CN109324007A - A kind of edible fungus species degeneration detection kit and detection method - Google Patents
A kind of edible fungus species degeneration detection kit and detection method Download PDFInfo
- Publication number
- CN109324007A CN109324007A CN201811346503.9A CN201811346503A CN109324007A CN 109324007 A CN109324007 A CN 109324007A CN 201811346503 A CN201811346503 A CN 201811346503A CN 109324007 A CN109324007 A CN 109324007A
- Authority
- CN
- China
- Prior art keywords
- reagent
- detection
- tpi
- edible fungus
- concentration
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000001514 detection method Methods 0.000 title claims abstract description 51
- 230000007850 degeneration Effects 0.000 title claims abstract description 23
- 241000233866 Fungi Species 0.000 title claims abstract description 20
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 62
- 108700015934 Triose-phosphate isomerases Proteins 0.000 claims abstract description 53
- 102100033598 Triosephosphate isomerase Human genes 0.000 claims abstract description 51
- 230000000694 effects Effects 0.000 claims abstract description 43
- GNGACRATGGDKBX-UHFFFAOYSA-N dihydroxyacetone phosphate Chemical compound OCC(=O)COP(O)(O)=O GNGACRATGGDKBX-UHFFFAOYSA-N 0.000 claims abstract description 30
- 238000006243 chemical reaction Methods 0.000 claims abstract description 28
- XJLXINKUBYWONI-NNYOXOHSSA-O NADP(+) Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-NNYOXOHSSA-O 0.000 claims abstract description 20
- 230000031700 light absorption Effects 0.000 claims abstract description 17
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims abstract description 14
- 102000006602 glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 claims abstract description 14
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 claims abstract description 14
- 238000005259 measurement Methods 0.000 claims abstract description 11
- 238000004458 analytical method Methods 0.000 claims abstract description 8
- 238000006911 enzymatic reaction Methods 0.000 claims abstract description 8
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims abstract description 7
- 229910001629 magnesium chloride Inorganic materials 0.000 claims abstract description 7
- 239000006228 supernatant Substances 0.000 claims abstract description 6
- 102000004190 Enzymes Human genes 0.000 claims description 29
- 108090000790 Enzymes Proteins 0.000 claims description 29
- 239000007788 liquid Substances 0.000 claims description 15
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 claims description 13
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 12
- 239000010453 quartz Substances 0.000 claims description 12
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 12
- 230000035484 reaction time Effects 0.000 claims description 11
- 238000004364 calculation method Methods 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 9
- 230000008033 biological extinction Effects 0.000 claims description 6
- 229910052757 nitrogen Inorganic materials 0.000 claims description 6
- 230000003287 optical effect Effects 0.000 claims description 6
- ACFIXJIJDZMPPO-NNYOXOHSSA-N NADPH Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](OP(O)(O)=O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 ACFIXJIJDZMPPO-NNYOXOHSSA-N 0.000 claims description 2
- 239000000470 constituent Substances 0.000 claims description 2
- 239000000284 extract Substances 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 238000012360 testing method Methods 0.000 abstract description 6
- 238000007619 statistical method Methods 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract description 2
- KLAKIAVEMQMVBT-UHFFFAOYSA-N p-hydroxy-phenacyl alcohol Natural products OCC(=O)C1=CC=C(O)C=C1 KLAKIAVEMQMVBT-UHFFFAOYSA-N 0.000 abstract 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 abstract 1
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 12
- 238000004321 preservation Methods 0.000 description 8
- 238000004519 manufacturing process Methods 0.000 description 6
- 238000010521 absorption reaction Methods 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- LXJXRIRHZLFYRP-VKHMYHEASA-N D-glyceraldehyde 3-phosphate Chemical compound O=C[C@H](O)COP(O)(O)=O LXJXRIRHZLFYRP-VKHMYHEASA-N 0.000 description 4
- 102000005924 Triose-Phosphate Isomerase Human genes 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 230000004060 metabolic process Effects 0.000 description 4
- 239000000843 powder Substances 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- LXJXRIRHZLFYRP-VKHMYHEASA-L (R)-2-Hydroxy-3-(phosphonooxy)-propanal Natural products O=C[C@H](O)COP([O-])([O-])=O LXJXRIRHZLFYRP-VKHMYHEASA-L 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerol Natural products OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 235000001603 Pleurotus ostreatus Nutrition 0.000 description 2
- 240000001462 Pleurotus ostreatus Species 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 230000034659 glycolysis Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000003412 degenerative effect Effects 0.000 description 1
- 238000001952 enzyme assay Methods 0.000 description 1
- 230000004110 gluconeogenesis Effects 0.000 description 1
- -1 glycerol aldehyde Chemical class 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000004108 pentose phosphate pathway Effects 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
Landscapes
- Physics & Mathematics (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
A kind of edible fungus species degeneration detection kit and detection method, kit include: one: Tris-HCl buffer of reagent, MgCl2；Reagent two: NADP；Reagent three: DHAP；Reagent four: GAPD；It after taking hypha of edible fungus broken, sets in centrifuge tube, is centrifuged, taking supernatant is TPI enzyme solution；100 μ L TPI enzyme solutions are added with kit to mix well, enzymatic reaction, UV spectrophotometry T1When light absorption value, the reaction was continued measurement T2When light absorption value, calculate TPI enzyme activity, with original strain be control, control with detection sample be respectively arranged 5 it is parallel, using statistics progress significance difference analysis be determined as degenerated strains as P < 0.05.Advantage is: detection kit high sensitivity, reproducible, low to detecting instrument dependence.Testing result can make the judgement whether strain has degenerated by conventional statistical method, and accuracy rate is high.
Description
Technical field
The present invention relates to a kind of edible fungus species degeneration detection kit and detection methods.
Background technique
The a large amount of strain of the development need of mushroom industry supports that strain is as the important means of production, to the hair of industry
Exhibition plays a crucial role, it might even be possible to say the success or failure for determining cultivation.But since strain belongs to asexual squamous subculture,
Algebra, storage conditions, poor environment factor and the dispersed mode of production formed for many years are cultivated, strain production lacks specification, bacterium
Kind production and quality frequently occur problem, as industry development problem becomes increasingly conspicuous, gradually have become obstruction Edible Fungi Industry Development
One of bottleneck.Spawn degeneration problem causes the underproduction reduction of income to happen occasionally, and without ideal detection means before fruiting harvesting
Judged.The development of industry is the development of mushroom industry there is an urgent need to carry out scientific and effective management and assessment to strain
Source is provided to ensure.
It is not isolated between each metabolic pathway in organism, pass through common mesostate or transition step phase
It contacts, complicated metabolism network can be formed each other, mutual turn between the coordination progress and each metabolin of metabolism is realized with this
Change.Triose phosphate is then the important node in this complicated metabolism network, can be by glycolysis, pentose phosphate pathway, gluconeogenesis and sweet
Oily phospholipid metabolism etc. is associated, and participates in the maintenance of body stable state.In some cases, triose phosphate metabolic disorder state can be with
Characterization organism is in abnormality.
Summary of the invention
It is an object of that present invention to provide a kind of edible fungus species degeneration detection kit and detection methods, by using detection
Kit is compared to after phosphotriose isomerase (TPI, TIM) Enzyme activity assay with original strain, the evaluable fructification for changing bacterial strain
Conversion ratio, therefore, it is determined that spawn degeneration degree.
A kind of edible fungus species degeneration detection kit, the reagent constituents and use concentration:
Reagent one: buffer, comprising:
Tris-HCl buffer, pH7.4, concentration 0.01mol/L；
MgCl2, concentration 0.5mmol/L；
Liquid component, 4 DEG C are protected from light preservation, and validity period 6 months；
Reagent two: nicotinamide-adenine dinucleotide phosphate (NADP), concentration 10mmol/L；
Liquid component, -80 DEG C are protected from light preservation, validity period 6 months, avoid multigelation；Or guarantor is protected from light using -20 DEG C of solid dry powder
Hiding, is configured to the concentration with pure water using preceding；
Reagent three: Dihydroxyacetone Phosphate (DHAP), concentration 10mmol/L；
Liquid component, -20 DEG C are protected from light preservation, validity period 6 months, avoid multigelation；
Reagent four: glyceraldehyde 3-phosphate dehydro-genase (GAPD), 1 μm of ol/L of concentration；
Liquid component, -80 DEG C are protected from light preservation, validity period 6 months, avoid multigelation；Or guarantor is protected from light using -20 DEG C of solid dry powder
Hiding, is configured to the concentration with pure water using preceding；
The reagent one, reagent two, reagent three and reagent four volume ratio be 6:1:1:1.
A method of it is degenerated using edible fungus species degeneration detection kit detection edible fungus species, specific steps are such as
Under: 1. mycelia are broken, TPI is extracted: after taking hypha of edible fungus 0.2g, liquid nitrogen grinding broken；Broken mycelia 0.1g is accurately weighed to set
In centrifuge tube, 4 DEG C, 10min is centrifuged under 8000g centrifugal force, taking supernatant is TPI enzyme solution, is set on ice, to be measured；
2. enzyme activity determination:
Ultraviolet specrophotometer adjusts wavelength to 340nm, 25 DEG C of preheating 20min；
One 600 μ L of reagent, reagent two, reagent three, each 100 μ L of reagent four are added in 1mL quartz colorimetric utensil, step 1 is added and extracts
100 μ L TPI enzyme solutions mix well, carry out enzymatic reaction at 25 DEG C, timing measures T1When light absorption value A1, the reaction was continued to when
Between T2, measure T2When light absorption value A2。
TPI enzyme activity calculates
TPI enzyme-activity unit definition: the NADPH that every gram of sample generates 1nmol per minute be defined as an enzyme activity unit (U,
nmol/min/g)；
TPI (U)=△ A ÷ (ε × d) × V ÷ W ÷ △ T
Wherein: △ A:A2- A1；
ε: NADPH molar extinction coefficient, 6.22 × 103L/mol/cm；
D: quartz colorimetric utensil optical path, 1cm；
V: reaction system total volume, 1mL；
W: the quality of sample to be tested, g in reaction system；
△ T:T2- T1, the system reaction time, min；
3. with original strain be control, control with detection sample be respectively arranged 5 it is parallel, measurement TPI enzyme activity numerical value after, using system
Meter learns progress significance difference analysis and is determined as degenerated strains as P value P < 0.05.
Further, the T1=1min, T2=10min.
Further, light absorption value A1And A2Detection range between 0.2-0.6, such as be higher than 0.6 need to dilute sample to be tested or
Shorten the reaction time, detection sample quality need to be increased lower than 0.2 or extends the reaction time.
TPI can be catalyzed reversible between two kinds of triose-phosphate isomerase bodies of dihydroxyacetone phosphate and D type glyceraldehyde-3-phosphate
Conversion participates in glycolysis and energy production.
Testing principle is that Dihydroxyacetone Phosphate can be converted to glyceraldehyde 3-phosphate, 3- phosphorus by phosphotriose isomerase
Acid glycerol aldehyde, which is reacted with nicotinamide-adenine dinucleotide phosphate by glyceraldehyde 3-phosphate dehydro-genase, generates 3-phoshoglyceric acid
There is absorption peak at 340nm with reduced nicotinamide adenine dinucleotide phosphate (NADPH), NADPH, 25 DEG C of enzymatic reactions,
Timing starts T by reaction1Terminate T with reaction2When light absorption value variation measurement TPI enzyme activity.
The present invention studies the normal strain of edible mushroom and degenerated strains difference by genomics, proteomics methodology, uses
The comparison of high-flux sequence genome difference, iTRQA differential protein analysis, proposition Testing index phosphotriose isomerase (TPI,
TIM) enzyme activity.In edible mushroom mycelium, when enzyme activity exception, the variation and loss of original good strains of seeds will lead to, have
Body shows as fruiting body yield decline, misshapen mushroom ratio increases, i.e., " spawn degeneration ".By to TPI enzyme quantitative analysis, Neng Goushi
The spawn degeneration detection of existing non-fruiting form, prejudges spawn degeneration, avoids losing.The beneficial effect is that:
(1), the industry problems that edible fungus species degeneration can not prejudge are solved；
(2), the spawn degeneration detection method determines that degenerated strains accuracy is high；
(3), the detection kit only needs conventional ultraviolet specrophotometer can be detected, low to detecting instrument dependence；
(4) detection kit includes that component simply easily obtains, and is detected swift and convenient to operate；
(5) the detection kit high sensitivity, TPI lowest detection is limited to 50mU/mL, and detection process is not introduced into other and may lead
The ingredient of testing result variation is caused, testing result repetitive rate is high.
Specific embodiment
Embodiment 1
Oyster mushroom P16 strain situation is detected using the detection method and kit:
(1) mycelia is crushed, TPI is extracted: the mushroom P16 that makes even control and each 1g of sample to be tested mycelia, liquid nitrogen grinding are broken；Accurately weigh
Broken mycelia 0.1g is set in centrifuge tube, and 4 DEG C, 8000g is centrifuged 10min, takes supernatant to set to be measured on ice, and 5 weights are arranged in every sample
It is multiple；
(2) enzyme activity determination:
Ultraviolet specrophotometer adjusts wavelength to 340nm, 25 DEG C of preheating 20min；
Reagent one: buffer, comprising:
Tris-HCl buffer, pH7.4, concentration 0.01mol/L；MgCl2, concentration 0.5mmol/L；
Liquid component, 4 DEG C are protected from light preservation, and validity period 6 months；
Reagent two: nicotinamide-adenine dinucleotide phosphate (NADP), concentration 10mmol/L；
Liquid component, -80 DEG C are protected from light preservation, validity period 6 months, avoid multigelation；Reagent three: Dihydroxyacetone Phosphate
(DHAP), concentration 10mmol/L；
Liquid component, -20 DEG C are protected from light preservation, validity period 6 months, avoid multigelation；
Reagent four: glyceraldehyde 3-phosphate dehydro-genase (GAPD), 1 μm of ol/L of concentration；
Liquid component, -80 DEG C are protected from light preservation, validity period 6 months, avoid multigelation；Or guarantor is protected from light using -20 DEG C of solid dry powder
Hiding, is configured to the concentration with pure water using preceding；
One 600 μ L of reagent, reagent two, reagent three, each 100 μ L of reagent four are added in 1mL quartz colorimetric utensil, it is to be measured that 100 μ L are added
Enzyme solution mixes well, and enzymatic reaction is carried out at 25 DEG C, and timing measures T1Light absorption value A when=1min1, the reaction was continued to 10min, inhales
Light value is lower than 0.2, extends the reaction time to T2=11min measures light absorption value A2；
TPI enzyme activity calculates:
TPI enzyme-activity unit definition: the NADPH that every gram of sample generates 1nmol per minute be defined as an enzyme activity unit (U,
nmol/min/g)。
Enzyme activity calculation formula: TPI (U)=△ A ÷ (ε × d) × V ÷ W ÷ △ T
Wherein: △ A:A2- A1；
ε: NADPH molar extinction coefficient, 6.22 × 103L/mol/cm；
D: quartz colorimetric utensil optical path, 1cm；
V: reaction system total volume, 1mL；
W: the quality of sample to be tested, g in reaction system；
△ T:T2- T1, the system reaction time, min；
3. with original strain be control, control with detection sample be respectively arranged 5 it is parallel, measurement TPI enzyme activity enzyme activity numerical value after, adopt
Significance difference analysis is carried out with statistics, as P value P < 0.05, is determined as degenerated strains.
Measure T1=1min and T2Numerical value is substituted into enzyme activity calculation formula, calculates TPI by=11min, the light absorption value of measurement
Enzyme activity, the statistical analysis significance of difference, the results are shown in Table 1.
Table 1
Control and sample to be tested significance of difference P value P < 0.05 determine that detection strain has been degenerated.
Parallel experiment in cultivation confirms that detection strain relatively control strain fructification conversion ratio reduces by 15%, degenerates and determines result
Really.
Embodiment 2
Mushroom L1 strain situation is detected using the detection method and kit:
(1) mycelia is crushed, TPI is extracted: taking mushroom L1 control and each 0.8g of sample to be tested mycelia, liquid nitrogen grinding is broken；It is accurate to claim
Broken mycelia 0.1g is taken to set in centrifuge tube, 4 DEG C, 8000g is centrifuged 10min, takes supernatant to set to be measured on ice, and every sample is arranged 5
It repeats；
(2) enzyme activity determination:
Ultraviolet specrophotometer adjusts wavelength to 340nm, 25 DEG C of preheating 20min；
Reagent one: buffer, comprising: Tris-HCl buffer, pH7.4, concentration 0.01mol/L；MgCl2, concentration 0.5mmol/
L；Reagent two: nicotinamide-adenine dinucleotide phosphate (NADP), concentration 10mmol/L；
Reagent three: Dihydroxyacetone Phosphate (DHAP), concentration 10mmol/L；
Reagent four: glyceraldehyde 3-phosphate dehydro-genase (GAPD), 1 μm of ol/L of concentration；
One 600 μ L of reagent, reagent two, reagent three, each 100 μ L of reagent four are added in 1mL quartz colorimetric utensil, it is to be measured that 100 μ L are added
Enzyme solution mixes well, and enzymatic reaction is carried out at 25 DEG C, and timing measures T1Light absorption value A when=1min1, the reaction was continued to T2=
6min measures light absorption value A2；
TPI enzyme activity calculates:
TPI enzyme-activity unit definition: the NADPH that every gram of sample generates 1nmol per minute be defined as an enzyme activity unit (U,
nmol/min/g)。
Enzyme activity calculation formula: TPI (U)=△ A ÷ (ε × d) × V ÷ W ÷ △ T
Wherein: △ A:A2- A1；
ε: NADPH molar extinction coefficient, 6.22 × 103L/mol/cm；
D: quartz colorimetric utensil optical path, 1cm；
V: reaction system total volume, 1mL；
W: the quality of sample to be tested, g in reaction system；
△ T:T2- T1, the system reaction time, min；
3. with original strain be control, control with detection sample be respectively arranged 5 it is parallel, measurement TPI enzyme activity enzyme activity numerical value after, adopt
Significance difference analysis is carried out with statistics, as P value P < 0.05, is determined as degenerated strains.
Measure T1=1min and T2Numerical value is substituted into enzyme activity calculation formula, calculates TPI enzyme by=6min, the light absorption value of measurement
Vigor, the statistical analysis significance of difference, the results are shown in Table 2.
Table 2
Control and sample to be tested significance of difference P value P < 0.05 determine that detection strain has been degenerated.
Parallel experiment in cultivation confirms that detection strain relatively control strain fructification conversion ratio reduces by 23%, and breeding time extends,
Misshapen mushroom quantity increases, and degenerates and determines real result.
Embodiment 3
The excellent white strain situation of needle mushroom F is detected using the detection method and kit:
[1] mycelia is crushed, TPI is extracted: taking the excellent white control of needle mushroom F and each 1g of sample to be tested mycelia, liquid nitrogen grinding broken；Accurately
It weighs broken mycelia 0.1g to set in centrifuge tube, 4 DEG C, 8000g is centrifuged 10min, takes supernatant to set to be measured on ice, every sample setting 5
A repetition；
[2] enzyme activity determination:
Ultraviolet specrophotometer adjusts wavelength to 340nm, 25 DEG C of preheating 20min；
Reagent one: buffer, comprising: Tris-HCl buffer, pH7.4, concentration 0.01mol/L；MgCl2, concentration 0.5mmol/
L；
Reagent two: nicotinamide-adenine dinucleotide phosphate (NADP), concentration 10mmol/L；
Reagent three: Dihydroxyacetone Phosphate (DHAP), concentration 10mmol/L；
Reagent four: glyceraldehyde 3-phosphate dehydro-genase (GAPD), 1 μm of ol/L of concentration；
One 600 μ L of reagent, reagent two, reagent three, each 100 μ L of reagent four are added in 1mL quartz colorimetric utensil, it is to be measured that 100 μ L are added
Enzyme solution mixes well, and enzymatic reaction is carried out at 25 DEG C, and timing measures T1Light absorption value A when=3min1, the reaction was continued to T2=
8min measures light absorption value A2；
TPI enzyme activity calculates:
TPI enzyme-activity unit definition: the NADPH that every gram of sample generates 1nmol per minute be defined as an enzyme activity unit (U,
nmol/min/g)。
Enzyme activity calculation formula: TPI (U)=△ A ÷ (ε × d) × V ÷ W ÷ △ T
Wherein: △ A:A2- A1；
ε: NADPH molar extinction coefficient, 6.22 × 103L/mol/cm；
D: quartz colorimetric utensil optical path, 1cm；
V: reaction system total volume, 1mL；
W: the quality of sample to be tested, g in reaction system；
△ T:T2- T1, the system reaction time, min；
3. with original strain be control, control with detection sample be respectively arranged 5 it is parallel, measurement TPI enzyme activity enzyme activity numerical value after, adopt
Significance difference analysis is carried out with statistics, as P value P < 0.05, is determined as degenerated strains.
Measure T1=1min and T2Numerical value is substituted into enzyme activity calculation formula, calculates TPI enzyme by=8min, the light absorption value of measurement
Vigor, the statistical analysis significance of difference, the results are shown in Table 3.
Table 3
Control and sample to be tested significance of difference P value P > 0.05 determine that detection strain is not degenerated.
The parallel experiment in cultivation of the factorial production confirms, detection strain with compare strain fructification conversion ratio without significant difference,
Determine real result.
Continuous squamous subculture detection test
Oyster mushroom P615, initial strains are labeled as P615-1, and continuous squamous subculture 10 times takes the 5th and the 10th passaged strain, point
Biao Ji not be and P615-10 that the detection method and kit is used to detect strain situation:
[1] mycelia is crushed, TPI is extracted: the mushroom P615-1 that makes even is P615-5 and P615-10 as control, sample to be tested, is taken respectively
Each 1g of mycelia, liquid nitrogen grinding are broken；It accurately weighs broken mycelia 0.1g to set in centrifuge tube, 4 DEG C, 8000g is centrifuged 10min, takes
Clear liquid sets to be measured on ice, 5 repetitions of every sample setting；
[2] enzyme activity determination:
Ultraviolet specrophotometer adjusts wavelength to 340nm, 25 DEG C of preheating 20min；
Reagent one: buffer, comprising: Tris-HCl buffer, pH7.4, concentration 0.01mol/L；MgCl2, concentration 0.5mmol/
L；
Reagent two: nicotinamide-adenine dinucleotide phosphate (NADP), concentration 10mmol/L；
Reagent three: Dihydroxyacetone Phosphate (DHAP), concentration 10mmol/L；
Reagent four: glyceraldehyde 3-phosphate dehydro-genase (GAPD), 1 μm of ol/L of concentration；
One 600 μ L of reagent, reagent two, reagent three, each 100 μ L of reagent four are added in 1mL quartz colorimetric utensil, it is to be measured that 100 μ L are added
Enzyme solution mixes well, and enzymatic reaction is carried out at 25 DEG C, and timing measures T1Light absorption value A when=1min1, the reaction was continued to T2=
11min measures light absorption value A2；
TPI enzyme activity calculates:
TPI enzyme-activity unit definition: the NADPH that every gram of sample generates 1nmol per minute be defined as an enzyme activity unit (U,
nmol/min/g)。
Enzyme activity calculation formula: TPI (U)=△ A ÷ (ε × d) × V ÷ W ÷ △ T
Wherein: △ A:A2- A1；
ε: NADPH molar extinction coefficient, 6.22 × 103L/mol/cm；
D: quartz colorimetric utensil optical path, 1cm；
V: reaction system total volume, 1mL；
W: the quality of sample to be tested, g in reaction system；
△ T:T2- T1, the system reaction time, min；
T will be measured1=1min and T2Numerical value is substituted into enzyme activity calculation formula, calculates TPI enzyme by=11min, the light absorption value of measurement
Vigor, the statistical analysis significance of difference, P615-1 and P615-5 passage statistical result is as shown in table 4, P615-5 and P615-10
It is as shown in table 5 to pass on statistical result.
Table 4
Table 5
After continuous passage, the parallel experiment in cultivation of fructification, conversion ratio drops to 114%, 94% by 130%, it was demonstrated that continuous passage
It will lead to spawn degeneration；TPI value also changes with passage number simultaneously, and P615-1 is with P615-5, P615-5 and P615-10 through counting
Credit analysis, shows the significant difference of downward trend, determines between generation from numerical value there are spawn degeneration phenomenon, determine with
It cultivates data to coincide, it was demonstrated that method is feasible.
Strain squamous subculture is vegetative propagation process, with energy exchange, the information exchange of mycelia individual and environment, mycelia
Reaction is responded to the variation adjustment of environment or condition, subtle quantitative change gradually accumulates, and causes kind of a property gradually to fail, merit
Separation weakens, and becomes spawn degeneration.Laboratory research discovery, between normal strains and degenerative strain, TPI vigor exists significant
Difference, and the lower spawn degeneration feature of TPI vigor is more obvious, and has strong correlation.
It selects TPI vigor to characterize spawn degeneration state, it is accurate feasible to be implemented verifying.
The above is only specific embodiments of the present invention, are not intended to restrict the invention, for those skilled in the art
For member, the invention may be variously modified and varied.All within the spirits and principles of the present invention, it is made it is any modification,
Equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Claims (4)
1. a kind of edible fungus species degeneration detection kit, it is characterized in that:
The reagent constituents and use concentration:
Reagent one: buffer, comprising: Tris-HCl buffer, pH7.4, concentration 0.01mol/L；MgCl2, concentration 0.5mmol/L；
Reagent two: nicotinamide-adenine dinucleotide phosphate (NADP), concentration 10mmol/L；
Reagent three: Dihydroxyacetone Phosphate (DHAP), concentration 10mmol/L；
Reagent four: glyceraldehyde 3-phosphate dehydro-genase (GAPD), 1 μm of ol/L of concentration；
The reagent one, reagent two, reagent three and reagent four volume ratio be 6:1:1:1.
2. a kind of method degenerated using the detection edible fungus species of edible fungus species degeneration detection kit described in claim 1,
It is characterized in that:
Specific step is as follows:
Mycelia is broken, TPI is extracted: after taking hypha of edible fungus 0.2g, liquid nitrogen grinding broken；Accurately weigh broken mycelia 0.1g set from
In heart pipe, 4 DEG C, 10min is centrifuged under 8000g centrifugal force, taking supernatant is TPI enzyme solution, is set on ice, to be measured；
Enzyme activity determination:
Ultraviolet specrophotometer adjusts wavelength to 340nm, 25 DEG C of preheating 20min；
One 600 μ L of reagent, reagent two, reagent three, each 100 μ L of reagent four are added in 1mL quartz colorimetric utensil, step 1 is added and extracts
100 μ L TPI enzyme solutions mix well, carry out enzymatic reaction at 25 DEG C, timing measures T1When light absorption value A1, the reaction was continued to when
Between T2, measure T2When light absorption value A2；
TPI enzyme activity calculation formula:
TPI (U)=△ A ÷ (ε × d) × V ÷ W ÷ △ T
Wherein: △ A:A2- A1；
ε: NADPH molar extinction coefficient, 6.22 × 103L/mol/cm；
D: quartz colorimetric utensil optical path, 1cm；
V: reaction system total volume, 1mL；
W: the quality of sample to be tested, g in reaction system；
△ T:T2- T1, the system reaction time, min；
With original strain be control, control with detection sample be respectively arranged 5 it is parallel, measurement TPI enzyme activity numerical value after, using statistics
It learns progress significance difference analysis and is determined as degenerated strains as P value P < 0.05.
3. the method degenerated according to claim 2 using edible fungus species degeneration detection kit detection edible fungus species,
It is characterized in that: the T1=1min, T2=10min.
4. the method degenerated according to claim 2 using edible fungus species degeneration detection kit detection edible fungus species,
It is characterized in that: light absorption value A1And A2Detection range between 0.2-0.6, if being higher than 0.6, dilute sample to be tested weight or contracting
Short reaction time；If being lower than 0.2, increases detection sample weight or extend the reaction time.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811346503.9A CN109324007A (en) | 2018-11-13 | 2018-11-13 | A kind of edible fungus species degeneration detection kit and detection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811346503.9A CN109324007A (en) | 2018-11-13 | 2018-11-13 | A kind of edible fungus species degeneration detection kit and detection method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109324007A true CN109324007A (en) | 2019-02-12 |
Family
ID=65260071
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811346503.9A Pending CN109324007A (en) | 2018-11-13 | 2018-11-13 | A kind of edible fungus species degeneration detection kit and detection method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109324007A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109916992A (en) * | 2019-04-15 | 2019-06-21 | 云南出入境检验检疫局检验检疫技术中心 | A kind of Mass Spectrometric Identification detection method of Wild Edible Fungi in Yunnan species |
| CN111424104A (en) * | 2020-01-21 | 2020-07-17 | 福建农林大学 | Method for rapidly judging degradation of pleurotus eryngii strain based on bacterial community composition and abundance and application of method |
-
2018
- 2018-11-13 CN CN201811346503.9A patent/CN109324007A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109916992A (en) * | 2019-04-15 | 2019-06-21 | 云南出入境检验检疫局检验检疫技术中心 | A kind of Mass Spectrometric Identification detection method of Wild Edible Fungi in Yunnan species |
| CN111424104A (en) * | 2020-01-21 | 2020-07-17 | 福建农林大学 | Method for rapidly judging degradation of pleurotus eryngii strain based on bacterial community composition and abundance and application of method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Murray et al. | Genetic improvement of sorghum as a biofuel feedstock: I. QTL for stem sugar and grain nonstructural carbohydrates | |
| Ratcliffe et al. | Revealing metabolic phenotypes in plants: inputs from NMR analysis | |
| Stackpole et al. | Genetic control of kraft pulp yield in Eucalyptus globulus | |
| US20100145625A1 (en) | Determination and prediction of the expression of traits of plants from the metabolite profile as a biomarker | |
| US20160319248A1 (en) | Fructose amino acid oxidase, preparation method and enzyme-containing kit for detecting glycated albumin | |
| Montero-Vargas et al. | Metabolic phenotyping for the classification of coffee trees and the exploration of selection markers | |
| CN109324007A (en) | A kind of edible fungus species degeneration detection kit and detection method | |
| Gong et al. | Metabolome and transcriptome integration reveals insights into flavor formation of ‘Crimson’watermelon flesh during fruit development | |
| Fernandez et al. | Metabolomic characterization of sunflower leaf allows discriminating genotype groups or stress levels with a minimal set of metabolic markers | |
| Edwards et al. | A simple microplate assay to quantify nonstructural carbohydrates of grapevine tissues | |
| Sui et al. | Genome‐wide association analysis of sucrose concentration in soybean (Glycine max L.) seed based on high‐throughput sequencing | |
| Liao et al. | Metabolome and transcriptome reveal novel formation mechanism of early mature trait in kiwifruit (Actinidia eriantha) | |
| Du et al. | Metabolic profiling of Pleurotus tuoliensis during mycelium physiological maturation and exploration on a potential indicator of mycelial maturation | |
| Zhang et al. | Metabolite profile of xylem sap in cotton seedlings is changed by K deficiency | |
| ElShamey et al. | Water deficit induced physiological and amino acid responses in some rice varieties using NMR‐metabolic analysis | |
| Simpson et al. | Experimental determination of group flux control coefficients in metabolic networks | |
| Strobbe et al. | A novel panel of yeast assays for the assessment of thiamin and its biosynthetic intermediates in plant tissues | |
| Ishikawa et al. | Measurement of chloroplastic NAD kinase activity and whole tissue NAD kinase assay | |
| Li et al. | Network analysis reveals the co-expression of sugar and aroma genes in the Chinese white pear (Pyrus bretschneideri) | |
| Mueller et al. | Shikimate accumulation in nine weedy species following glyphosate application | |
| Sanada et al. | Inheritance of the concentration of water‐soluble carbohydrates and its relationship with the concentrations of fibre and crude protein in herbage of cocksfoot (Dactylis glomerata L.) | |
| Farma | Assay Optimization of Lactate Levels in Athletes Using Nanophotometry Methods | |
| CN111650136A (en) | Detection method for activity of polyphenol oxidase of wheat grains and application of detection method | |
| Albertson et al. | Application of high performance anion exchange-pulsed amperometric detection to measure the activity of key sucrose metabolising enzymes in sugarcane | |
| CN110779887B (en) | Method for determining phosphoglycerate kinase activity |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |