occurs the patient of serious underlying diseases, its poor prognosis, case fatality rate height, and the moniliosis case fatality rate is 30%～40%, the aspergillosis case fatality rate is up to 50%～100%.Infectivity fungi infestation has become one of important factor of death in big organ transplantation patient.
According to relevant bibliographical information in recent years, the fungi infestation lift velocity is so fast, major cause have following some: 1 wide spectrum and the out-of-control use of super Broad spectrum antibiotics, destroyed the normal microecological balance of human body and caused fungi infestation; 2 immunocompromised crowd ratios increase, and as ICU ward patient, tumour, hemopathy, acquired immune deficiency syndrome (AIDS) and gerontal patient are high-risk ward, high-risk sick kind, high risk population; 3 interventional therapies and operating improvement have improved patient's survival rate, have also created condition for invasive fungi infestation simultaneously.And the abuse of various antifungal drugs has more aggravated the recurrence of fungi infestation and has increased the treatment difficulty.Therefore, to setting up quick fungi separation and Culture clinically, identifying and to seem particularly important, can improve clinical therapeutic efficacy greatly.
Mycotic diagnosis is at present main to be relied on conventional directly microscopy and cultivation and morphology to identify to find and confirms pathogenic bacteria, its complex operation, time-consuming and need the expertise of height.Especially to be differentiated the very difficulty that seems by traditional phenotypic characteristic authentication method for special, the plesiomorphic bacterial strain of those growth conditionss.Directly microscopy is easy and simple to handle, quick, practical, positive findings can be determined fungi infestation, but it is very low to detect positive rate, negative findings can not be got rid of infection, need to be used in combination with culture method, often can establish the diagnosis of deep mycosis as discovery fungi composition in the direct microscopy of aseptic body fluid, but have the bacterium position then to have only a large amount of hypha,hyphaes of discovery just to have diagnostic significance.Cultivate test procedure and can further improve positive rate, but its complicated operation wastes time and energy, incubation time was generally for 4 weeks, and indivedual poky bacterium then need more of a specified duration, and the positive rate of culture method is not high yet.Immunological technique diagnoses clinical fungi infestation because of the antibody test cross reaction is serious, specific antigens detects same false negative and the false positive problem that exists and limited its application.Though biochip technology can be carried out the evaluation of multiple fungi simultaneously, but the bacterial classification face of containing is narrow, powerless to non-common bacteria, mutant bacteria or novel bacterial, cost is too high also to be the important factor of its development of restriction, and hybridization mode inherent limitation has influenced its specificity.This year, mass-spectrometric technique with its fast and accurately advantage be used to microorganism identification must be to cultivate the back to divide pure mono-clonal bacterium colony because mass spectrum is identified, and apparatus expensive, operation easier is high to have limited its application.
In its tangible real research work, based on sequential analysis to length polymorphism and the sequence polymorphism of rDNA-ITS, be used for reacting biological sibship and classification situation owing to obtain enough relatively information in can never oversize nucleotide sequence, thereby become the focus of classification of fungi and identification research, be widely used between the genus kind of fungi at present and part plant in the Phylogenetic Studies of cohort level.Conserved sequence on the fungi rDNA (rDNA) is extensive, and the regional sequence of different evolution levels is arranged, and the classification of fungi that can be used for different grades is identified.Internal transcribed spacer district ITS on the rDNA is the zone that is present between 18S rDNA, 5.8S rDNA and the 28S rDNA, this zone is subjected to the influence of external environment factor little, compare with the coding region and to have the fast characteristics of rate of evolution, high conservative between the different strains in planting, change greatly and between the fungi kind, exist, show great sequence polymorphism, can provide prolific hereditary information for mycologic research.Therefore based on fungi rDNA sequential analysis, the conservative characteristic that has based on internal transcribed spacer region sequence mark, satisfy the evaluation of clinical disease fungal pathogens, within a short period of time just can be to the pathogenic fungi evaluation of classifying, identify the unknown or difficult fungi of cultivating, be convenient to clinical disease is carried out early diagnosis and implements effectively treatment.
Tetra-sodium order-checking (Pyrosequencing) technology is a dna sequence analysis technology of new generation, and this technology need not be carried out electrophoresis, and dna fragmentation also need not fluorescent mark, operates very easy.The tetra-sodium sequencing technologies is by the enzyme cascade chemiluminescence reaction in 4 kinds of enzymatic same reaction systems, take turns in the sequencing reaction at each, only add a kind of dNTP, if this dNTP and template are matched, polysaccharase just can be incorporated into it in primer strand and the tetra-sodium group (PPi) of mole number such as discharge.PPi can finally be converted into visible light signal, and is converted into a peak value by Pyrogram TM.The height of each peak value is directly proportional with the Nucleotide number that mixes in the reaction.Add a kind of dNTP down then, continue the synthetic of DNA chain.
The tetra-sodium sequencing technologies can carry out the short dna sequential analysis quickly and accurately, and flux height, easy to operate is convenient to make up the normalizing operation flow process, therefore favored by the investigator, and has been applied to the somatotype evaluation of Clinical microorganism aspect much.Gharizaden etc. (2004) come yeast is carried out somatotype with 40 base sequences that the tetra-sodium sequencing technologies is measured yeast 18S RNA mutation district by a pair of universal primer among the rDNA-ITS for the first time.Jason etc. (2005) carry out sequencing analysis with the tetra-sodium sequencing technologies to 231 routine colpitis mycoticaes by real-time quantitative PCR with ITS2 district universal primer, just can identify most pathogenic bacterium in first circulation of checking order.Leaw etc. (2006) utilize the internal transcribed spacer district to identify the important Saccharomycodes pathomycete of medical science by the tetra-sodium sequencing analysis, by two groups of different Auele Specific Primers successfully increase ITS1 and ITS2 district, successful standard of perfection bacterial strain and medical science pathogenic bacterium arrive the level of planting through checking order, this research is also found: the ITS1 district has more specificity on the identification level than the ITS2 district in planting, therefore, this method Candida parapsilosis I-III group that can also just have a high conservative makes a distinction.Bobby etc. (2007) use rDNA ITS2 section gene test candiyeast pathogenic bacterium by the tetra-sodium sequencing technologies, and the result compares with biochemical and morphologic detection, has shown 100% consistence.Andrew etc. (2008) utilize tetra-sodium sequencing technologies Rapid identification to go out the non-common fungi of curing the disease equally, and can identify the fungi that causes that mixed type infects.Kang etc. (2009) are further divided into 8 genotype according to the difference of ITS nucleotide sequence regiospecificity base with 3 mutation of cryptococcus neoformans.
This research by design software to common clinical fungi sequencing primer subsequently the sequence of 40bp carried out permutation and combination, produced the tetra-sodium order-checking Virtualization Mode database of multiple different fungi infestations, clinical sample is through DNA extracting, universal primer amplification, use universal primer as sequencing primer then, type is judged in sequencing result and the comparison of tetra-sodium order-checking Virtualization Mode database.This method can once sequencing be distinguished common clinical fungi, can be used for the clinical diagnosis and the resistance monitoring of fungi infestation.The blank that adopts can effectively be deducted contingent pollution in type is judged, solved high sensitivity and must bring a unmanageable pollution difficult problem.The significance of fungi kind is in the clinical samples such as fast and reliable evaluation phlegm, urine, secretory product: 1) reasonable application of antibiotics, avoid invalid medication; 2) improve prognosis; 3) slow down the acquisition resistance; 4) reduce medical expense 5): the specificity and the susceptibility of height, 6): shortened detection time greatly.Improper microbiotic is used and is usually caused the treatment failure, and hospital stay is prolonged, complication, consequences such as resistance and survey fee increase.
Summary of the invention
Technical problem to be solved by this invention provides a kind of rapid and reliable fungi quick detection kit.
The technical problem that the present invention also will solve provides the detection method of mentioned reagent box.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
A kind of fungi quick detection kit, it comprises:
(1) amplification fungi 18S, the universal primer of 5.8S internal transcribed spacer district ITSI: its nucleotide sequence primer shown in SEQ ID NO:1 and SEQ ID NO:2 is right;
(2) sequencing primer: its nucleotide sequence is shown in SEQ ID NO:3;
(3) magnetic bead of avidin mark;
Wherein, SEQ ID NO:1 is at its 5 ' end mark vitamin H;
In one-time detection, use jointly.
Above-mentioned fungi quick detection kit specifically comprises following reagent:
(1) DNA extraction reagent: Buffer I, Buffer II, Buffer III; Buffer TE;
Wherein, Buffer I is the 9g/LNaCl aqueous solution;
Wherein, Buffer II is 10g/L SDS, 2% (v/v) Triton X-100, and 100mmol/L NaCl, 10mmolTris-HCl (pH 8.0), 1mmol/L EDTA, solvent are water;
Wherein, Buffer III is the saturated NaCl aqueous solution (about 6mol/L);
Wherein, Buffer TE is 10mmol/L Tris-HCl (pH 8.0), and 1mmol/L EDTA, solvent are water;
(2) reaction solution: PCR Buffer, universal primer SEQ ID NO:1～2,2mM MgCl
2, 0.2mM dNTPs, 2U/ μ L Taq archaeal dna polymerase;
Wherein, PCR Buffer is specially: 0.1% (v/v) NP-40,0.02% (v/v) gelatin, 0.06%g/mL BSA, 0.1% (v/v) Tween-20,0.06M pH8.9 Tricine.
(3) strand purified reagent: 75% (v/v) ethanolic soln, 0.2M NaOH, 10mM pH 7.6 Tris-Acetate solution, binding buffer liquid, annealing buffer;
Wherein, binding buffer liquid is specially: 10mM Tris-HCl, 2M NaCl, 1mM EDTA, 0.1% (v/v) Tween-20; Annealing buffer is specially: 20mM pH 7.6 Tris-Acetate, 2mM magnesium acetate.
(4) sequencing reagent: archaeal dna polymerase, the ATP sulfurylase, luciferase, apyrase, substrate A PS, fluorescein and dNTP (dNTP is dATP S, dTTP, and dCTP, dGTP).
A kind of fungi quick detection kit, it comprises the complementary base sequences thereof of all above-mentioned nucleotide sequences, just comprises:
(1) amplification fungi 18S, the universal primer of internal transcribed spacer district ITS1 between the 5.8S: its nucleotide sequence primer shown in SEQID NO:4 and SEQ ID NO:5 is right; Synthetic by the handsome bio tech ltd in Shanghai.
(2) sequencing primer: its nucleotide sequence is shown in SEQ ID NO:6; Synthetic by the handsome bio tech ltd in Shanghai.
(3) magnetic bead of avidin mark;
Wherein, SEQ ID NO:4 is at its 5 ' end mark vitamin H; Synthetic by the handsome bio tech ltd in Shanghai.
In one-time detection, use jointly.
Above-mentioned fungi quick detection kit specifically comprises following reagent:
(1) DNA extraction reagent: Buffer I, Buffer II, Buffer III; Buffer TE;
Wherein, Buffer I is the 9g/LNaCl aqueous solution;
Wherein, Buffer II is 10g/L SDS, 2% (v/v) Triton X-100, and 100mmol/L NaCl, 10mmolTris-HCl (pH 8.0), 1mmol/L EDTA, solvent are water;
Wherein, Buffer III is the saturated NaCl aqueous solution (about 6mol/L);
Wherein, Buffer TE is 10mmol/L Tris-HCl (pH 8.0), and 1mmol/L EDTA, solvent are water;
(2) reaction solution: PCR Buffer, universal primer SEQ ID NO:3～4,2mM MgCl
2, 0.2mM dNTPs, 2U/ μ L Taq archaeal dna polymerase;
Wherein, PCR Buffer is specially: 0.1% (v/v) NP-40,0.02% (v/v) gelatin, 0.06% (w/v) g/mL BSA, 0.1% (v/v) Tween-20,0.06M pH8.9 Tricine.
(3) strand purified reagent: 75% (v/v) ethanolic soln, 0.2 MNaOH, 10mM pH 7.6 Tris-Acetate solution, binding buffer liquid, annealing buffer;
Wherein, binding buffer liquid is specially: 10mM Tris-HCl, 2M NaCl, 1mM EDTA, 0.1% (v/v) Tween-20; Annealing buffer is specially: 20mM pH 7.6 Tris-Acetate, 2mM magnesium acetate.
(4) sequencing reagent: archaeal dna polymerase, the ATP sulfurylase, luciferase, apyrase, substrate A PS, fluorescein and dNTP (dNTP is dATP S, dTTP, and dCTP, dGTP).
The fungi that the present invention detected is the fungi bacterium of narrow sense, comprises Candida, genera cryptococcus, Coccidioides, Histoplasma, Blastomyces, Eurotium, mucor, Rhizopus.
The detection method of above-mentioned fungi quick detection kit comprises the steps:
(1) extracts fungal DNA in the clinical sample;
(2) be template with step (1) gained DNA, utilize universal primer to carry out fungi 18S, the pcr amplification of the internal transcribed spacer district ITS1 of 5.8S;
(3) pcr amplification product that step (2) is obtained combines with the magnetic bead of avidin mark and carries out the strand purifying;
(4) the strand purified product that step (3) is obtained carries out the tetra-sodium order-checking;
(5) the fungi kind is judged in the comparison of sequencing result and database.
In the step (2), described pcr amplification system is: 10 * PCR buffer, 5 μ L, SEQ ID NO:10.3 μ L, SEQ ID NO:20.3 μ L, Template DNA 2 μ L, 0.2mM dNTPs, 2mM MgCl
2, Taq archaeal dna polymerase 2U adds sterilized water to 50 μ L; The pcr amplification condition is: 94 ℃ of 5min; 40 circulations, 94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 30s; 72 ℃ of 5min.Perhaps SEQ ID NO:4 is replaced above-mentioned SEQ ID NO:1, simultaneously SEQID NO:54 is replaced above-mentioned SEQ ID NO:2, other pcr amplification system is identical with condition.
Beneficial effect: test kit of the present invention utilizes the sequence and the database comparison of the ITS1 backward section of tetra-sodium sequencing technologies mensuration to judge the fungi kind.Use this test kit and can once sequencing identify clinical sample fungi kind.Can be used for clinical diagnosis.Not only for clinic diagnosis has won valuable rescue time, and have with low cost, easy to operate, the advantage of high specificity.The inventive method can make clinical sample fungi kind qualification time shorten (about 4～5 hours of detection time of the inventive method) greatly, in addition, identify that by sequence alignment fungi is the ultimate method of strain identification, particularly to some difficult evaluation bacterium, sequence alignment is identified the advantage that has more.
Description of drawings
Fig. 1 is 1 routine negative detection result of specimen figure, and sequencing result is negative.
Fig. 2 is 1 routine fungi infestation detection result of specimen figure, and sequencing result is GAAAGTTTTG ACTATTTAGTAATAATCTGG TG, is judged to be the Candida albicans bacterium with the database comparison.
Fig. 3 is 1 routine fungi infestation detection result of specimen figure, and sequencing result is GAAAGTTTT GAAGTTGTTTTCATATCATAAAAAGAAATTCGTTTGG, is judged to be Candida glabrata with the database comparison.
Fig. 4 is 1 routine fungi infestation detection result of specimen figure, and sequencing result GAAAGTTTTGACTATTGTAATAATAAATCAAGTTTG is judged to be candida tropicalis with the database comparison.
Fig. 5 is 1 routine fungi infestation detection result of specimen figure, and sequencing result is GAAAGTTTT ATTATTGTTATAATAAGATT ACATTC, is judged to be Cryptococcus neoformans with the database comparison.
Fig. 6 is 1 routine fungi infestation detection result of specimen figure, and sequencing result is GAAAGTTTTGATTTAGTTTGTTAGAATAAAATTTATTTTG, is judged to be Candida lusitaniae with the database comparison.
Fig. 7 is 1 routine fungi infestation detection result of specimen figure, and sequencing result is GAAGTTTTGACTATTAGTTAATCAAGTTGAC, is judged to be Candida parapsilosis with the database comparison.
Embodiment
According to following embodiment, the present invention may be better understood.Yet, those skilled in the art will readily understand that embodiment only is used to illustrate the present invention, and should also can not limit the present invention described in detail in claims.
Embodiment 1: the preparation method of test kit.
(1) DNA extraction reagent:
Buffer I is the 9g/L NaCl aqueous solution;
Buffer II is 10g/L SDS, 2% (v/v) Triton X-100, and 100mmol/L NaCl, 10mmol Tris-HCl (pH 8.0), 1mmol/L EDTA, solvent are water;
Buffer III is the saturated NaCl aqueous solution (about 6mol/L);
Buffer TE is 10mmol/L Tris-HCl (pH 8.0), and 1mmol/L EDTA, solvent are water;
Wherein: NaCl (purchasing) in Shishewei Chemical Co., Ltd., Shanghai, SDS (purchasing Sigma company) in the U.S., EDTA (purchasing) in Hangzhou chemical reagent company limited, (Tris-base purchases the Sigma company in the U.S. to Tris-HCl, hydrochloric acid is purchased in Hangzhou chemical reagent company limited), Triton X-100 (purchasing Sigma company) in the U.S.
(2) reaction solution:
PCRBuffer:0.1% (v/v) NP-40 (purchasing Sigma company) in the U.S., 0.02% (v/v) gelatin (purchasing Sigma company) in the U.S., 0.06%g/mL BSA (purchasing Sigma company) in the U.S., 0.1% (v/v) Tween-20 (purchasing Sigma company) in the U.S., 0.06M pH8.9Tricine (purchase in German Merck company)
Universal primer SEQ ID NO:1～2 or SEQ ID NO:4～5, synthetic by the handsome bio tech ltd in Shanghai.
MgCl
2: purchase Sigma company in the U.S.,
0.2mM dNTPs: purchase in Shanghai ancient cooking vessel state Bioisystech Co., Ltd,
2U/ μ L Taq archaeal dna polymerase: available from U.S. Fermentas company.
(3) strand purified reagent:
75% (v/v) ethanolic soln: purchase in Hangzhou Long March chemical reagent company limited,
0.2M NaOH: purchase in Shishewei Chemical Co., Ltd., Shanghai,
10mM Tris-Acetate (pH 7.6): Tris-base purchases the Sigma company in the U.S., and anhydrous acetic acid is purchased in Hangzhou chemical reagent company limited,
Binding buffer liquid: (Tris-base purchases the Sigma company in the U.S. by 10mM Tris-HCl, hydrochloric acid is purchased in Hangzhou chemical reagent company limited), 2M NaCl (purchasing) in Shishewei Chemical Co., Ltd., Shanghai, 1mM EDTA (purchasing) in Hangzhou chemical reagent company limited, 0.1% (v/v) Tween 20 (purchasing the Sigma company in the U.S.) forms
Annealing buffer: by 20mM Tris-Acetate (pH 7.6) (Tris-base purchases the Sigma company in the U.S., and anhydrous acetic acid is purchased in Hangzhou chemical reagent company limited), 2mM magnesium acetate (purchasing in Shishewei Chemical Co., Ltd., Shanghai) is formed.
The magnetic bead of avidin mark (purchasing AB) in GE healthcare Bioscience.
(4) sequencing reagent:
Archaeal dna polymerase, ATP sulfurylase, luciferase and apyrase: purchase company in QIAGEN,
Substrate A PS and fluorescein: purchase company in QIAGEN,
Four kinds of dNTP (dATP S, dTTP, dCTP, dGTP): purchase company in QIAGEN.
Sequencing primer SEQ ID NO:3, SEQ ID NO:6, synthetic by the handsome bio tech ltd in Shanghai.
Embodiment 2: detection method.
Instrument: Bio-Rad S1000 PCR instrument, the desk-top micro-refrigerated centrifuge of Beckman Microfuge 22R, clear gel imaging system is trained in Beijing 61 agarose gel electrophoresis instrument, Shanghai, QIAGEN PyroMark Q96ID sequenator.
(1) extracting fungal DNA, is example with clinical urine specimen, specifically comprises the steps:
(1a) get clinical urine specimen 2ml in centrifuge tube, 12000rpm high speed centrifugation 10min;
(1b) abandon supernatant, add 800ul BufferI, whirlpool concussion 30 seconds;
(1c) 12000rpm high speed centrifugation 5min;
(1d) abandon supernatant, be inverted as far as possible and blot, add 300ul Buffer II, high-speed oscillator concussion 10min;
(1e) 300ul Buffer III will be added in the centrifuge tube, whirlpool concussion 30s;
(1f) the centrifugal 15min of 4000rpm;
(1g) with (1f) gained supernatant liquor, place another clean centrifuge tube, add 2 times of volume dehydrated alcohols, the mixing that overturns gently up and down leaves standstill 2min;
(1h) the centrifugal 10min of 12000rpm;
(1i) abandon supernatant, will precipitate oven dry, add 100ul TE and hatch 30min for 65 ℃, as pcr template.
(2) be template with step (1) gained DNA, utilize universal primer to carry out fungi 18S, the pcr amplification of 5.8S internal transcribed spacer district ITS1;
Wherein, described pcr amplification system is: 10 * PCR buffer, 5 μ L, SEQ ID NO:10.3 μ L, SEQ IDNO:20.3 μ L, Template DNA 2 μ l, 0.2mM dNTPs, 2mM MgCl
2, Taq archaeal dna polymerase 2U adds sterilized water to 50 μ L; The pcr amplification condition is: 94 ℃ of 5min; 40 circulations (94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 30s); 72 ℃ of 5min.
(3) pcr amplification product that step (2) is obtained combines with the magnetic bead of avidin mark and carries out the strand purifying:
● before use, guarantee that all solution all reach room temperature;
● add 45 μ l annealing buffer in PSQ 96 plates, every then hole adds SEQ ID NO:2 sequencing primer (10uM) 0.5uL;
● use Vertex mixing Sepharose beads, the sepharoe beads total amount (every sample 3 μ L) that needs use is transferred in the Eppendorf pipe, in sepharose bead, add 47ulbinding buffer, make average each sample that the volume of 50 μ L be arranged approximately, with the mixture mixing;
● above mixture is added in the PCR product (50 μ L reaction volume), and every sample 50 μ L shook mixing 10 minutes down with PCR product normal temperature, made beads combine with vitamin H;
● in Vacuum prep workstation, add 180mL high purity water, 70% ethanol, washing buffer and 120ml Denaturation buffer in four sample panel successively;
● open the pump of vacuum prep workstation, vacuum prep tool was cleaned in high purity water 30 seconds, then vacuum prep tool is moved on in the PCR plate, grasp sepharose beads, vacuum prep tool was put into 70% ethanol 5 seconds, moved on to then among the denatureation buffer 5 seconds, move on to again among the washing buffer and cleaned 10 seconds, suction nozzle is placed on the corresponding top of containing the plate hole of sequencing primer, do not contact liquid level, turn off pump, vacuum prep tool is put into the plate that contains sequencing primer, shake, discharge sepharose beads;
● use high purity water to clean vacuum prep tool.
With PSQ 96 plates that are placed with sample be placed on be heated on the ThermoPlate 85 ℃ 2 minutes, put into sequenator behind the cool to room temperature again.
(4) the strand purified product that step (3) is obtained carries out the tetra-sodium order-checking;
(5) the fungi kind is judged in the comparison of sequencing result and American National biology information technology center (NCBI) database.
The inventive method is used for the evaluation of 7 routine clinical cultivation positive samples, and sequencer map is shown in Fig. 2-7, and wherein Fig. 1 is 1 routine radiolucent table table qualification result.The inventive method qualification result compares with the microbial identification system of French biological Mei Liai after pure with dividing through microorganism culturing, pathogenic bacterium, as a result unanimity.
Claims (7)
1. fungi quick detection kit is characterized in that it comprises:
(1) universal primer of the internal transcribed spacer district ITS1 of amplification fungi: its nucleotide sequence primer shown in SEQ ID NO:1 and SEQ ID NO:2 is right;
(2) sequencing primer: its nucleotide sequence is shown in SEQ ID NO:3;
(3) magnetic bead of avidin mark;
Wherein, SEQ IDNO:1 is at its 5 ' end mark vitamin H;
In one-time detection, use jointly.
2. fungi quick detection kit according to claim 1 is characterized in that it comprises following reagent:
(1) DNA extraction reagent: Buffer I, Buffer II, Buffer III; Buffer TE;
(2) reaction solution: PCR Buffer, universal primer SEQ ID NO:1～2,2mM MgCl
2, 0.2mM dNTPs, 2U/ μ L Taq archaeal dna polymerase;
(3) strand purified reagent: 75% (v/v) ethanolic soln, 0.2MNaOH, 10mM pH 7.6 Tris-Acetate solution, binding buffer liquid, annealing buffer;
(4) sequencing reagent: archaeal dna polymerase, ATP sulfurylase, luciferase, apyrase, substrate A PS, fluorescein and dNTP.
3. fungi quick detection kit is characterized in that it comprises:
(1) universal primer of the internal transcribed spacer district ITS1 of amplification fungi: its nucleotide sequence primer shown in SEQ ID NO:4 and SEQ ID NO:5 is right;
(2) sequencing primer: its nucleotide sequence is shown in SEQ ID NO:6;
(3) magnetic bead of avidin mark;
Wherein, SEQ IDNO:4 is at its 5 ' end mark vitamin H;
In one-time detection, use jointly.
4. fungi quick detection kit according to claim 3 is characterized in that it comprises following reagent:
(1) DNA extraction reagent: Buffer I, Buffer II, Buffer III; Buffer TE;
(2) reaction solution: PCR Buffer, universal primer SEQ ID NO:3～4,2mM MgCl
2, 0.2mM dNTPs, 2U/ μ L Taq archaeal dna polymerase;
(3) strand purified reagent: 75% (v/v) ethanolic soln, 0.2MNaOH, 10mM pH 7.6Tris-Acetate solution, binding buffer liquid, annealing buffer;
(4) sequencing reagent: archaeal dna polymerase, ATP sulfurylase, luciferase, apyrase, substrate A PS, fluorescein and dNTP.
5. the detection method of a fungi quick detection kit is characterized in that it comprises the steps:
(1) extracts fungal DNA in the clinical sample;
(2) be template with step (1) gained DNA, utilize universal primer to carry out fungi 18S, the pcr amplification of the internal transcribed spacer district ITS1 of 5.8S;
(3) pcr amplification product that step (2) is obtained combines with the magnetic bead of avidin mark and carries out the strand purifying;
(4) the strand purified product that step (3) is obtained carries out the tetra-sodium order-checking;
(5) the fungi kind is judged in the comparison of sequencing result and database.
6. the detection method of fungi quick detection kit according to claim 5, it is characterized in that the pcr amplification system described in the step (2) is: 10 * PCR buffer, 5 μ L, SEQ ID NO:10.3 μ L, SEQ ID NO:20.3 μ L, Template DNA 2 μ L, 0.2mM dNTPs, 2mM MgCl
2, Taq archaeal dna polymerase 2U adds sterilized water to 50 μ L; The pcr amplification condition is: 94 ℃ of 5min; 40 circulations, 94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 30s; 72 ℃ of 5min.
7. the detection method of fungi quick detection kit according to claim 5, it is characterized in that the pcr amplification system described in the step (2) is: 10 * PCR buffer, 5 μ L, SEQ ID NO:40.3 μ L, SEQ ID NO:50.3 μ L, Template DNA 2 μ L, 0.2mM dNTPs, 2mM MgCl
2, Taq archaeal dna polymerase 2U adds sterilized water to 50 μ L; The pcr amplification condition is: 94 ℃ of 5min; 40 circulations, 94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 30s; 72 ℃ of 5min.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103343168A (en) * | 2013-07-26 | 2013-10-09 | 中国农业科学院油料作物研究所 | Rapid identification method for pathogenic fungi |
| CN106987626A (en) * | 2017-03-27 | 2017-07-28 | 杭州迪安生物技术有限公司 | For a variety of fungies of quick detection and identify primer and probe and its application of strain |
| CN107058559A (en) * | 2017-05-15 | 2017-08-18 | 华南农业大学 | The molecular detecting method and kit of a kind of plant pathogenic fungi |
| CN109722485A (en) * | 2018-11-20 | 2019-05-07 | 上海派森诺生物科技股份有限公司 | A method of Rapid identification Human Fungi is sequenced based on sanger |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103343168A (en) * | 2013-07-26 | 2013-10-09 | 中国农业科学院油料作物研究所 | Rapid identification method for pathogenic fungi |
| CN103343168B (en) * | 2013-07-26 | 2015-03-11 | 中国农业科学院油料作物研究所 | Rapid identification method for pathogenic fungi |
| CN106987626A (en) * | 2017-03-27 | 2017-07-28 | 杭州迪安生物技术有限公司 | For a variety of fungies of quick detection and identify primer and probe and its application of strain |
| CN106987626B (en) * | 2017-03-27 | 2020-12-15 | 杭州迪安生物技术有限公司 | Primer and probe for rapidly detecting various fungi and identifying strains and application thereof |
| CN107058559A (en) * | 2017-05-15 | 2017-08-18 | 华南农业大学 | The molecular detecting method and kit of a kind of plant pathogenic fungi |
| CN107058559B (en) * | 2017-05-15 | 2020-10-23 | 华南农业大学 | Molecular detection method and kit for plant pathogenic fungi |
| CN109722485A (en) * | 2018-11-20 | 2019-05-07 | 上海派森诺生物科技股份有限公司 | A method of Rapid identification Human Fungi is sequenced based on sanger |
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| Publication number | Publication date |
|---|---|
| CN101974640B (en) | 2012-11-28 |
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