CN101402995B - Method for technical evaluation of hedgehog fungus bacterial Houwang with molecular marker technology - Google Patents
Method for technical evaluation of hedgehog fungus bacterial Houwang with molecular marker technology Download PDFInfo
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- CN101402995B CN101402995B CN2008100721368A CN200810072136A CN101402995B CN 101402995 B CN101402995 B CN 101402995B CN 2008100721368 A CN2008100721368 A CN 2008100721368A CN 200810072136 A CN200810072136 A CN 200810072136A CN 101402995 B CN101402995 B CN 101402995B
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Abstract
The invention relates to a method for identifying the strain of a hericium erinaceus king by making use of a molecular marker technology, which comprises the culture and collection of mycelium, the extraction of genomic DNA, the detection establishment of SCAR-PCR molecular markers and the detection of SCAR-PCR products. The invention adopts two pairs of different primer kings: F1/R1 and F2/R2, to detect the SCAR-PCR molecular markers of the strains to be tested and the SCAR-PCR products; when the king F1/R1 is used for detecting the primers, the molecular weight of the detection result of the SCAR-PCR products shown in a DNA map is a specific 1012bp DNA band which is the symbol of the strain of the hericium erinaceus king; when the king F2/R2 is used for detecting the primers, the molecular weight of the detection result of the SCAR-PCR products shown in a DNA map is a specific 371bp DNA band which is the symbol of the strain of the hericium erinaceus king.
Description
Technical field the present invention relates to the authentication method of a kind of microorganism, is specifically related to utilize molecular marking technique to identify the method for hedgehog fungus bacterial Monkey King, belongs to biological technical field.
Background technology Hericium erinaceus (Bull. Ex Fr.) Pers. (Hericium erinaceus) meat is tender delicious, nutritious.A long time ago, people are listed as it and bear's paw, sea cucumber, shark fin in " four big famous dishes ", and the laudatory title of " mountain delicacy hedgehog hydnum, seafood delights bird's nest " is arranged, and are used as tribute in ancient times.9 seed amino acids of needed by human, Hericium erinaceus (Bull. Ex Fr.) Pers. all contains.It is reported that the nutritive ingredient that Hericium erinaceus (Bull. Ex Fr.) Pers. contained is compared with present tame other edible fungus variety and all occupied first and second position, thereby seem precious more.
China's domestic fungus resource is abundant, and kind is many, but since part producing person have a mind to or rename unintentionally, cause the cultivation strain of Hericium erinaceus (Bull. Ex Fr.) Pers. the to exist confusion phenomena of " xenogenesis different name of the same name or of the same race ".This phenomenon has greatly been damaged the breeder and the producer's interests, brings obstacle not only for scientific research and academic exchange, and the formulation of standard production and target level of product quality is brought very big difficulty.
The contribution of good quality strain in Hericium erinaceus (Bull. Ex Fr.) Pers. per unit area yield and quality is very important, and this has determined the critical role of hedgehog fungus bacterial in the Hericium erinaceus (Bull. Ex Fr.) Pers. industry.Along with the appearance of large-scale factory culture mode, more and more higher to the requirement of Hericium erinaceus (Bull. Ex Fr.) Pers. cultivated strain quality, need more easy, identification of strains technology fast and accurately, with guarantee every batch with kind all accurate.
Summary of the invention the purpose of this invention is to provide a kind of method for quick that utilizes molecular marking technique to identify the hedgehog fungus bacterial Monkey King.
The method of utilizing molecular marking technique to identify the hedgehog fungus bacterial Monkey King of the present invention has 2 kinds, and 2 kinds of authentication methods all comprise mycelium culture and collection, the extraction of genomic dna, the detection foundation of SCAR-PCR molecule marker and the detection of SCAR-PCR product;
Method one is characterised in that
1, the detection of SCAR-PCR molecule marker, the detection primer of use is Monkey King F1/R1, wherein, Monkey King F1 is 5 '-CTCCTCCCTTCACAATAAATAGCCA-3 ' ', Monkey King R1 is 5 '-TCCCTGAACTTCTTTGTCTTCCCGT-3 ';
2, the detection of SCAR-PCR molecule marker is set up: the SCAR-PCR amplification system is 10 * PCRbuffer, 2.5 μ l, 25mmol/L MgCL
21 μ l, 2.5m mol/L dNTP 2 μ l, 5U/ul TaqDNA Polymerase 0.3 μ l, 10 μ mol/L detect each 1 μ l of primer, 1ng～10ng/ μ l template DNA 1 μ l, ddH
2O 16.2 μ l;
The SCAR-PCR reaction conditions is 94 ℃ of 1min; 94 ℃ of 45second, 62 ℃ of 45second, 72 ℃ of 1min, 30 circulations; 72 ℃ of 5min;
3, the detected result of SCAR-PCR product: show that in the DNA of electrophoresis detection collection of illustrative plates the molecular weight that amplifies is the specific DNA band of 1012bp, is the sign of hedgehog fungus bacterial Monkey King.
Method two is characterised in that:
1, the detection of SCAR-PCR molecule marker, the detection primer of use is Monkey King F2/R2, wherein, Monkey King F2 is 5 '-CCCTCCACAACCACCACAAGATAAT-3 ', Monkey King R2 is 5 '-CTCCTCGCCGTCTGCATAAGAACTC-3 ';
2, the detection of SCAR-PCR molecule marker is set up: the SCAR-PCR amplification system is 10 * PCRbuffer, 2.5 μ l, 25mmol/L MgCL
21 μ l, 2.5m mol/L dNTP 2 μ l, 5U/ul TaqDNA Polymerase 0.3 μ l, 10 μ mol/L detect each 1 μ l of primer, 1ng～10ng/ μ l template DNA 1 μ l, ddH
2O 16.2 μ l;
The SCAR-PCR reaction conditions is 94 ℃ of 1min; 94 ℃ of 45second, 67 ℃ of 45second, 72 ℃ of 1min, 30 circulations; 72 ℃ of 5min;
3, the detected result of SCAR-PCR product: show that in the DNA of electrophoresis detection collection of illustrative plates the molecular weight that amplifies is the specific DNA band of 371bp, is the sign of hedgehog fungus bacterial Monkey King.
The method of utilizing molecular marking technique to identify hedgehog fungus bacterial provided by the invention, have highly sensitive, needed DNA consumption is few, compares with conventional morphologic detection, antagonistic effect, fruiting experiment, has short, advantages such as accuracy is high, easy judgement, good reproducibility detection time.Specific as follows:
1, detection time is short: this detection method required time only needs 2-3 days, and conventional antagonistic effect required time needs two time-of-weeks at least, and conventional morphologic detection and fruiting experiment then need at least 2 months time.
2, accuracy height, judgement easily, and good reproducibility: this detection method is material with the genomic dna, DNA is stable genetic material, be not subject to the influence of extraneous factor etc., its 1012bp or the dominant marker of 371bp judge very clear simultaneously, reduce the artificial deviation of judging, improved accuracy greatly; In the conventional antagonistic effect, the antagonism manifestation has 3 kinds at least, even more, sometimes manifestation is not obvious, also is subjected to the influence of culture environment, and the antagonism performance not only can occur between the different varieties in planting, also can show between the bacterial strain between planting simultaneously, giving accurately, judgement causes difficulty; The easier influence that is subjected to each side's factors such as environment, time of fruiting experiment, poor repeatability has strengthened detection difficult; Diacritic feature is few between the different varieties of morphologic detection in same genus kind, some in addition do not have what difference, also need judgement person to have rich knowledge and experience simultaneously, can make accurately and to judge.
3, the dominant marker that obtains of this method can reduce the workload that the Hericium erinaceus (Bull. Ex Fr.) Pers. new variety are assert in addition, this method only needs 1 positive strain and 1 negative strain contrast just can compare fast during detection, and assert new variety with conventional morphology, antagonistic effect, fruiting experiment method, need all interior different varietiess of the same race are taken out of relatively together, just can make correct identification, this will very big man power and material, may make identification to carry out when wide in variety.
The present invention provides more efficiently strain identification system to the utilization of Hericium erinaceus (Bull. Ex Fr.) Pers. germ plasm resource and the registration work of new variety, and strengthens all significant to the protection work of China's Hericium erinaceus (Bull. Ex Fr.) Pers. germ plasm resource.
Description of drawings Fig. 1 detects the DNA collection of illustrative plates of hedgehog fungus bacterial amplification for adopting first method.Wherein 1～M is the swimming lane numbering; The molecular weight of the numeral dna fragmentation on right side, arrow indication are Monkey King bacterial strain specific markers.
Fig. 2 detects the DNA collection of illustrative plates of hedgehog fungus bacterial amplification for adopting second method.Wherein M～17 are the swimming lane numbering; The molecular weight of the numeral dna fragmentation in left side, the arrow indication is a Monkey King bacterial strain specific marker.
Embodiment is illustrated below in conjunction with embodiment in order fully to disclose the method for utilizing molecular marking technique to identify the hedgehog fungus bacterial Monkey King of the present invention.
Embodiment 1: a kind of method of utilizing molecular marking technique to identify the hedgehog fungus bacterial Monkey King
A kind of method of utilizing molecular marking technique to identify the hedgehog fungus bacterial Monkey King may further comprise the steps: one, mycelium culture and collection
(1) if confession was tested the preservation time of Hericium erinaceus (Bull. Ex Fr.) Pers. bacterial strain less than 6 months, then mycelium culture is carried out as follows:
1, with the broken PDA substratum that contains hericium mycelium of inoculation rake rake, the 250ml triangular flask is gone in switching, contains in the 100ml PDA liquid nutrient medium;
2, be placed on 25 ℃ of shaking tables, rotating speed 90～130r/min cultivates 7～10d;
3, with the cultured mycelia of filtered through gauze, distilled water flushing, filter paper blots;
4, take by weighing 0.5～1.3g mycelia, wrap with filter paper, be stored in-20 ℃ standby.
(2) if supply the test Hericium erinaceus (Bull. Ex Fr.) Pers. bacterial strain preservation time to be not less than 6 months, then mycelium culture adopts following method:
1, gets hedgehog fungus bacterial beans piece size and be transferred on the PDA inclined-plane, cultivated 7～10 days for 25 ℃;
2, with the broken PDA substratum that contains hericium mycelium of inoculation rake rake, the 250ml triangular flask is gone in switching, contains in the 100ml PDA liquid nutrient medium;
3, be placed on 25 ℃ of shaking tables, rotating speed 90～130r/min cultivates 7～10d;
4, with the cultured mycelia of filtered through gauze, distilled water flushing, filter paper blots;
5, take by weighing 0.5～1.3g mycelia, wrap with filter paper, be stored in-20 ℃ standby.
Two, the extraction of genomic dna, described genomic dna can extract from the mycelium of bacterial strain or sporophore.
1, go bail for and deposit standby hericium mycelium 0.5～1.3g, or sporophore 0.5～1.3g, put into mortar, add the rapid grind into powder of liquid nitrogen, change the centrifuge tube of 7ml over to;
2, the extract that adds 65 ℃ of preheatings of 2.5mL adds 50 μ l mercaptoethanols simultaneously, and concussion makes protein denaturation precipitation up and down;
3,65 ℃ of water-bath 1h are every 10min vibration 1 time;
4, the chloroform isoamyl alcohol mixing that adds 2.5ml removes deproteinize;
5,8000r/min, 4 ℃ of centrifugal 10min get supernatant to the 7ml centrifuge tube;
6, the CTAB/NaCl mixing that adds 65 ℃ of preheatings of 1/5 volume, the chloroform isoamyl alcohol mixing of adding 2.5ml;
7,10000r/min, 4 ℃ of centrifugal 10min get supernatant;
8, add the CTAB precipitated liquid of 65 ℃ of preheatings of 2.5ml, put upside down mixing, as seen 65 ℃ of water-bath 1h maybe can spend the night to precipitating;
9,12000r/min behind 4 ℃ of centrifugal 10min, carefully removes supernatant liquor;
10, with 0.5ml TE solution or sterilized water dissolution precipitation 10min;
11, add saturated phenol of 0.25ml and 0.25m L chloroform isoamyl alcohol, mixing;
12,12000r/min, 4 ℃ of centrifugal 10min get supernatant, add the dehydrated alcohol of 2 times of volume precoolings, mixing;
13, place-20 ℃ of refrigerator 1h or spend the night;
14,12000r/min, 4 ℃ of centrifugal 10min abandon supernatant;
15, natural air drying precipitation, with 30 μ lTE solution or aseptic deionized water dissolution precipitation ,-20 ℃ of preservations are standby.
Three, the detection of SCAR-PCR molecule marker is set up
SCAR-PCR amplification system: 10 * PCR buffer, 2.5 μ l, 25mmol/L MgCL
21 μ l, 2.5m mol/L dNTP 2 μ l, 5U/ul Taq DNA Polymerase 0.3 μ l, 10 μ mol/L special primer for checking Monkey King F1/R1,1 μ l, 1ng～10ng/ μ l template DNA 1 μ l, ddH
2O 16.2 μ l.
SCAR-PCR reaction conditions: 94 ℃ of 1min; 94 ℃ of 45second, 62 ℃ of 45second, 72 ℃ of 1min, 30 circulations; 72 ℃ of 5min.
Described special primer for checking Monkey King F1/R1 is to adopt round pcr, through a large amount of shaker tests, has obtained the specific DNA segment of hedgehog fungus bacterial Monkey King, based on this segmental dna sequence dna, and the special detection primer of design Monkey King bacterial classification.Described special primer for checking Monkey King F1/R1 particular content is: Monkey King F1 is 5 '-CTCCTCCCTTCACAATAAATAGCCA-3 ', Monkey King R1 is 5 '-TCCCTGAACTTCTTTGTCTTCCCGT-3 '.
Four, pcr amplification product detects
Specifically detection method is, gets pcr amplification product 8 μ l and adds 1.5 μ l, 6 * tetrabromophenol sulfonphthalein sample-loading buffer, and mixing at 1.0% sepharose, contains Goldview
TMThe DNA dyestuff carries out electrophoresis, and 5V/cm constant voltage electrophoresis 50min observes and takes pictures by the ultraviolet gel imaging system.Perhaps, get pcr amplification product 8 μ l, with 1.5 μ l, 6 * tetrabromophenol sulfonphthalein sample-loading buffer mixing, on the fine jade vinegar sugar gel of point sample in 1%, in 0.5 X tbe buffer liquid, 5V/cm constant voltage electrophoresis 0.5～1h, after electrophoresis finishes,, on the gel imaging instrument, take a picture then with EB dyeing.
The pcr amplification product detected result as shown in Figure 1, adopts detection primer Monkey King F1/R1 that the Hericium erinaceus (Bull. Ex Fr.) Pers. bacterial strain is carried out SCAR-pcr amplification, has only the Monkey King bacterial strain can amplify the DNA band that molecular weight is 1012bp.
Embodiment 2: a kind of method of utilizing molecular marking technique to identify the hedgehog fungus bacterial Monkey King
A kind of method of utilizing molecular marking technique to identify the hedgehog fungus bacterial Monkey King may further comprise the steps:
One, mycelium culture and collection, concrete grammar is identical with the step 1 of embodiment 1;
Two, the extraction of genomic dna, described genomic dna can extract from the mycelium of bacterial strain or sporophore; Concrete grammar is identical with the step 2 of embodiment 1;
Three, the detection of SCAR-PCR molecule marker is set up, and wherein, the SCAR-PCR amplification system is identical with the amplification system of embodiment 1 step 3; The SCAR-PCR reaction conditions is: 94 ℃ of 1min; 94 ℃ of 45second, 67 ℃ of 45second, 72 ℃ of 1min, 30 circulations; 72 ℃ of 5min.
Described special primer for checking Monkey King F2/R2 is to adopt round pcr, through a large amount of shaker tests, has obtained the specific DNA segment of hedgehog fungus bacterial Monkey King, based on this segmental dna sequence dna, and the special detection primer of design Monkey King bacterial classification.Described special primer for checking Monkey King F2/R2 particular content is: Monkey King F2 is 5 '-CCCTCCACAACCACCACAAGATAAT-3 ', Monkey King R2 is 5 '-CTCCTCGCCGTCTGCATAAGAACTC-3 '.
Four, pcr amplification product detects, and concrete grammar is identical with the concrete detection method of embodiment 1 step 4.The pcr amplification product detected result as shown in Figure 2, adopts detection primer Monkey King F2/R2 that the Hericium erinaceus (Bull. Ex Fr.) Pers. bacterial strain is carried out SCAR-pcr amplification, has only the Monkey King bacterial strain can amplify the DNA band that molecular weight is 371bp.
Main agents used in the present invention is as follows: (all chemical reagent are analytical pure)
1, CTAB extract: 100mmol/L Tris-HCl, 2.0%CTAB, 20mmol/LEDTA, 1.4mol/L NaCl, pH8.0;
2, CTAB precipitated liquid: 50mmol/L Tris-HCl, 1.0%CTAB, 10mmol/LEDTA, pH8.0;
3、CTAB/NaCl：0.7mol/L?NaCl，10％?CTAB；
4, TE damping fluid: 10mmol/L Tris HCl, 1mmol/L EDTA;
5、0.5×TBE：44.5mmol/L?Tris，50mmol/L?HBO
3、1mmol/LEDTA；
6, sample-loading buffer: 0.1% tetrabromophenol sulfonphthalein, 40% sucrose;
7, EB:10mg/ml ethidium bromide;
8, pcr amplification reagent is available from the precious biotech firm in Dalian.
Key instrument used in the present invention is as follows: the dual-purpose clean work station of the single horizontal vertical of SW-CJ-1FB type: Purifying Equipment Co., Ltd., Suzhou;
Portable Autoclave: three Shens, Shanghai;
The full temperature of HYG-A is shaken a bottle cabinet: the laboratory, Taicang;
Refrigerated centrifuge: Sigma 3K30;
Pcr amplification instrument: Eppendorf AG22331 Hamburg;
Palm type whizzer: the kylin medical apparatus Lx-100 of factory of Haimen City, Jiangsu palm type whizzer;
Gel imaging system: TANON-2008.
Sequence table
SEQUENCE?LISTING
＜110〉University Of Agriculture and Forestry In Fujian
＜120〉utilize molecular marking technique to identify the method for hedgehog fungus bacterial Monkey King
<130>
<160>4
<170>PatentIn?version?3.1
<210>1
<211>25
<212>DNA
＜213〉Hericium erinaceus (Bull. Ex Fr.) Pers.
<400>1
<210>2
<211>25
<212>DNA
＜213〉Hericium erinaceus (Bull. Ex Fr.) Pers.
<400>2
<210>3
<211>25
<212>DNA
＜213〉Hericium erinaceus (Bull. Ex Fr.) Pers.
<400>3
<210>4
<211>25
<212>DNA
＜213〉Hericium erinaceus (Bull. Ex Fr.) Pers.
<400>4
Claims (1)
1. a method of utilizing molecular marking technique to identify Hericium erinaceus (Bull. Ex Fr.) Pers. (Hericium erinaceus) bacterial classification Monkey King comprises the extraction of mycelium culture and collection, genomic dna, the detection foundation of SCAR-PCR molecule marker and the detection of SCAR-PCR product; It is characterized in that:
(1) detection of SCAR-PCR molecule marker, the detection primer of use is Monkey King F1/R1, wherein, Monkey King F1 is 5 '-CTCCTCCCTTCACAATAAATAGCCA-3 ', Monkey King R1 is 5 '-TCCCTGAACTTCTTTGTCTTCCCGT-3 ';
(2) detection of SCAR-PCR molecule marker is set up: the SCAR-PCR amplification system is 10 * PCRbuffer, 2.5 μ l, 25mmol/L MgCL
21 μ l, 2.5m mol/L dNTP 2 μ l, 5U/ul TaqDNA Polymerase 0.3 μ l, 10 μ mol/L detect each 1 μ l of primer, 1ng～10ng/ μ l template DNA 1 μ l, ddH
2O 16.2 μ l;
The SCAR-PCR reaction conditions is 94 ℃ of 1min; 94 ℃ of 45second, 62 ℃ of 45second, 72 ℃ of 1min, 30 circulations; 72 ℃ of 5min;
(3) detected result of SCAR-PCR product: show that in the DNA of electrophoresis detection collection of illustrative plates the molecular weight that amplifies is the specific DNA band of 1012bp, is the sign of hedgehog fungus bacterial Monkey King.
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| CN2008100721368A CN101402995B (en) | 2008-11-18 | 2008-11-18 | Method for technical evaluation of hedgehog fungus bacterial Houwang with molecular marker technology |
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| CN2010105395324A Division CN101985657B (en) | 2008-11-18 | 2008-11-18 | Method for identifying hericium erinaceus strain king by utilizing molecule marker technology |
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| CN101402995B true CN101402995B (en) | 2011-04-06 |
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Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105567844B (en) * | 2016-02-05 | 2018-12-11 | 杭州市农业科学研究院 | Identify the primer pair and method of Hericium erinaceus monkey outstanding person No. 2 or big hedgehog hydnum |
| CN105567842B (en) * | 2016-02-05 | 2019-01-15 | 杭州市农业科学研究院 | Identify Hericium erinaceus monkey outstanding No. 2 primer pairs and method |
| CN106244701B (en) * | 2016-08-25 | 2019-10-22 | 黑龙江省林副特产研究所 | A kind of building of Hericium erinaceus DNA bar code and identification method |
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