CN109295174A - A kind of reagent set for detecting fungal infection, kit and detection method - Google Patents
A kind of reagent set for detecting fungal infection, kit and detection method Download PDFInfo
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Abstract
Reagent set, kit and the detection method that the invention discloses a kind of for detecting fungal infection, are related to field of biotechnology.The reagent set includes the combination of one or both of following reagent: PCR buffer and complex enzyme formulation；Wherein, PCR buffer contains following ingredient: potassium acetate, MgCl2, glycerol, DMSO, Tween-20 and PEG-200；Complex enzyme formulation contains following ingredient: glusulase, lysozyme, papain and laccase.Directly sample such as blood can be detected using the reagent set, do not need have many advantages, such as that high sensitivity, specificity are good and detection time is short to sample progress nucleic acid extraction purification process.
Description
Technical field
The present invention relates to field of biotechnology, in particular to a kind of reagent set for detecting fungal infection, reagent
Box and detection method.
Background technique
Malignant hematologic disease refers to Malignancy, including various types leukaemia, Huppert's disease, pernicious leaching
Bar tumor, myelodysplastic syndrome etc..This kind of patient in body's immunity disorder and therapeutic process due to being widely used
Glucocorticoid, cytotoxic drug, immune formulation, the use of broad-spectrum antibiotic and invasion operation increase in addition, so that invading
The incidence of attacking property nosomycosis (invasive fungal disease, IFD) is in gradually increase trend.The country is recent more than one
Epidemiological study statistics in center shows that the disease incidence of patient accounts for about 8%~15% in intensive care unit, organ transplant recipients
Disease incidence be 20%~40%, the disease incidence of hematological system tumor patient is up to about 31%.
Studies of invasive fungal infections, also known as invasive infections with fungi refer to that fungi invades tissue, blood, and give birth to wherein
Long breeding leads to the pathological change and pathophysiological process of histologic lesion, organ dysfunction and inflammatory reaction.
One of the most common infection pathogen is that the yeast-like fungi based on candida albicans and the filiform based on aspergillus are true
Bacterium accounts for 70%~90% and 10%~20% respectively.However, due to fungal infection atypism clinical manifestation, laboratory and
Imageological examination limit by specificity and sensibility, and early diagnosis is difficult, easy Misdiagnosis, delay treatment so that IFD at
For one of the important death cause of patients with malignant hematological diseases.
Early diagnosis is the key that successful treatment fungal infection, makes a definite diagnosis the goldstandard of studies of invasive fungal infections at present in addition to hidden ball
It is all traditional detection method other than the latex agglutination test of bacterium.And rely on traditional microorganism or pathology method diagnosis invasion
Nosomycosis, detection sensitivity is low, can not effectively be early diagnosed.Therefore, it for the early diagnosis of studies of invasive fungal infections, need to use up
Quick, special and easily operated detection method and reagent are developed fastly, are that clinically disease fungus detection research is urgent at present
The major issue for needing to solve.
Bio-molecular analysis is the hot spot of diagnosing fungal infections and research both at home and abroad at present, high specificity, high sensitivity,
It is the main trend of deep fungal infection early diagnosis.PCR diagnosis is in terms of the diagnosis of Invasive candidiasis than blood culture sensitivity
Fast, while again the false positive and false negative of GM testing inspection be can overcome the disadvantages that.But molecular biology method higher cost, operation are multiple
It is miscellaneous, do not standardize, it is also extensive for clinic.Currently, being mostly on the market cultivation or fungi (1-3)-about fungal detection
Callose detection kit, the temporary mature kit without other related detecting methods, wherein about cultivation detection reagent
Box, as a result accuracy rate is high, but time-consuming, can not carry out quickly having detection to subject；Fungi (1-3)-callose detection
Kit, that is, what is detected is the cell wall constituent of fungi, and this method can be applied to the detection of IFD early infection, but in following situation
Easily there is false positive (1) and carries out hemodialysis, sample or patient is exposed to gauze or other contain glucan using cellulose membrane in detection
Material；(2) Intravenous immunoglobulin, albumin, coagulation factor or blood product；(3) strepticemia；(4) it operates
There is pollution when handling sample in person.In addition, mucosa injury caused by polysaccharide anticancer drug, chemicotherapy is used to cause in food
The candida albicans of glucan or field planting, which enters blood etc. through gastrointestinal tract, may also cause false positive, edible fungi, such as mushroom food
It can lead to false positive.Thus testing result error is larger, and false positive rate is high, with a low credibility.
Based on this, the present invention is proposed.
Summary of the invention
The purpose of the present invention is to provide a kind of for detecting the reagent set of fungal infection, can be directly right using the reagent set
Sample such as blood is detected, without having high sensitivity, specificity to sample progress nucleic acid extraction purification process
The advantages that good and detection time is short.
Another object of the present invention is to provide a kind of kits for detecting fungal infection, can be directly right using the kit
Sample such as blood is detected, and is not needed to sample progress nucleic acid extraction purification process, with traditional detection method
It compares, has easy to operate, high sensitivity, specificity good and detection time is short, can also primarily determine fungi sense in positive sample
The type of dye, has many advantages, such as significant advantage, and practical application of being more convenient for is promoted.
Another object of the present invention is to provide a kind of detection method of fungal infection, the detection method directly with sample into
Row detection does not need have high sensitivity, specificity good and detection time sample progress nucleic acid extraction purification process
The advantages that short.
The present invention is implemented as follows:
On the one hand, the present invention provides a kind of for detecting the reagent set of fungal infection comprising one in following reagent
Kind or two kinds of combination: PCR buffer and complex enzyme formulation；
Wherein, the PCR buffer contains following ingredient: potassium acetate, MgCl2, glycerol, DMSO, Tween-20 and
PEG-200；
The complex enzyme formulation contains following ingredient: glusulase, lysozyme, papain and laccase.
Further, in some embodiments of the present invention, the PCR buffer contains following ingredient: 190-210mM
Potassium acetate, 19-21mM MgCl2, 45%-55% (v/v, ml/ml) glycerol, 24%-26% (v/v, ml/ml) DMSO,
0.45%-0.55% (v/v, ml/ml) Tween-20 and 3%-7% (v/v, ml/ml) PEG-200；PH is 8.2-8.4.
Further, in some embodiments of the present invention, the PCR buffer contains following ingredient: 200mM acetic acid
Potassium, 20mM MgCl2, 50% glycerol, 25%DMSO, 0.5%Tween-20 and 5%PEG-200；PH is 8.3.
Further, in some embodiments of the present invention, by volume percentage, the complex enzyme formulation contains:
20%-30% snail enzyme solution (i.e. every 100ml complex enzyme formulation snail containing 20-30ml enzyme solution), 15%-25% bacteriolyze enzyme solution,
10%-20% Papain enzyme solution and 5%-15% laccase liquid.
Wherein, the mass concentration of snail enzyme solution, bacteriolyze enzyme solution, Papain enzyme solution and laccase liquid is 10mg/ml, such as
Every ml snail enzyme solution contains 10mg glusulase pulvis.
Wherein, the Rate activity of glusulase and papain are as follows: 5.0 ten thousand U/g；The Rate activity of lysozyme and laccase are as follows: 2.0
Ten thousand U/g.
Further, in some embodiments of the present invention, by volume percentage, the complex enzyme formulation contains:
20% snail enzyme solution, 15% bacteriolyze enzyme solution, 10% Papain enzyme solution and 5% laccase liquid.
Glusulase in the complex enzyme formulation, can effective lytic cell wall, be released effectively cell inclusion；Pawpaw egg
White enzyme, the degradable protein in conjunction with nucleic acid, catalyzing hydrolysis multiple polypeptides key promote the separation of nucleic acid；Papain and
Glusulase be for the cracking of cell wall it is highly important, the two use in conjunction can be improved broken wall efficiency, shorten incubation time；
Lysozyme can be catalyzed β-(1,4) key water between the proteoglycan N-acetyl-glucosamine of bacteria cell wall and -acetylmuramic acid residue
Solution.
Above-mentioned PCR buffer and complex enzyme formulation are used in combination, and archaeal dna polymerase in sample such as blood can be made to press down
Object processed is denaturalized (such as: ferroheme, protein and fat), reduces the inhibiting effect to fluorescent PCR, and to occurring in blood sample
Fungi Direct Pyrolysis, while enhancing DNA polymerase activity.
Further, in some embodiments of the present invention, the complex enzyme formulation also contains following ingredient: Tris,
EDTA-2Na (disodium ethylene diamine tetraacetate), Triton X-100 (Triton X-100) and sodium citrate.
Further, in some embodiments of the present invention, the complex enzyme formulation contains following ingredient: 10mmol/L
Tris-HCL, pH8.0；10mmol/L EDTA-2Na；0.6% Triton X-100；2mmol/L sodium citrate.
When carrying out fluorescent quantitative PCR for blood for the prior art, need to carry out nucleic acid extraction to blood in advance
The problem of cross contamination and toxic reagent use largely is lost so as to cause nucleic acid and increased to purification process.Hair of the invention
Bright people carries out reasonably optimizing and scientific design by the ingredient to PCR buffer and complex enzyme formulation, so that provided by the invention
Reagent set for detecting fungal infection can carry out fluorescent PCR directly against the fungal nucleic acid in sample such as blood sample, with
Whether test sample deposits fungal infection, when using the reagent set test sample, does not need to carry out at nucleic acid extraction purifying sample
Reason and etc..It due to not needing additional nucleic acid extraction step, and then can avoid nucleic acid loss, reduce the risk of cross contamination,
The reliability and accuracy for improving testing result also improve the convenience of detection and shorten detection time.Entirely detected
Journey only needs 90min or so.
On the other hand, the present invention provides a kind of kits for detecting fungal infection comprising above-mentioned is true for detecting
The reagent set of bacterium infection.
Further, in some embodiments of the present invention, the kit further includes one had in following nucleic acid group
Kind or several combinations:
Fungal detection nucleic acid group, aspergillus detection nucleic acid group and candida albicans detect nucleic acid group；
Wherein, the fungal detection nucleic acid group includes: fungal detection primer pair shown in SEQ ID NO.1-2 and SEQ ID
Fungal detection probe shown in NO.3；The fungal detection nucleic acid group can detect clinical common deep disease fungus, including cigarette song
Mould, aspergillus flavus, aspergillus niger, candida albicans, Candida parapsilosis, Candida tropicalis, candida krusei, Candida glabrata, new life
Cryptococcus.Wherein, above-mentioned fungal detection primer pair and detection probe are degenerate primer and detection probe.
Aspergillus detection nucleic acid group include: aspergillus detection primer shown in SEQ ID NO.4-5 to and SEQ ID NO.6
Shown in aspergillus detection probe；Aspergillus detection nucleic acid group can detect aspergillus fumigatus, aspergillus flavus, aspergillus niger.
Candida albicans detection nucleic acid group include: candida albicans detection primer shown in SEQ ID NO.7-8 to and SEQ ID
Candida albicans detection probe shown in NO.9.Candida albicans detection nucleic acid group can detect candida albicans, Candida parapsilosis, torrid zone thought
Pearl bacterium, candida krusei, Candida glabrata.
Above-mentioned 3 detections nucleic acid group ensure that every kind of fungi primed probe and amplified production and other fungies, bacterium, virus
And human genome sequencing is without compared with high homology.
There is good consistency between this 3 detection nucleic acid groups, detection can be obtained final in same pipe detection architecture
As a result, and the advantages that the detection architecture high sensitivity, specificity are good and detection time is short.
Further, in some embodiments of the present invention, above-mentioned fungal detection probe, aspergillus detection probe and thought
5 ' ends of pearl bacterium detection probe are marked with different fluorophors, and 3 ' ends are marked with quenching group.
Such as 5 ' end flag F AM of fungal detection probe, 5 ' end label VIC of aspergillus detection probe, candida albicans detection are visited
5 ' end label CY3 of needle.Certainly, the classification of fluorophor can select according to the actual situation, as long as the fluorogene of three
Difference.The quenching group at 3 ' ends is NFQ.
Further, in some embodiments of the present invention, the kit further includes thermal starting taq DNA polymerization
Enzyme.
The kit that the present invention provides can be used for the early detection of Common fungi infection, improve the specificity and spirit of detection
Sensitivity.
On the other hand, the present invention provides a kind of detection methods of fungal infection comprising: by sample to be tested and institute as above
The reagent set mixing for the detection fungal infection stated.
Further, in some embodiments of the present invention, above-mentioned detection method is for the purpose of being diagnosed by non-disease.
Further, in some embodiments of the present invention, molten in the sample to be tested and the mixing of the reagent set
Thermal starting taq archaeal dna polymerase, primer pair, probe and dNTPs are also added in liquid.
Wherein, primer pair includes: fungal detection primer pair shown in SEQ ID NO.1-2, shown in SEQ ID NO.4-5
Aspergillus detection primer to and SEQ ID NO.7-8 shown in candida albicans detection primer pair；
Probe includes: fungal detection probe shown in SEQ ID NO.3, aspergillus detection probe shown in SEQ ID NO.6
With candida albicans detection probe shown in SEQ ID NO.9.
5 ' ends of above-mentioned fungal detection probe, aspergillus detection probe and candida albicans detection probe be marked with 3 kinds it is different
Fluorophor.
Further, in some embodiments of the present invention, the sample to be tested and the mixing of the reagent set is molten
Enzymolysis processing 15-25min under the conditions of liquid is placed in 36-38 DEG C.
Further, in some embodiments of the present invention, after enzymatic treatment, above-mentioned detection method includes: that fluorescence is fixed
Measure PCR step.
Further, in some embodiments of the present invention, the program of quantitative fluorescent PCR is as follows: 95 DEG C 15 minutes it is pre-
Denaturation；94 DEG C of 15s, 57 DEG C of 30s, 40 circulations；CY3, FAM and VIC signal are collected at 57 DEG C.
Application of the invention is more fast facilitated compared with traditional detection methods such as culture, microscopies, and above-mentioned detection method can operate
Property it is strong, as a result interpretation is convenient, and solving clinically fungi deep infection, to make a definite diagnosis the period long, requires laboratory and testing staff
The problems such as high, provides convenience for detection fungal infection.
Detailed description of the invention
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached
Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair
The restriction of range for those of ordinary skill in the art without creative efforts, can also be according to this
A little attached drawings obtain other relevant attached drawings.
Figure 1A is the fluorescent PCR amplification of sample 1 and sample 2 in experimental example 1.
Figure 1B is the fluorescent PCR amplification of sample 3 in experimental example 1.
Fig. 1 C is the fluorescent PCR amplification of sample 4 in experimental example 1.
Fig. 1 D is the fluorescent PCR amplification of sample 5 and sample 6 in experimental example 1.
Fig. 2 is the fluorescent PCR amplification of experimental example 2, and curve is CY3 fluorescence curve in figure.
Fig. 3 is the fluorescent PCR amplification of experimental example 3, and curve is CY3 fluorescence curve in figure.
Fig. 4 is the fluorescent PCR amplification of experimental example 4, and curve is CY3 fluorescence curve in figure.
Specific embodiment
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds
The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional products that can be obtained by commercially available purchase.
Feature and performance of the invention are described in further detail with reference to embodiments.
Embodiment 1
The kit of detection fungal infection provided in this embodiment comprising: thermal starting taq archaeal dna polymerase, 2 × PCR
Buffer, primer pair, probe and complex enzyme formulation；
Wherein, 2 × PCR buffer contains: 200mM potassium acetate, 20mM MgCl2, 50% glycerol, 25%DMSO, 0.5%
Tween-20 and 5%PEG-200；PH is 8.3.
Complex enzyme formulation includes: 20% glusulase, 15% lysozyme, 10% papain, 5% laccase, 10mmol/L
Tris-HCL, 10mmol/L EDTA-2Na, 0.6%Triton X-100 and 2mmol/L sodium citrate, pH7.2.
Complex enzyme formulation configuration step:
1,30% Triton X-100 stock solution is prepared: taking triton X-100 28.2ml and 0.1mol/l PBS
(ph7.3) 72.8mL is mixed, and sets 2~3h in 37 DEG C~40 DEG C water-baths, it is made sufficiently to dissolve mixing.
2,0.05M Tris-hydrochloride buffer is prepared: 50mL0.1M trishydroxymethylaminomethane (Tris) solution with
After 44.7mL 0.1M hydrochloric acid mixes, distilled water is added to be settled to 100 milliliters.
3,0.1mol/L EDTA-2Na solution is prepared: disodium EDTA 37.7g is weighed in a beaker,
The distilled water that 600-700mL is heated to 60-70 DEG C is added, stirring is poured into 1000mL volumetric flask after cooling and added to being completely dissolved
Enter distilled water constant volume, i.e. acquisition 0.1mol/L EDTA-2Na solution.
4, complex enzyme formulation buffer configures: weighing Citric Acid Mono (molecular weight 210.14) 21.01g in 50mL beaker
In, after the dissolution of a small amount of distilled water, 30% Triton X-100 stock solution 2mL, 0.05M Tris-hydrochloride buffer is added
After (0.05M, 25 DEG C) 2mL, 0.1mol/L EDTA-2Na solution 1mL, moves into 100mL volumetric flask and be settled to distilled water
100mL。
5, complex enzyme formulation buffer 100mL is taken to dissolve glusulase, lysozyme, papain and the thick enzyme powder of laccase respectively
The enzyme solution that corrresponding quality concentration is 10mg/ml is prepared after (4 DEG C) of the low temperature mixings that are vortexed in 1.0g.
6, the preparation (100ml) of complex enzyme formulation: taking the complex enzyme formulation buffer 50ml of pH7.2, and it is above-mentioned that 20ml is added
Snail enzyme solution, the bacteriolyze enzyme solution of 15ml, 10ml Papain enzyme solution 10ml and 5ml laccase enzyme solution.It obtains containing volume hundred
Divide the complex enzyme than being 20% snail enzyme solution, 15% bacteriolyze enzyme solution, 10% Papain enzyme solution and 5% laccase enzyme solution
Preparation.Wherein, specific activity of enzyme used in complex enzyme formulation is as follows:
Glusulase, papain (being purchased from prosperity Bioisystech Co., Ltd of Beijing ancient cooking vessel state) Rate activity are as follows: 5.0 ten thousand U/g；
Lysozyme (brand: Solarbio；Article No.: L8120), laccase (Shanghai Shi Feng Biotechnology Co., Ltd) Rate activity are as follows: 2.0
Ten thousand U/g.
Primer pair includes: fungal detection primer pair shown in SEQ ID NO.1-2, aspergillus shown in SEQ ID NO.4-5
Detection primer to and SEQ ID NO.7-8 shown in candida albicans detection primer pair, sequence is shown in Table 1.
Probe includes: fungal detection probe shown in SEQ ID NO.3, aspergillus detection probe shown in SEQ ID NO.6
With candida albicans detection probe shown in SEQ ID NO.9, sequence is shown in Table 1.5 ' end flag F AM of fungal detection probe, aspergillus inspection
5 ' end label VIC of probing needle, 5 ' end label CY3 of candida albicans detection probe.The quenching group at 3 ' ends of each probe is NFQ.
Table 1
Note: being degeneracy base, R=a or g, S=g or c, D=a, g or t at the sequence underscore of table 1.
Method using kit detection fungal infection is as follows:
(1) prepare subject's blood sample to be detected；
(2) each PCR system is 50 μ l total volumes: 1 μ l of thermal starting Taq DNA enzymatic, 5 μ l of complex enzyme formulation, 2 × blood are straight
25 μ l of PCR buffer, primer pair (concentration of every primer is 0.5 μM) 2 μ l, probe are connect to (2 μM of the concentration of every middle probe) 4
μ l, dNTPs (200 μM) 2 μ l, blood sample 10 μ l and ddH2O polishing is to 50 μ l.
(3) configured PCR system is put into fluorescence quantitative PCR instrument, carries out Real-time PCR detection；
Reaction condition are as follows:
First stage: 37 DEG C of 20 minutes complex enzyme formulation enzymolysis processings；
Second stage: 95 DEG C of 15 minutes initial denaturations；
Phase III: 94 DEG C of 15s, 57 DEG C of 30s, 40 circulations.
Signal collection: FAM, CY3 and VIC signal are collected at 57 DEG C of fourth stage.
After completing above-mentioned PCR reaction, result judgement is performed as follows in acquired results:
The detection judgement general for fungi: using Ct value no more than 35 as the positive, the Ct value of clinical samples is 25~35；
Aspergillus and candida albicans detection judgement: using Ct value no more than 40 as the positive, the Ct value of clinical samples is 30~40.
Experimental example 1
Experimental group:
Experimental group: using the kit of embodiment 1, by the application method in embodiment 1 to 6 parts of fungi known infection sun
Property sample (sample 1-6) carry out fluorescent PCR augmentation detection；
Control group: using regular-PCR buffer (10 ×) replace control group PCR buffer (2 ×) as compare, other
Same experimental group；The ingredient of regular-PCR buffer (10 ×) is as follows: 50mmol/L KCl, 2.5mmol/LMgCl2, 30mmol/L
(NH4)2SO4, 20 mmol/L Tris-HCl, pH8.3.
The result is shown in Figure 1 A- Fig. 1 D, Tu1AZhong: curve 1,5 and 7 is respectively the fungi Positive fluorescence of experimental group detection sample 1
(FAM) amplification curve, candida albicans Positive fluorescence amplification curve (CY3) and aspergillus negative signal fluorescence (VIC) amplification curve；
Curve 2,4 and 7 is respectively fungi Positive fluorescence (FAM) amplification curve of experimental group detection sample 2, candida albicans sun
Property fluorescent amplification curve (CY3) and aspergillus negative signal fluorescence (VIC) amplification curve；
Curve 3 and 7 is respectively fungi Positive fluorescence (FAM) amplification curve of control group detection sample 1, and candida albicans feminine gender is glimmering
Light amplification curve (CY3) and aspergillus negative signal fluorescence (VIC) amplification curve；
Curve 6 and 7 is fungi Positive fluorescence (FAM) amplification curve according to group detection sample 2, candida albicans feminine gender amplified fluorescence
Curve (CY3) and aspergillus negative signal fluorescence (VIC) amplification curve；
In Figure 1B: curve 1,3 and 4 is respectively fungi Positive fluorescence (FAM) amplification curve, the song of experimental group detection sample 3
Mould positive signal fluorescence (VIC) amplification curve and candida albicans feminine gender fluorescent amplification curve (CY3)；
Curve 2 and 4 is respectively fungi Positive fluorescence (FAM) amplification curve, the aspergillus negative signal of control group detection sample 3
Fluorescence (VIC) amplification curve and candida albicans feminine gender fluorescent amplification curve (CY3).
In Fig. 1 C: curve 1,2 and 3 is respectively fungi Positive fluorescence (FAM) amplification curve of experimental group detection sample 4, thought
Pearl bacterium Positive fluorescence (CY3) amplification curve and aspergillus positive signal fluorescence (VIC) amplification curve；Curve 4 and 5 is respectively control group
Detect fungi Positive fluorescence (FAM) amplification curve, aspergillus negative signal fluorescence (VIC) amplification curve and candida albicans yin of sample 4
Property fluorescent amplification curve (CY3)
In Fig. 1 D: curve 1 and 6 is respectively fungi Positive fluorescence (FAM) amplification curve, the aspergillus of experimental group detection sample 5
Negative signal fluorescence (VIC) amplification curve and candida albicans feminine gender fluorescent amplification curve (CY3)；Curve 2 and 6 is experimental group detection
Fungi Positive fluorescence (FAM) amplification curve of sample 6, aspergillus negative signal fluorescence (VIC) amplification curve and candida albicans feminine gender are glimmering
Light amplification curve (CY3)；Curve 3 and 4 is respectively fungi feminine gender fluorescence (FAM) amplification curve, the aspergillus of control group detection sample 5
Negative signal fluorescence (VIC) amplification curve and candida albicans feminine gender fluorescent amplification curve (CY3)；Curve 3 and 5 is respectively according to group inspection
Fungi feminine gender fluorescence (FAM) amplification curve, aspergillus negative signal fluorescence (VIC) amplification curve and the candida albicans of test sample sheet 6 are negative
Fluorescent amplification curve (CY3).
As can be seen that the fluorescent amplification curve inflection point of experimental group and Exponential growth stage are obvious in Figure 1A-Fig. 1 D, and control group
Without obvious " S " type amplification curve, illustrate as a result, embodiment 1 kit can directly to the fungal DNA in blood sample into
Row amplification, without nucleic acid extraction purifying, still available good amplification curve.
Experimental example 2
According to the form below 2 combines shown in 1-6, configures different composition complex enzyme formulation (preparation method and realities using same buffer
Apply that example 1 is identical, prepared according to 2 ratio of table using different enzyme solution volumes), to same fungi known to be checked infect positive sample into
Row detection, detection method step and other reagents used are same as Example 1, after to be detected, as a result see Fig. 2 (figure
2 kinds of curve 1-13 respectively represent combination 1-13), by distinguishing the optimum range of complex enzyme formulation configuration known to interpretation of result
Are as follows: glusulase (15-25%), lysozyme (10-20%), papain (5-15%), laccase (0-10%)；Wherein most preferably match
Than for combination 3: glusulase (20%), lysozyme (15%), papain (10%), laccase (5%).
The comparison of enzyme system using effect separately is mixed in order to investigate this 4 kinds mixing enzyme systems with 3 kinds, 2 kinds, test combinations 7- is set
12, the complex enzyme formulation of different compositions is configured, above-mentioned detecting step is repeated to same detection sample, after to be detected, is obtained
Experimental result is shown in Fig. 2, by comparative analysis it is found that containing 4 kinds of enzymes (glusulase (20%), lysozyme (15%), the papain
(10%) and laccase (5%)) mixing enzyme system be Optimal system.
Table 2
3 sensitivity technique of experimental example
To inquire into kit detection sensitivity of the present invention, the sensitivity of kit detection is studied respectively, through testing
Verifying discovery, kit of the present invention can reach 1 × 10 to the detection sensitivity of candida albicans and Aspergillus0CFU/ml, detection spirit
Sensitivity is strong, as follows for Candida albicans sensitivity technique, and the sensitivity of other candida albicans and aspergillus detection is not another herein
It repeats:
By taking the detection of the type strain of Candida albicans (ATCC10231) as an example, the Candida albicans of logarithmic growth phase is given birth to
It manages salt water and carries out 10 times of doubling dilutions at 1 × 107~100CFU/ml, identical Healthy People blood is added in totally eight gradients, difference equivalent
In liquid sample, this experimental example is obtained standard curve after to be detected, is passed through using kit and the method detection of embodiment 1
The size of type strain fluorescence curve shape and each concentration fluorescence curve correlation to each various concentration determines detection architecture
The range of linearity.
As a result as shown in Figure 3, it can be seen that various concentration standard from the type strain testing result curve graph of Candida albicans
Strain (107~100It CFU/ml) is in parallel standard serpentine amplification curve.Down to 1 × 100Still can under the concentration of CFU/ml
It realizes amplification, illustrates that the kit of embodiment 1 and method have good sensitivity as a result,.
4 specific detection of experimental example
To inquire into kit detection specificity of the present invention, the specificity of kit detection bacterial strain is studied respectively, is passed through
Verification experimental verification discovery, kit of the present invention all has good specificity to the detection of candida albicans and Aspergillus, to Candida albicans
As follows for bacterium specific detection, the specificity of other candida albicans and aspergillus detection does not repeat separately herein:
With Klebsiella Pneumoniae (ATCC700603) (sample 1), staphylococcus aureus (ATCC6538) (sample 3), white
Color candida albicans (ATCC10231) (sample 2) type strain detection for, by type strain storing liquid normal saline dilution at
107CFU/ml, equivalent is added in identical healthy human blood's sample respectively, is configured to sample to be examined (1-3), this experimental example is using real
Kit and the method detection for applying example 1, after to be detected, analyze experimental result by detection curve.
As a result as shown in Figure 4, three sample to be examined are after kit of the present invention and detection method detection, and only sample 2 is i.e. white
Color candida albicans occurs obvious detection curve, and other samples do not have testing result, illustrates kit and method tool of the invention
There is good specificity.
Experimental example 5
Using the kit and commercial reagent box of embodiment 1, (fungi (1,3)-callose immue quantitative detection reagent box is (aobvious
Color method), Dan Na Bioisystech Co., Ltd) and directly Microscopical Method For Detection identical 20 samples are detected, separately using goldstandard examine
Survey method (i.e. microculture detection method) is rechecked, and the kit of embodiment 1 is detected according to the step of embodiment 1, commercially available
Mannan antigen immunity detection reagent is carried out according to product description correlation step, direct Microscopical Method For Detection and microculture inspection
Survey method illustrates that step carries out referring to conventional microbiological examination criteria.It the results are shown in Table 3.
Table 3
In table :+indicate that pattern detection result is the positive, i.e., detect that fungi exists in sample；Indicate pattern detection result
For feminine gender, i.e., fungi is not detected in sample.
By the Analysis of test results to upper table 3 it is found that the kit of embodiment 1 passes through the testing result accuracy of sample
Traditional microbiological culture detects goldstandard verifying, accuracy rate of testing result 100%, the tradition culture detection compared to time-consuming several days
Method, kit of the present invention substantially reduce detection cycle and testing cost, together while ensure that testing result accuracy
When, the kit of the embodiment of the present invention 1 is compared with common G test detecting reagent kit and direct Microscopical Method For Detection, the standard of testing result
True property highest, while being detected using the kit of embodiment 1, it can also primarily determine the type of fungal infection in positive sample, have
There is significant advantage, practical application of being more convenient for is promoted.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
SEQUENCE LISTING
<110>Surexam Biotechnology Co., Ltd.
<120>a kind of reagent set for detecting fungal infection, kit and detection method
<130> 250
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Claims (10)
1. a kind of for detecting the reagent set of fungal infection, which is characterized in that it includes one or both of following reagent
Combination: PCR buffer and complex enzyme formulation；
Wherein, the PCR buffer contains following ingredient: potassium acetate, MgCl2, glycerol, DMSO, Tween-20 and PEG-200；
The complex enzyme formulation contains following ingredient: glusulase, lysozyme, papain and laccase.
2. according to claim 1 for detecting the reagent set of fungal infection, which is characterized in that the PCR buffer contains
There is following ingredient: 190-210mM potassium acetate, 19-21mM MgCl2, 45%-55% glycerol, 24%-26%DMSO, 0.45%-
0.55%Tween-20 and 3%-7%PEG-200；PH is 8.2-8.4.
3. according to claim 2 for detecting the reagent set of fungal infection, which is characterized in that the PCR buffer contains
There is following ingredient: 200mM potassium acetate, 20mM MgCl2, 50% glycerol, 25%DMSO, 0.5%Tween-20 and 5%PEG-
200；PH is 8.3.
4. according to claim 1-3 for detecting the reagent set of fungal infection, which is characterized in that press volume hundred
Divide than meter, the complex enzyme formulation contains: 20%-30% snail enzyme solution, 15%-25% bacteriolyze enzyme solution, 10%-20% pawpaw egg
White enzyme solution and 5%-15% laccase liquid.
5. according to claim 4 for detecting the reagent set of fungal infection, which is characterized in that volume percentage is pressed,
The complex enzyme formulation contains: 20% snail enzyme solution, 15% bacteriolyze enzyme solution, 10% Papain enzyme solution and 5% laccase liquid.
6. according to claim 5 for detecting the reagent set of fungal infection, which is characterized in that the complex enzyme formulation is also
Contain following ingredient: Tris, EDTA-2Na, Triton X-100 and sodium citrate.
7. a kind of kit for detecting fungal infection, which is characterized in that it includes described in any one of claims 1-6 for examining
Survey the reagent set of fungal infection.
8. it is according to claim 7 detection fungal infection kit, which is characterized in that the kit further include just like
The combination of one or more of lower nucleic acid group:
Fungal detection nucleic acid group, aspergillus detection nucleic acid group and candida albicans detect nucleic acid group；
Wherein, the fungal detection nucleic acid group includes: fungal detection primer pair shown in SEQ ID NO.1-2 and SEQ ID
Fungal detection probe shown in NO.3；
Aspergillus detection nucleic acid group include: aspergillus detection primer shown in SEQ ID NO.4-5 to and SEQ ID NO.6 shown in
Aspergillus detection probe；
Candida albicans detection nucleic acid group include: candida albicans detection primer shown in SEQ ID NO.7-8 to and SEQ ID NO.9
Shown in candida albicans detection probe.
9. a kind of detection method of fungal infection, characterized in that it comprises: by any one of sample to be tested and claim 1-6
The reagent set mixing of the detection fungal infection.
10. detection method according to claim 9, which is characterized in that by the mixed of the sample to be tested and the reagent set
Close enzymolysis processing 15-25min under the conditions of solution is placed in 36-38 DEG C.
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Cited By (1)
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|---|---|---|---|---|
| WO2021042323A1 (en) * | 2019-09-05 | 2021-03-11 | 中国石油大学(北京) | Method for extracting heavy oil genomic deoxyribonucleic acid, and kit and application thereof |
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