CN101402994B - Method for technical evaluation of hedgehog fungus Houjie bacterial with molecule labeling technology - Google Patents
Method for technical evaluation of hedgehog fungus Houjie bacterial with molecule labeling technology Download PDFInfo
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- CN101402994B CN101402994B CN2008100721353A CN200810072135A CN101402994B CN 101402994 B CN101402994 B CN 101402994B CN 2008100721353 A CN2008100721353 A CN 2008100721353A CN 200810072135 A CN200810072135 A CN 200810072135A CN 101402994 B CN101402994 B CN 101402994B
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Abstract
The invention relates to a method for identifying the houjie strain of hericium erinaceus 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 method provided by the invention for identifying the houjie strain of the hericium erinaceus by making use of the molecular marker technology is high in sensitivity and less in the required quantity of DNA. Compared with the conventional morphologic detection, antagonistic test and fruiting test, the method has the advantages of short detection time, high accuracy, easy judgment, good repetition and the like, provides a rapid and effective system of bacteria identification for the utilization of the germplasm resources of the hericium erinaceus and the registration work of new species, and is very important for scientific research and production.
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 the outstanding bacterial classification of Hericium erinaceus (Bull. Ex Fr.) Pers. monkey, belongs to biological technical field.
Background technology Hericium erinaceus (Bull. Ex Fr.) Pers. (Hericium erinaceus) Hericium erinaceus (Bull. Ex Fr.) Pers. is one of eight famous big mountains delicacy, is listed as four your name's meat and fish dishes with bird's nest, shark's fin, sea cucumber.The tradition traditional Chinese medical science is thought: Hericium erinaceus (Bull. Ex Fr.) Pers. property is flat, flavor is sweet, aid digestion, sharp the five internal organs, the function of profit six lungs and the effect of tonifying deficiency are arranged, can improve the human immunological competence, multiple digestive tract diseases such as chronic gastritis, duodenal ulcer, stomach ulcer and neurasthenia are all had better curative effect.Hericium erinaceus (Bull. Ex Fr.) Pers. is nutritious, is a kind of high protein, lower fat, is rich in multiple amino acids, polypeptide, polysaccharide and aliphatic acid amides material and the multivitamin good protective foods of needed by human.
China's domestic fungus resource is abundant, and kind is many, in recent years, because the bacterial classification management system is unsound, causes the hedgehog fungus bacterial confusion, the phenomenon of homonym, synonym occurs.A large amount of confused strain names not only makes the industrial technology standard of having no way of, the improved seeds that China is not almost selected by the The Breeding of Edible Mushroom technical specifications over nearly 10 years.And a large amount of majorities that use are separate tissue things without screening system and test of seed selection before the more than ten years.The bacterial classification of these non-kinds is again often by titled with newname, and its distinctiveness, consistence and stability have no way of finding out about it.This obscure with confusion not only bring inconvenience to production link, also scientific research and the academic exchange to edible mushrooms causes obstacle.
The artificial culture of Hericium erinaceus (Bull. Ex Fr.) Pers. forms fairly large gradually, has created favorable economic benefit.The fine hedgehog fungus bacterial is to obtain high per unit area yield and high-quality prerequisite and basis, and this has just determined the critical role of hedgehog fungus bacterial in the Hericium erinaceus (Bull. Ex Fr.) Pers. industry.Therefore for guarantee every batch with kind all accurate, reduce unnecessary loss, need easy, the identification of strains technology fast and accurately of exploitation.
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 outstanding bacterial classification of Hericium erinaceus (Bull. Ex Fr.) Pers. monkey.
The method of utilizing molecular marking technique to identify the outstanding bacterial classification of Hericium erinaceus (Bull. Ex Fr.) Pers. monkey of the present invention 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, the detection of SCAR-PCR molecule marker, the detection primer of use are the outstanding F/R of monkey, wherein, the outstanding F of monkey is 5 '-CGCAACCAAAACAAAAACGACAATG-3 ' ', the outstanding R of monkey is 5 '-CGTTCCACCCCTACGTTTAGCCTCA-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 414bp, be the sign of the outstanding bacterial classification of Hericium erinaceus (Bull. Ex Fr.) Pers. monkey.
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 using the outstanding F/R of primer monkey.Wherein M～17 are the swimming lane numbering; The molecular weight of the numeral dna fragmentation in left side, arrow indication are the outstanding bacterial strain specific markers of monkey.
Embodiment is illustrated below in conjunction with embodiment in order fully to disclose the method for utilizing molecular marking technique to identify the outstanding bacterial classification of Hericium erinaceus (Bull. Ex Fr.) Pers. monkey of the present invention.
Embodiment 1: a kind of method of utilizing molecular marking technique to identify the outstanding bacterial classification of Hericium erinaceus (Bull. Ex Fr.) Pers. monkey
A kind of method of utilizing molecular marking technique to identify the outstanding bacterial classification of Hericium erinaceus (Bull. Ex Fr.) Pers. monkey 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 l TE solution or aseptic deionized water dissolution precipitations ,-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, the outstanding F/R 1 μ l of 10 μ mol/L special primer for checking monkeys, 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.
The outstanding F/R of described special primer for checking monkey is to adopt round pcr, through a large amount of shaker tests, has obtained the specific DNA segment of the outstanding bacterial classification of Hericium erinaceus (Bull. Ex Fr.) Pers. monkey, based on this segmental dna sequence dna, and the special detection primer of the outstanding bacterial classification of design monkey.The outstanding F/R particular content of described special primer for checking monkey is: the outstanding F of monkey is 5 '-CGCAACCAAAACAAAAACGACAATG-3 ', the outstanding R of monkey is 5 '-CGTTCCACCCCTACGTTTAGCCTCA-3 '.
Four, pcr amplification product detects
Specifically detection method is, gets pcr amplification product 8 μ l and adds 1.5 μ l6 * 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 adopts the outstanding F/R of detection primer monkey that the Hericium erinaceus (Bull. Ex Fr.) Pers. bacterial strain is carried out SCAR-pcr amplification, has only the outstanding bacterial strain of monkey can amplify the DNA band that molecular weight is 414bp.
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.
Untitled1.ST25
SEQUENCE?LISTING
＜110〉University Of Agriculture and Forestry In Fujian
＜120〉utilize the method for having divided labeling technique to identify the outstanding bacterial classification of Hericium erinaceus (Bull. Ex Fr.) Pers. monkey
<130>
<160>2
<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
Claims (1)
1. a method of utilizing molecular marking technique to identify the outstanding bacterial classification of Hericium erinaceus (Bull. Ex Fr.) Pers. monkey 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 are the outstanding F/R of monkey, wherein, the outstanding F of monkey is 5 '-CGCAACCAAAACAAAAACGACAATG-3 ' ', the outstanding R of monkey is 5 '-CGTTCCACCCCTACGTTTAGCCTCA-3 ';
(2) detection of SCAR-PCR molecule marker is set up, the SCAR-PCR amplification system is 10 * PCR buffer, 2.5 μ l, 25mmol/L MgCL2 1 μ l, 2.5m mol/L dNTP 2 μ l, 5U/ulTaq DNA 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 be 94 ℃ 1 minute; 94 ℃ 45 seconds, 62 ℃ 45 seconds, 72 ℃ 1 minute, 30 circulations; 72 ℃ 5 minutes;
(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 414bp, be the sign of the outstanding bacterial classification of Hericium erinaceus (Bull. Ex Fr.) Pers. monkey.
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| 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 |
| 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 |
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Non-Patent Citations (4)
| Title |
|---|
| Ling Lu et al..PCR-Based Sensitive Detection of Medicinal Fungi Hericium Species from Ribosomal Internal Transcribed Spacer(ITS) Sequences.《Biological & Pharmaceutical Bulletin》.2002,第25卷(第8期),975-980. |
| Ling Lu et al..PCR-Based Sensitive Detection of Medicinal Fungi Hericium Species from Ribosomal Internal Transcribed Spacer(ITS) Sequences.《Biological & * |
| Pharmaceutical Bulletin》.2002,第25卷(第8期),975-980. * |
| 陶佳喜.鄂东地区猴头菇菌株的筛选实验.《华中农业大学学报》.2004,第23卷(第3期),311-313. * |
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