WO2019178901A1 - 一种蝉花菌株的scar分子标记鉴定方法 - Google Patents

一种蝉花菌株的scar分子标记鉴定方法 Download PDF

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WO2019178901A1
WO2019178901A1 PCT/CN2018/082481 CN2018082481W WO2019178901A1 WO 2019178901 A1 WO2019178901 A1 WO 2019178901A1 CN 2018082481 W CN2018082481 W CN 2018082481W WO 2019178901 A1 WO2019178901 A1 WO 2019178901A1
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pcr result
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PCT/CN2018/082481
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English (en)
French (fr)
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李春如
钱泽勇
王玉芹
戴梓飞
路文遥
董建飞
谭悠久
李增智
孙长胜
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浙江泛亚生物医药股份有限公司
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Publication of WO2019178901A1 publication Critical patent/WO2019178901A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi

Definitions

  • the invention relates to a method for identifying a strain of sassafras, belonging to the technical field of molecular biology.
  • the scented flower is a product of some nymphs that are parasitized by scorpion flowers and is a kind of worm complex.
  • This bacterium was named Miquel in 1838 by Isaria cicadae. Since then, a variety of synonymous names have emerged, such as Cordyceps cicadae, Sphaeria sinclairii, Cordyceps sinclairii, Isaria sinclairii, Isaria sinclairii, genus Cassia Torubia caespitosa, Isaria hariottii, Cordyceps sobolifera, Isaria mokanshawii and Isaria arbuscula.
  • the medicinal material scorpion flower is mainly a worm-like complex after parasitizing flowers.
  • the silk flower was cultured by taking the preserved sample of the safflower 17-7-1 and other different regions of the scented flower from the liquid nitrogen, and connecting it to the PSA slant medium on a clean bench, 22 ° C Incubate at -25 °C for 10 days, then connect the long squid in the PSA slant medium to the SAAY liquid medium on the ultra-clean workbench, shake the bacteria at 22 °C -25 °C for 24 h, collect the hyphae and freeze the liquid nitrogen, -80 °C save spare.
  • the main morphological characteristics of ⁇ 17-7-1 were as follows: cultured on PDA medium for 10 days at 25 °C, the colony was round, flat, initial flocculent, powdery, white to pale yellow, and the diameter was 5-6 cm.
  • the mycelium is colorless, branched, and has a membrane and is 1.2-2.5 ⁇ m wide.
  • the conidial spores are multi-branched, 3.0-5.5 ⁇ m wide, and consist of 2 to 5 sporulated cells per round.
  • the sporulated cells are in the shape of a bottle, the base is spherically expanded, and the upper is narrower and narrower, 4.5 to 6.5 x 2.5 to 3.5 ⁇ m.
  • the culture has the characteristics of easy cultivation and high yield. It has anti-tumor, immune regulation, blood sugar lowering, blood fat reduction, blood pressure lowering, eyesight, anti-radiation, antipyretic and analgesic. It has the functions of sedative hypnosis, nourishing and strengthening, and improving kidney function, so it has a wide range of industrial application values.
  • the object of the present invention is to provide a method for identifying a strain of Lycium bark 17-7-1.
  • a method for identifying a strain of Lycium chinensis 17-7-1 comprising the following steps:
  • Step 1 extracting DNA of the strain to be detected
  • Step 2 using the following primers for PCR amplification of the DNA of the strain to be detected obtained in step 1,
  • S11-2-F3 GTAGACCCGTGTTGTATGACAAACT
  • S11-2-R3 GTAGACCCGTAAGGAGACGGAGGAT
  • Step 3 the amplified product obtained in step 2 is subjected to agarose gel electrophoresis to obtain an electrophoresis band spectrum of the strain to be detected;
  • step 4 the map of the strain to be detected obtained in step 3 is analyzed to obtain a determination result.
  • the primer of step 2 can also be:
  • S11-2-F4 CCTCCCGTCGCTCATTGTT
  • PCR amplification reaction system used in the step 2 is as follows:
  • the PCR amplification reaction conditions used in the step 2 are as follows: first 95 ° C for 5 minutes; then 95 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 2.5 minutes, a total of 35 cycles; then 72 ° C extension 10 Minutes, finally cool down to 4 ° C to save.
  • the SCAR band spectrum obtained in the step 3 is analyzed, and it is found that the single 17-7-1 has a specific band, and in other different regions, the flower has no such specific band, and the flower is identified.
  • the strain is single 17-7-1.
  • the scorpion flower strain involved in the present invention is owned by Zhejiang Pan Asia Biopharmaceutical Co., Ltd., and the serial number 17-7-1 has been registered on November 18, 2009 at the General Microbiology Center of China Microbial Culture Collection Management Committee (CGMCC). Deposit, the deposit number is CGMCC No.3453.
  • the present invention finally obtains the primer of the present invention by screening a large number of random primers, and the primer has a very high specificity and has a unique specific band (the length of the fragments is 2414 bp and 2222 bp, respectively), which can well distinguish the production strain single 17- 7-1 and other different areas of flowers.
  • the SCAR molecular marker technology of the invention can obtain the result only by PCR and electrophoresis, and has the characteristics of short time-consuming, low price and simple operation.
  • Figure 1 is a SCAR band map corresponding to primers S11-2-F1 and S11-2-R1.
  • Figure 2 is a SCAR band map corresponding to primers S11-2-F2 and S11-2-R2.
  • Figure 3 is a SCAR band map corresponding to primers S11-10-F1 and S11-10-R1.
  • Figure 4 is a SCAR band map corresponding to primers S11-10-F2 and S11-10-R2.
  • Figure 5 is a SCAR band map corresponding to primers S11-7-F2 and S11-7-R2.
  • Figure 6 is a SCAR band map corresponding to primers S11-10-F3 and S11-10-R3.
  • Figure 7 is a SCAR band map corresponding to primers S11-11-F2 and S11-11-R2.
  • Figure 8 is a SCAR band map corresponding to primers S11-2-F3 and S11-2-R3.
  • Figure 9 is a SCAR band map corresponding to primers S11-2-F4 and S11-2-R4.
  • lane 1 is the PCR result of single 17-7-1
  • lane 2 is the PCR result of 15JJ8
  • lane 3 is the PCR result of 15JJ10
  • lane 4 is the PCR result of 15JJ14
  • lane 5 is the PCR of 15JJ18.
  • lane 6 is the PCR result of 15AX27
  • lane 7 is Marker
  • lane 8 is the PCR result of single 17-7-1
  • lane 9 is the PCR result of 15AX30
  • lane 10 is the PCR result of 15AX36
  • lane 11 is the PCR of 15AX39.
  • lane 12 is the PCR result of 15JGS35
  • lane 13 is the PCR result of 15JGS38;
  • lane 1 is the PCR result of single 17-7-1
  • lane 2 is the PCR result of 15JJ5
  • lane 3 is the PCR result of 15JJ7
  • lane 4 is the PCR result of 15JJ8, and
  • lane 5 is the PCR of 15JJ10.
  • lane 6 is the PCR result of 15JJ14
  • lane 7 is Marker
  • lane 8 is the PCR result of single 17-7-1
  • lane 9 is the PCR result of 15JJ17
  • lane 10 is the PCR result of 15JJ18
  • lane 11 is the PCR of 15AX22.
  • lane 12 was the PCR result of 15AX27
  • lane 13 was the PCR result of 15AX30.
  • Figure 10 is a SCAR band map corresponding to primers S11-2-F3 and S11-2-R3;
  • Lane 1 is the PCR result of single 17-7-1
  • lane 2 is the PCR result of 15JJ5
  • lane 3 is the PCR result of 15JJ7
  • lane 4 is the PCR result of 15JJ8
  • lane 5 is the PCR result of 15JJ10
  • lane 6 is the result of 15JJ14.
  • PCR results Lane 7 is Marker
  • Lane 8 is the PCR result of single 17-7-1
  • Lane 9 is the PCR result of 15JJ17
  • Lane 10 is the PCR result of 15JJ18
  • Lane 11 is the PCR result of 15AX22
  • Lane 12 is the 15AX27.
  • Lane 13 is the PCR result of 15AX30, wherein the lane 7 Marker bands are in order of molecular weight from top to bottom: 10000 bp, 5000 bp, 3000 bp, 2000 bp, 1500 bp, 1000 bp, 750 bp, 500 bp, 250 bp, 100 bp;
  • Lane 1 is a single 17-7-1 strip size of 2414 bp;
  • the lane size of lane 2 is 15JJ5 is estimated to be none;
  • the lane size of lane 3 is 15JJ7 is estimated to be none;
  • the lane size of lane 4 is 15JJ8 is estimated to be none;
  • the lane size of lane 5 is 15JJ10 is estimated to be none;
  • the lane size of lane 6 is estimated to be about 3000 bp for 15JJ14;
  • Lane 8 is a single 17-7-1 strip size of 2414 bp;
  • the lane size of lane 9 is 15JJ17 is estimated to be none;
  • Lane 10 is estimated to be 15JJ18 strip size
  • the lane size of lane 11 for 15AX22 is estimated to be none;
  • the lane size of lane 12 is 15AX27 is estimated to be none;
  • the lane size of lane 13 for 15AX30 is estimated to be none.
  • Figure 11 is a SCAR band map corresponding to primers S11-2-F3 and S11-2-R3;
  • Lane 1 is the PCR result of single 17-7-1
  • lane 2 is the PCR result of 15AX33
  • lane 3 is the PCR result of 15AX34
  • lane 4 is the PCR result of 15AX36
  • lane 5 is the PCR result of 15AX38
  • lane 6 is the result of 15AX39.
  • PCR results Lane 7 is Marker
  • Lane 8 is the PCR result of single 17-7-1
  • Lane 9 is the PCR result of 15JGS2
  • Lane 10 is the PCR result of 15JGS4
  • Lane 11 is the PCR result of 15JGS11
  • Lane 12 is the 15JGS35.
  • Lane 13 is the PCR result of 15JGS38, wherein the lane 7 Marker bands are in order of molecular weight from top to bottom: 10000 bp, 5000 bp, 3000 bp, 2000 bp, 1500 bp, 1000 bp, 750 bp, 500 bp, 250 bp, 100 bp;
  • Lane 1 is a single 17-7-1 strip size of 2414 bp;
  • the lane size of lane 2 is 15AX33 is estimated to be none;
  • the lane size of lane 3 is 15AX34 is estimated to be none;
  • the lane size of lane 4 is 15AX36 is estimated to be none;
  • the lane size of lane 5 is 15AX38 is estimated to be none;
  • the lane size of lane 6 is 15AX39 is estimated to be none;
  • Lane 8 is a single 17-7-1 strip size of 2414 bp;
  • the lane size of lane 9 is 15JGS2 is estimated to be none;
  • Lane 10 is estimated to be 15JGS4 strip size
  • Lane 11 is estimated to be 15JGS11 strip size
  • Lane 12 is estimated to be 15JGS35 strip size
  • the lane size of lane 13 for 15JGS38 is estimated to be none.
  • Figure 12 is a SCAR band map corresponding to primers S11-2-F3 and S11-2-R3;
  • Lane 1 Marker Lane 2 is the PCR result of single 17-7-1
  • Lane 3 is the PCR result of 15FY8
  • Lane 4 is the PCR result of 15FY13
  • Lane 5 is the PCR result of 15FY14
  • Lane 6 is the PCR result of 14DBS50
  • Lane 7 For the PCR result of 15LQ4, Lane 8 is a negative control, wherein the lane 1 Marker bands are in order of molecular weight from top to bottom: 10000 bp, 5000 bp, 3000 bp, 2000 bp, 1500 bp, 1000 bp, 750 bp, 500 bp, 250 bp, 100 bp;
  • Lane 2 is a single 17-7-1 strip size of 2414 bp;
  • the lane size of lane 3 is 15FY8 is estimated to be none;
  • the lane size of lane 4 is 15FY13 is estimated to be none;
  • the lane size of lane 5 is 15FY14 is estimated to be none;
  • Lane 6 is estimated to be 14DBS50 strip size
  • the lane size of lane 7 for 15LQ4 is estimated to be none;
  • Lane 8 is a negative control with no bands.
  • Figure 13 is a SCAR band map corresponding to primers S11-2-F4 and S11-2-R4;
  • Lane 1 is the PCR result of single 17-7-1
  • lane 2 is the PCR result of 15JJ5
  • lane 3 is the PCR result of 15JJ7
  • lane 4 is the PCR result of 15JJ8
  • lane 5 is the PCR result of 15JJ10
  • lane 6 is the result of 15JJ14.
  • PCR results Lane 7 is Marker
  • Lane 8 is the PCR result of single 17-7-1
  • Lane 9 is the PCR result of 15JJ17
  • Lane 10 is the PCR result of 15JJ18
  • Lane 11 is the PCR result of 15AX22
  • Lane 12 is the 15AX27.
  • Lane 13 is the PCR result of 15AX30, wherein the lane 7 Marker bands are in order of molecular weight from top to bottom: 10000 bp, 5000 bp, 3000 bp, 2000 bp, 1500 bp, 1000 bp, 750 bp, 500 bp, 250 bp, 100 bp;
  • Lane 1 is a single 17-7-1 strip size of 2222 bp;
  • the lane size of lane 2 is 15JJ5 is estimated to be none;
  • the lane size of lane 3 is 15JJ7 is estimated to be none;
  • the lane size of lane 4 is 15JJ8 is estimated to be none;
  • the lane size of lane 5 is 15JJ10 is estimated to be none;
  • the lane size of lane 6 is 15JJ14 is estimated to be none;
  • Lane 8 is a single 17-7-1 strip size of 2222 bp;
  • the lane size of lane 9 is 15JJ17 is estimated to be none;
  • Lane 10 is estimated to be 15JJ18 strip size
  • the lane size of lane 11 for 15AX22 is estimated to be none;
  • the lane size of lane 12 is 15AX27 is estimated to be none;
  • the lane size of lane 13 for 15AX30 is estimated to be none.
  • Figure 14 is a SCAR band map corresponding to primers S11-2-F4 and S11-2-R4;
  • Lane 1 is the PCR result of single 17-7-1
  • lane 2 is the PCR result of 15AX33
  • lane 3 is the PCR result of 15AX34
  • lane 4 is the PCR result of 15AX36
  • lane 5 is the PCR result of 15AX38
  • lane 6 is the result of 15AX39.
  • PCR results Lane 7 is Marker
  • Lane 8 is the PCR result of single 17-7-1
  • Lane 9 is the PCR result of 15JGS2
  • Lane 10 is the PCR result of 15JGS4
  • Lane 11 is the PCR result of 15JGS11
  • Lane 12 is the 15JGS35.
  • Lane 13 is the PCR result of 15JGS38, wherein the lane 7 Marker bands are in order of molecular weight from top to bottom: 10000 bp, 5000 bp, 3000 bp, 2000 bp, 1500 bp, 1000 bp, 750 bp, 500 bp, 250 bp, 100 bp;
  • Lane 1 is a single 17-7-1 strip size of 2222 bp;
  • the lane size of lane 2 is 15AX33 is estimated to be none;
  • the lane size of lane 3 is 15AX34 is estimated to be none;
  • the lane size of lane 4 is 15AX36 is estimated to be none;
  • the lane size of lane 5 is 15AX38 is estimated to be none;
  • the lane size of lane 6 is 15AX39 is estimated to be none;
  • Lane 8 is a single 17-7-1 strip size of 2222 bp;
  • the lane size of lane 9 is 15JGS2 is estimated to be none;
  • Lane 10 is estimated to be 15JGS4 strip size
  • Lane 11 is estimated to be 15JGS11 strip size
  • Lane 12 is estimated to be 15JGS35 strip size
  • the lane size of lane 13 for 15JGS38 is estimated to be none.
  • Figure 15 is a SCAR band map corresponding to primers S11-2-F4 and S11-2-R4;
  • Lane 1 Marker Lane 2 is the PCR result of single 17-7-1
  • Lane 3 is the PCR result of 15FY8
  • Lane 4 is the PCR result of 15FY13
  • Lane 5 is the PCR result of 15FY14
  • Lane 6 is the PCR result of 14DBS50
  • Lane 7 For the PCR result of 15LQ4, Lane 8 is a negative control, wherein the lane 1 Marker bands are in order of molecular weight from top to bottom: 10000 bp, 5000 bp, 3000 bp, 2000 bp, 1500 bp, 1000 bp, 750 bp, 500 bp, 250 bp, 100 bp;
  • Lane 2 is a single 17-7-1 strip size of 2222 bp;
  • the lane size of lane 3 is 15FY8 is estimated to be none;
  • the lane size of lane 4 is 15FY13 is estimated to be none;
  • the lane size of lane 5 is 15FY14 is estimated to be none;
  • Lane 6 is estimated to be 14DBS50 strip size
  • the lane size of lane 7 for 15LQ4 is estimated to be none;
  • Lane 8 is a negative control with no bands.
  • Figure 16 is a SCAR band map corresponding to primers S11-2-F3 and S11-2-R3.
  • Lane 1 is the PCR result of single 17-7-1
  • lane 2 is the PCR result of 15JJ5
  • lane 3 is the PCR result of 15JJ7
  • lane 4 is the PCR result of 15JJ8
  • lane 5 is the PCR result of 15JJ10
  • lane 6 is the result of 15JJ14.
  • PCR results Lane 7 is Marker
  • Lane 8 is the PCR result of single 17-7-1
  • Lane 9 is the PCR result of 15JJ17
  • Lane 10 is the PCR result of 15JJ18
  • Lane 11 is the PCR result of 15AX22
  • Lane 12 is the 15AX27.
  • Lane 13 is the PCR result of 15AX30, wherein the lane 7 Marker bands are in order of molecular weight from top to bottom: 10000 bp, 5000 bp, 3000 bp, 2000 bp, 1500 bp, 1000 bp, 750 bp, 500 bp, 250 bp, 100 bp.
  • Lane 1 is a single 17-7-1 strip size of 2414 bp.
  • the lane size of lane 2 for 15JJ5 is estimated to be none.
  • the lane size of lane 3 for 15JJ7 is estimated to be none.
  • the lane size of lane 4 for 15JJ8 is estimated to be none.
  • the lane size of lane 5 for 15JJ10 is estimated to be none.
  • the lane size of lane 6 is 15JJ14 and is estimated to be about 3000 bp.
  • Lane 8 is a single 17-7-1 strip size of 2414 bp.
  • the lane size of lane 9 for 15JJ17 is estimated to be none.
  • the lane size of lane 10 for 15JJ18 is estimated to be none.
  • the lane size of lane 11 for 15AX22 is estimated to be none.
  • the strip size of lane 12 for 15AX27 is estimated to be none.
  • the lane size of lane 13 for 15AX30 is estimated to be none.
  • Figure 17 is a SCAR band map corresponding to primers S11-2-F3 and S11-2-R3.
  • Lane 1 is the PCR result of single 17-7-1
  • lane 2 is the PCR result of 15AX33
  • lane 3 is the PCR result of 15AX34
  • lane 4 is the PCR result of 15AX36
  • lane 5 is the PCR result of 15AX38
  • lane 6 is the result of 15AX39.
  • PCR results Lane 7 is Marker
  • Lane 8 is the PCR result of single 17-7-1
  • Lane 9 is the PCR result of 15JGS2
  • Lane 10 is the PCR result of 15JGS4
  • Lane 11 is the PCR result of 15JGS11
  • Lane 12 is the 15JGS35.
  • Lane 13 is the PCR result of 15JGS38, wherein the lane 7 Marker bands are in order of molecular weight from top to bottom: 10000 bp, 5000 bp, 3000 bp, 2000 bp, 1500 bp, 1000 bp, 750 bp, 500 bp, 250 bp, 100 bp.
  • Lane 1 is a single 17-7-1 strip size of 2414 bp.
  • the lane size of lane 2 for 15AX33 is estimated to be none.
  • the lane size of lane 3 for 15AX34 is estimated to be none.
  • the lane size of lane 4 for 15AX36 is estimated to be none.
  • the lane size of lane 5 for 15AX38 is estimated to be none.
  • the lane size of lane 6 for 15AX39 is estimated to be none.
  • Lane 8 is a single 17-7-1 strip size of 2414 bp.
  • the lane size of lane 9 for 15JGS2 is estimated to be none.
  • the lane size of lane 10 for 15JGS4 is estimated to be none.
  • the lane size of lane 11 for 15JGS11 is estimated to be none.
  • the lane size of lane 12 for 15JGS35 is estimated to be none.
  • the lane size of lane 13 for 15JGS38 is estimated to be none.
  • Figure 18 is a SCAR band map corresponding to primers S11-2-F3 and S11-2-R3.
  • Lane 1 Marker Lane 2 is the PCR result of single 17-7-1
  • Lane 3 is the PCR result of 15FY8
  • Lane 4 is the PCR result of 15FY13
  • Lane 5 is the PCR result of 15FY14
  • Lane 6 is the PCR result of 14DBS50
  • Lane 7 For the PCR result of 15LQ4, Lane 8 is a negative control, wherein the lane 1 Marker bands are in order of molecular weight from top to bottom: 10000 bp, 5000 bp, 3000 bp, 2000 bp, 1500 bp, 1000 bp, 750 bp, 500 bp, 250 bp, 100 bp.
  • Lane 2 is a single 17-7-1 strip size of 2414 bp.
  • the strip size of lane 3 for 15FY8 is estimated to be none.
  • the lane size of lane 4 for 15FY13 is estimated to be none.
  • the lane size of lane 5 for 15FY14 is estimated to be none.
  • the lane size of lane 6 for 14DBS50 is estimated to be none.
  • the lane size of lane 7 for 15LQ4 is estimated to be none.
  • Lane 8 is a negative control with no bands.
  • Figure 19 is a SCAR band map corresponding to primers S11-2-F4 and S11-2-R4.
  • Lane 1 is the PCR result of single 17-7-1
  • lane 2 is the PCR result of 15JJ5
  • lane 3 is the PCR result of 15JJ7
  • lane 4 is the PCR result of 15JJ8
  • lane 5 is the PCR result of 15JJ10
  • lane 6 is the result of 15JJ14.
  • PCR results Lane 7 is Marker
  • Lane 8 is the PCR result of single 17-7-1
  • Lane 9 is the PCR result of 15JJ17
  • Lane 10 is the PCR result of 15JJ18
  • Lane 11 is the PCR result of 15AX22
  • Lane 12 is the 15AX27.
  • Lane 13 is the PCR result of 15AX30, wherein the lane 7 Marker bands are in order of molecular weight from top to bottom: 10000 bp, 5000 bp, 3000 bp, 2000 bp, 1500 bp, 1000 bp, 750 bp, 500 bp, 250 bp, 100 bp.
  • Lane 1 is a single 17-7-1 strip size of 2222 bp.
  • the lane size of lane 2 for 15JJ5 is estimated to be none.
  • the lane size of lane 3 for 15JJ7 is estimated to be none.
  • the lane size of lane 4 for 15JJ8 is estimated to be none.
  • the lane size of lane 5 for 15JJ10 is estimated to be none.
  • the lane size of lane 6 for 15JJ14 is estimated to be none.
  • Lane 8 is a single 17-7-1 strip size of 2222 bp.
  • the lane size of lane 9 for 15JJ17 is estimated to be none.
  • the lane size of lane 10 for 15JJ18 is estimated to be none.
  • the lane size of lane 11 for 15AX22 is estimated to be none.
  • the strip size of lane 12 for 15AX27 is estimated to be none.
  • the lane size of lane 13 for 15AX30 is estimated to be none.
  • Figure 20 is a SCAR band map corresponding to primers S11-2-F4 and S11-2-R4.
  • Lane 1 is the PCR result of single 17-7-1
  • lane 2 is the PCR result of 15AX33
  • lane 3 is the PCR result of 15AX34
  • lane 4 is the PCR result of 15AX36
  • lane 5 is the PCR result of 15AX38
  • lane 6 is the result of 15AX39.
  • PCR results Lane 7 is Marker
  • Lane 8 is the PCR result of single 17-7-1
  • Lane 9 is the PCR result of 15JGS2
  • Lane 10 is the PCR result of 15JGS4
  • Lane 11 is the PCR result of 15JGS11
  • Lane 12 is the 15JGS35.
  • Lane 13 is the PCR result of 15JGS38, wherein the lane 7 Marker bands are in order of molecular weight from top to bottom: 10000 bp, 5000 bp, 3000 bp, 2000 bp, 1500 bp, 1000 bp, 750 bp, 500 bp, 250 bp, 100 bp.
  • Lane 1 is a single 17-7-1 strip size of 2222 bp.
  • the lane size of lane 2 for 15AX33 is estimated to be none.
  • the lane size of lane 3 for 15AX34 is estimated to be none.
  • the lane size of lane 4 for 15AX36 is estimated to be none.
  • the lane size of lane 5 for 15AX38 is estimated to be none.
  • the lane size of lane 6 for 15AX39 is estimated to be none.
  • Lane 8 is a single 17-7-1 strip size of 2222 bp.
  • the lane size of lane 9 for 15JGS2 is estimated to be none.
  • the lane size of lane 10 for 15JGS4 is estimated to be none.
  • the lane size of lane 11 for 15JGS11 is estimated to be none.
  • the lane size of lane 12 for 15JGS35 is estimated to be none.
  • the lane size of lane 13 for 15JGS38 is estimated to be none.
  • Figure 21 is a SCAR band map corresponding to primers S11-2-F4 and S11-2-R4.
  • Lane 1 Marker Lane 2 is the PCR result of single 17-7-1
  • Lane 3 is the PCR result of 15FY8
  • Lane 4 is the PCR result of 15FY13
  • Lane 5 is the PCR result of 15FY14
  • Lane 6 is the PCR result of 14DBS50
  • Lane 7 For the PCR result of 15LQ4, Lane 8 is a negative control, wherein the lane 1 Marker bands are in order of molecular weight from top to bottom: 10000 bp, 5000 bp, 3000 bp, 2000 bp, 1500 bp, 1000 bp, 750 bp, 500 bp, 250 bp, 100 bp.
  • Lane 2 is a single 17-7-1 strip size of 2222 bp.
  • the strip size of lane 3 for 15FY8 is estimated to be none.
  • the lane size of lane 4 for 15FY13 is estimated to be none.
  • the lane size of lane 5 for 15FY14 is estimated to be none.
  • the lane size of lane 6 for 14DBS50 is estimated to be none.
  • the lane size of lane 7 for 15LQ4 is estimated to be none.
  • Lane 8 is a negative control with no bands.
  • the safflower single 17-7-1 strain used in the following examples was registered and deposited with the General Microbiology Center of China Microbial Culture Collection Management Committee (CGMCC) on November 18, 2009, and the deposit number is CGMCC No. 3453; other different regions ⁇ The flowers were collected from Anxi (numbers: 15AX22, 15AX27, 15AX30, 15AX33, 15AX34, 15AX36, 15AX38, 15AX39), Jiujiang (numbered: 15JJ5, 15JJ7, 15JJ8, 15JJ10, 15JJ14, 15JJ17, 15JJ18), Jinggangshan ( The numbers are: 15JGS2, 15JGS4, 15JGS11, 15JGS35, 15JGS38), Fuyang (number: 15FY8, 15FY13, 15FY14), Dabie Mountain (number: 14DBS50), Yueqing (number: 15LQ4).
  • the upper strains are all spores.
  • the CTAB method was used to extract the DNA of Lycium barbarum L. 17-7-1 and different regions, and the above primers were used for polymerase chain reaction amplification, followed by agarose electrophoresis to detect the electrophoresis bands.
  • the PCR reaction conditions are as follows:
  • test results are shown in Figure 1-9. It can be seen from the electropherogram that, except for the primers S11-2-F3 and S11-2-R3 (Fig. 8), S11-2-F4 and S11-2-R4 (Fig. 9), different regions in the electropherogram of other primers are shown. Both the scutellaria and the single 17-7-1 strain have one or more identical electrophoresis bands, and the S11-7-F1 and S11-7-R1, S11-11-F1 and S11-11-R1 PCR results have no bands.
  • ⁇ 17-7-1 has a specific band with a fragment length of It is 2414 bp and 2222 bp, while other flowers in different regions do not have, and can clearly distinguish ⁇ 17-7-1 from other different regions.
  • the primers S11-2-F3 and S11-2-R3, S11-2-F4 and S11-2-R4 obtained by screening in Example 1 were used to establish a single flower 17-7-1. Identification method.
  • the CTAB method was used to extract the same batch of safflower single 17-7-1 and different regions of scutellaria DNA, and the primers were synthesized by primers S11-2-F3 and S11-2-R3, S11-2-F4 and S11-2-R4. Enzyme chain reaction amplification, followed by agarose electrophoresis, detection of electrophoresis bands.
  • Electrophoresis patterns obtained by PCR using S11-2-F4 and S11-2-R4 as primers to extract PCR of the same batch of scutellaria single 17-7-1 and different regions of scutellaria DNA as templates (Fig. 13 and Fig. 14) Figure 15 shows).
  • the DNA extracted in the same batch was used as a template and repeated three times, and the main amplified fragments were identical.
  • CTAB method was used to extract different batches of scutellariae 17-7-1 and different regions of scorpion DNA, using primers S11-2-F3 and S11-2-R3, S11-2-F4 and S11-2-R4 for polymerization. Enzyme chain reaction amplification, followed by agarose electrophoresis, detection of electrophoresis bands.
  • Electrophoresis patterns obtained by PCR using S11-2-F3 and S11-2-R3 as primers to extract PCR from different batches of Lycium chinensis 17-7-1 and different regions of scutellaria (Figure 16, Figure 17) Figure 18 shows).
  • Electrophoresis patterns obtained by PCR using S11-2-F4 and S11-2-R4 as primers to extract PCR from different batches of Lycium chinensis 17-7-1 and different regions of scutellaria (Figure 19, Figure 20) Figure 21).
  • the DNA extracted from different batches was used as a template and repeated three times, and the main amplified fragments were basically identical.
  • SCAR markers are a stable and reliable method for detecting DNA polymorphisms in different samples.

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Abstract

提供一种蝉花单17-7-1菌株的鉴定方法，包括提取待检测菌株的DNA；对待检测菌株DNA采用引物S11-2-F3和S11-2-R3或S11-2-F4和S11-2-R4进行PCR扩增；获得的扩增产物经琼脂糖凝胶电泳后获得待检测菌株的SCAR条带图谱；将获得的待检测菌株SCAR图谱进行分析获得判定结果。

Description

一种蝉花菌株的SCAR分子标记鉴定方法 技术领域
本发明涉及一种蝉花菌株的鉴定方法，属于分子生物学技术领域。
背景技术
蝉花，别名虫花，是某些蝉若虫受蝉花寄生后的产物，是一种菌虫复合体。此菌于1838年由Miquel定名为蝉棒束孢霉Isaria cicadae。此后出现多种同物异名，如蝉草Cordyceps cicadae，辛克莱球壳孢Sphaeria sinclairii，辛克莱虫草Cordyceps sinclairii，辛克莱棒束孢Isaria sinclairii，基生棒束孢Isaria basili，丛生虫壳菌Torrubia caespitosa，哈里棒束孢Isaria hariottii，小蝉茸Cordyceps sobolifera，蒙克苏棒束孢Isaria mokanshawii和多枝棒束孢Isaria arbuscula等等。
常采到的是蝉花及其寄主山蝉若虫寄生后的复合体。根据历史产区浙江的1467份样品的调查，药材蝉花主要是蝉花寄生后的虫菌复合体。蝉花属半知菌类真菌，其子实体自虫体头部长出，单生或丛聚成束，浅黄色，长1.5～8.0cm，前端膨大，呈纺锤形或呈鸡冠花状。
该蝉花采用如下方法进行培养：从液氮中取出蝉花单17-7-1和其他不同地域蝉花的保藏样品，在超净工作台上将其接到PSA斜面培养基上，22℃-25℃培养10d，然后在超净工作台将PSA斜面培养基中长好的蝉花接到SAAY液体培养基中，22℃-25℃摇菌24h，收集菌丝并液氮速冻，-80℃保存备用。
蝉花单17-7-1的主要形态特征为：在PDA培养基上25℃培养10d，菌落圆形，平展，初絮状，后粉状，白色至浅黄色，直径5～6cm。菌丝无色，分枝，具有隔膜，宽1.2～2.5μm。分生孢子梗多分枝，宽3.0～5.5μm，由每轮2～5个个产孢细胞组成轮状分枝。产孢细胞瓶形，基部球状膨大，向上变细窄，4.5～6.5×2.5～3.5μm。分生孢子圆柱形，单细胞，无色，平滑，链生，直或弯曲，6.5～8.8(～11.0)×2.5～3.5μm。
蝉花单17-7-1经实验验证，其培养物具有易于培养、产量高的特点，具有抗肿瘤、调节免疫、降血糖、降血脂、降血压、明目、抗辐射、解热镇痛、镇静催眠、滋补强壮、改善肾功能等功能，因此具有广泛的工业化应用价值。
由于地理来源不同、寄主不同的蝉花，其次生代谢产物方面具有较大的变异性。本专利的目的是利用SCAR分子标记技术鉴定蝉花单17-7-1菌株和其他不同地域蝉花的区别。
发明内容
本发明的目的在于提供蝉花单17-7-1菌株的鉴定方法。
本发明的目的是通过如下技术方案实现的：
一种蝉花单17-7-1菌株的鉴定方法，该方法包括如下步骤：
步骤1，提取待检测菌株的DNA；
步骤2，对步骤1获得的待检测菌株DNA采用下列引物进行PCR扩增，
S11-2-F3：GTAGACCCGTGTTGTATGACAAACT；
S11-2-R3：GTAGACCCGTAAGGAGACGGAGGAT；
步骤3，将步骤2获得的扩增产物经琼脂糖凝胶电泳后获得待检测菌株的电泳条带图谱；
步骤4，将步骤3获得的待检测菌株图谱进行分析获得判定结果。
其中，步骤2的引物还可以是：
S11-2-F4：CCTCCCGTCGCTCATTGTT；
S11-2-R4：TCGGGCAAATTTCAAGCGCC A；
进一步的，所述步骤2所采用的PCR扩增反应体系如下：
Figure PCTCN2018082481-appb-000001
进一步的，所述步骤2所采用的PCR扩增反应条件如下：先95℃5分钟； 然后依次95℃30秒，55℃30秒，72℃2.5分钟，共循环35次；然后72℃延伸10分钟，最后降温至4℃保存。
进一步的，所述步骤4中，将步骤3获得的SCAR条带图谱进行分析，发现单17-7-1具有特异条带，而其他不同地域蝉花没有该特异条带，则鉴定出蝉花菌株单17-7-1。
本发明涉及的蝉花菌株为浙江泛亚生物医药股份有限公司所有，编号为单17-7-1已于2009年11月18日在中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC)注册保藏，保藏号为CGMCC No.3453。
本发明有益效果：
本发明通过筛选大量的随机引物最终获得本发明引物，该引物具有非常高的特异性，具有唯一的特异性条带(片段长度分别为2414bp和2222bp)，能很好的区分生产菌株单17-7-1和其他不同地域蝉花。
由于目前其他菌株并未进行过全基因组测序，如果要从整个基因组区分不同地域蝉花分子水平的不同，则需要对其他不同地域蝉花进行重测序(每个样至少还需要三次重复)，再进行个体差异性分析，需要消耗大量的金钱，大量的时间。相对于以往的蝉花全基因组测序结果，本发明SCAR分子标记技术只需通过PCR和电泳就能得到结果，具有耗时短，价格低，操作简便的特点。
附图说明
图1为引物S11-2-F1和S11-2-R1对应的SCAR条带图谱。
图2为引物S11-2-F2和S11-2-R2对应的SCAR条带图谱。
图3为引物S11-10-F1和S11-10-R1对应的SCAR条带图谱。
图4为引物S11-10-F2和S11-10-R2对应的SCAR条带图谱。
图5为引物S11-7-F2和S11-7-R2对应的SCAR条带图谱。
图6为引物S11-10-F3和S11-10-R3对应的SCAR条带图谱。
图7为引物S11-11-F2和S11-11-R2对应的SCAR条带图谱。
图8为引物S11-2-F3和S11-2-R3对应的SCAR条带图谱。
图9为引物S11-2-F4和S11-2-R4对应的SCAR条带图谱。
以上图1-7中，泳道1为单17-7-1的PCR结果，泳道2为15JJ8的PCR结果，泳道3为15JJ10的PCR结果，泳道4为15JJ14的PCR结果，泳道5为15JJ18的PCR结果，泳道6为15AX27的PCR结果，泳道7为Marker，泳道8为单17-7-1的PCR结果，泳道9为15AX30的PCR结果，泳道10为15AX36的PCR结果，泳道11为15AX39的PCR结果，泳道12为15JGS35的PCR结果，泳道13为15JGS38的PCR结果；
以上图8-9中，泳道1为单17-7-1的PCR结果，泳道2为15JJ5的PCR结果，泳道3为15JJ7的PCR结果，泳道4为15JJ8的PCR结果，泳道5为15JJ10的PCR结果，泳道6为15JJ14的PCR结果，泳道7为Marker，泳道8为单17-7-1的PCR结果，泳道9为15JJ17的PCR结果，泳道10为15JJ18的PCR结果，泳道11为15AX22的PCR结果，泳道12为15AX27的PCR结果，泳道13为15AX30的PCR结果。
图10为引物S11-2-F3和S11-2-R3对应的SCAR条带图谱；
泳道1为单17-7-1的PCR结果，泳道2为15JJ5的PCR结果，泳道3为15JJ7的PCR结果，泳道4为15JJ8的PCR结果，泳道5为15JJ10的PCR结果，泳道6为15JJ14的PCR结果，泳道7为Marker，泳道8为单17-7-1的PCR结果，泳道9为15JJ17的PCR结果，泳道10为15JJ18的PCR结果，泳道11为15AX22的PCR结果，泳道12为15AX27的PCR结果，泳道13为15AX30的PCR结果，其中泳道7Marker条带按照分子量大小从上往下顺序依次为：10000bp，5000bp，3000bp，2000bp，1500bp，1000bp，750bp，500bp，250bp，100bp；
泳道1为单17-7-1的条带大小为2414bp；
泳道2为15JJ5的条带大小估算为无；
泳道3为15JJ7的条带大小估算为无；
泳道4为15JJ8的条带大小估算为无；
泳道5为15JJ10的条带大小估算为无；
泳道6为15JJ14的条带大小估算为3000bp左右；
泳道8为单17-7-1的条带大小为2414bp；
泳道9为15JJ17的条带大小估算为无；
泳道10为15JJ18的条带大小估算为无；
泳道11为15AX22的条带大小估算为无；
泳道12为15AX27的条带大小估算为无；
泳道13为15AX30的条带大小估算为无。
图11为引物S11-2-F3和S11-2-R3对应的SCAR条带图谱；
泳道1为单17-7-1的PCR结果，泳道2为15AX33的PCR结果，泳道3为15AX34的PCR结果，泳道4为15AX36的PCR结果，泳道5为15AX38的PCR结果，泳道6为15AX39的PCR结果,泳道7为Marker，泳道8为单17-7-1的PCR结果，泳道9为15JGS2的PCR结果，泳道10为15JGS4的PCR结果，泳道11为15JGS11的PCR结果，泳道12为15JGS35的PCR结果，泳道13为15JGS38的PCR结果，其中泳道7Marker条带按照分子量大小从上往下顺序依次为：10000bp，5000bp，3000bp，2000bp，1500bp，1000bp，750bp，500bp，250bp，100bp；
泳道1为单17-7-1的条带大小为2414bp；
泳道2为15AX33的条带大小估算为无；
泳道3为15AX34的条带大小估算为无；
泳道4为15AX36的条带大小估算为无；
泳道5为15AX38的条带大小估算为无；
泳道6为15AX39的条带大小估算为无；
泳道8为单17-7-1的条带大小为2414bp；
泳道9为15JGS2的条带大小估算为无；
泳道10为15JGS4的条带大小估算为无；
泳道11为15JGS11的条带大小估算为无；
泳道12为15JGS35的条带大小估算为无；
泳道13为15JGS38的条带大小估算为无。
图12为引物S11-2-F3和S11-2-R3对应的SCAR条带图谱；
泳道1Marker，泳道2为单17-7-1的PCR结果，泳道3为15FY8的PCR结果，泳道4为15FY13的PCR结果，泳道5为15FY14的PCR结果，泳道6为14DBS50的PCR结果，泳道7为15LQ4的PCR结果，泳道8为阴性对照，其中泳道1Marker条带按照分子量大小从上往下顺序依次为：10000bp，5000bp，3000bp，2000bp，1500bp，1000bp，750bp，500bp，250bp，100bp；
泳道2为单17-7-1的条带大小为2414bp；
泳道3为15FY8的条带大小估算为无；
泳道4为15FY13的条带大小估算为无；
泳道5为15FY14的条带大小估算为无；
泳道6为14DBS50的条带大小估算为无；
泳道7为15LQ4的条带大小估算为无；
泳道8为阴性对照，无条带。
图13为引物S11-2-F4和S11-2-R4对应的SCAR条带图谱；
泳道1为单17-7-1的PCR结果，泳道2为15JJ5的PCR结果，泳道3为15JJ7的PCR结果，泳道4为15JJ8的PCR结果，泳道5为15JJ10的PCR结果，泳道6为15JJ14的PCR结果，泳道7为Marker，泳道8为单17-7-1的PCR结果，泳道9为15JJ17的PCR结果，泳道10为15JJ18的PCR结果，泳道11为15AX22的PCR结果，泳道12为15AX27的PCR结果，泳道13为15AX30的PCR结果，其中泳道7Marker条带按照分子量大小从上往下顺序依次为：10000bp，5000bp，3000bp，2000bp，1500bp，1000bp，750bp，500bp，250bp，100bp；
泳道1为单17-7-1的条带大小为2222bp；
泳道2为15JJ5的条带大小估算为无；
泳道3为15JJ7的条带大小估算为无；
泳道4为15JJ8的条带大小估算为无；
泳道5为15JJ10的条带大小估算为无；
泳道6为15JJ14的条带大小估算为无；
泳道8为单17-7-1的条带大小为2222bp；
泳道9为15JJ17的条带大小估算为无；
泳道10为15JJ18的条带大小估算为无；
泳道11为15AX22的条带大小估算为无；
泳道12为15AX27的条带大小估算为无；
泳道13为15AX30的条带大小估算为无。
图14为引物S11-2-F4和S11-2-R4对应的SCAR条带图谱；
泳道1为单17-7-1的PCR结果，泳道2为15AX33的PCR结果，泳道3为15AX34的PCR结果，泳道4为15AX36的PCR结果，泳道5为15AX38的PCR结果，泳道6为15AX39的PCR结果,泳道7为Marker，泳道8为单17-7-1的PCR结果，泳道9为15JGS2的PCR结果，泳道10为15JGS4的PCR结果，泳道11为15JGS11的PCR结果，泳道12为15JGS35的PCR结果，泳道13为15JGS38的PCR结果，其中泳道7Marker条带按照分子量大小从上往下顺序依次为：10000bp，5000bp，3000bp，2000bp，1500bp，1000bp，750bp，500bp，250bp，100bp；
泳道1为单17-7-1的条带大小为2222bp；
泳道2为15AX33的条带大小估算为无；
泳道3为15AX34的条带大小估算为无；
泳道4为15AX36的条带大小估算为无；
泳道5为15AX38的条带大小估算为无；
泳道6为15AX39的条带大小估算为无；
泳道8为单17-7-1的条带大小为2222bp；
泳道9为15JGS2的条带大小估算为无；
泳道10为15JGS4的条带大小估算为无；
泳道11为15JGS11的条带大小估算为无；
泳道12为15JGS35的条带大小估算为无；
泳道13为15JGS38的条带大小估算为无。
图15为引物S11-2-F4和S11-2-R4对应的SCAR条带图谱；
泳道1Marker，泳道2为单17-7-1的PCR结果，泳道3为15FY8的PCR结果，泳道4为15FY13的PCR结果，泳道5为15FY14的PCR结果，泳道6为14DBS50的PCR结果，泳道7为15LQ4的PCR结果，泳道8为阴性对照，其中泳道1Marker条带按照分子量大小从上往下顺序依次为：10000bp，5000bp，3000bp，2000bp，1500bp，1000bp，750bp，500bp，250bp，100bp；
泳道2为单17-7-1的条带大小为2222bp；
泳道3为15FY8的条带大小估算为无；
泳道4为15FY13的条带大小估算为无；
泳道5为15FY14的条带大小估算为无；
泳道6为14DBS50的条带大小估算为无；
泳道7为15LQ4的条带大小估算为无；
泳道8为阴性对照，无条带。
图16为引物S11-2-F3和S11-2-R3对应的SCAR条带图谱
泳道1为单17-7-1的PCR结果，泳道2为15JJ5的PCR结果，泳道3为15JJ7的PCR结果，泳道4为15JJ8的PCR结果，泳道5为15JJ10的PCR结果，泳道6为15JJ14的PCR结果，泳道7为Marker，泳道8为单17-7-1的PCR结果，泳道9为15JJ17的PCR结果，泳道10为15JJ18的PCR结果，泳道11为15AX22的PCR结果，泳道12为15AX27的PCR结果，泳道13为15AX30的PCR结果，其中泳道7Marker条带按照分子量大小从上往下顺序依次为：10000bp，5000bp，3000bp，2000bp，1500bp，1000bp，750bp，500bp，250bp，100bp。
泳道1为单17-7-1的条带大小为2414bp。
泳道2为15JJ5的条带大小估算为无。
泳道3为15JJ7的条带大小估算为无。
泳道4为15JJ8的条带大小估算为无。
泳道5为15JJ10的条带大小估算为无。
泳道6为15JJ14的条带大小估算为3000bp左右。
泳道8为单17-7-1的条带大小为2414bp。
泳道9为15JJ17的条带大小估算为无。
泳道10为15JJ18的条带大小估算为无。
泳道11为15AX22的条带大小估算为无。
泳道12为15AX27的条带大小估算为无。
泳道13为15AX30的条带大小估算为无。
图17为引物S11-2-F3和S11-2-R3对应的SCAR条带图谱
泳道1为单17-7-1的PCR结果，泳道2为15AX33的PCR结果，泳道3为15AX34的PCR结果，泳道4为15AX36的PCR结果，泳道5为15AX38的PCR结果，泳道6为15AX39的PCR结果,泳道7为Marker，泳道8为单17-7-1的PCR结果，泳道9为15JGS2的PCR结果，泳道10为15JGS4的PCR结果，泳道11为15JGS11的PCR结果，泳道12为15JGS35的PCR结果，泳道13为15JGS38的PCR结果，其中泳道7Marker条带按照分子量大小从上往下顺序依次为：10000bp，5000bp，3000bp，2000bp，1500bp，1000bp，750bp，500bp，250bp，100bp。
泳道1为单17-7-1的条带大小为2414bp。
泳道2为15AX33的条带大小估算为无。
泳道3为15AX34的条带大小估算为无。
泳道4为15AX36的条带大小估算为无。
泳道5为15AX38的条带大小估算为无。
泳道6为15AX39的条带大小估算为无。
泳道8为单17-7-1的条带大小为2414bp。
泳道9为15JGS2的条带大小估算为无。
泳道10为15JGS4的条带大小估算为无。
泳道11为15JGS11的条带大小估算为无。
泳道12为15JGS35的条带大小估算为无。
泳道13为15JGS38的条带大小估算为无。
图18为引物S11-2-F3和S11-2-R3对应的SCAR条带图谱
泳道1Marker，泳道2为单17-7-1的PCR结果，泳道3为15FY8的PCR结果，泳道4为15FY13的PCR结果，泳道5为15FY14的PCR结果，泳道6为14DBS50的PCR结果，泳道7为15LQ4的PCR结果，泳道8为阴性对照，其中泳道1Marker条带按照分子量大小从上往下顺序依次为：10000bp，5000bp，3000bp，2000bp，1500bp，1000bp，750bp，500bp，250bp，100bp。
泳道2为单17-7-1的条带大小为2414bp。
泳道3为15FY8的条带大小估算为无。
泳道4为15FY13的条带大小估算为无。
泳道5为15FY14的条带大小估算为无。
泳道6为14DBS50的条带大小估算为无。
泳道7为15LQ4的条带大小估算为无。
泳道8为阴性对照，无条带。
图19为引物S11-2-F4和S11-2-R4对应的SCAR条带图谱
泳道1为单17-7-1的PCR结果，泳道2为15JJ5的PCR结果，泳道3为15JJ7的PCR结果，泳道4为15JJ8的PCR结果，泳道5为15JJ10的PCR结果，泳道6为15JJ14的PCR结果，泳道7为Marker，泳道8为单17-7-1的PCR结果，泳道9为15JJ17的PCR结果，泳道10为15JJ18的PCR结果，泳道11为15AX22的PCR结果，泳道12为15AX27的PCR结果，泳道13为15AX30的PCR结果，其中泳道7Marker条带按照分子量大小从上往下顺序依次为：10000bp，5000bp，3000bp，2000bp，1500bp，1000bp，750bp，500bp，250bp，100bp。
泳道1为单17-7-1的条带大小为2222bp。
泳道2为15JJ5的条带大小估算为无。
泳道3为15JJ7的条带大小估算为无。
泳道4为15JJ8的条带大小估算为无。
泳道5为15JJ10的条带大小估算为无。
泳道6为15JJ14的条带大小估算为无。
泳道8为单17-7-1的条带大小为2222bp。
泳道9为15JJ17的条带大小估算为无。
泳道10为15JJ18的条带大小估算为无。
泳道11为15AX22的条带大小估算为无。
泳道12为15AX27的条带大小估算为无。
泳道13为15AX30的条带大小估算为无。
图20为引物S11-2-F4和S11-2-R4对应的SCAR条带图谱
泳道1为单17-7-1的PCR结果，泳道2为15AX33的PCR结果，泳道3为15AX34的PCR结果，泳道4为15AX36的PCR结果，泳道5为15AX38的PCR结果，泳道6为15AX39的PCR结果,泳道7为Marker，泳道8为单17-7-1的PCR结果，泳道9为15JGS2的PCR结果，泳道10为15JGS4的PCR结果，泳道11为15JGS11的PCR结果，泳道12为15JGS35的PCR结果，泳道13为15JGS38的PCR结果，其中泳道7Marker条带按照分子量大小从上往下顺序依次为：10000bp，5000bp，3000bp，2000bp，1500bp，1000bp，750bp，500bp，250bp，100bp。
泳道1为单17-7-1的条带大小为2222bp。
泳道2为15AX33的条带大小估算为无。
泳道3为15AX34的条带大小估算为无。
泳道4为15AX36的条带大小估算为无。
泳道5为15AX38的条带大小估算为无。
泳道6为15AX39的条带大小估算为无。
泳道8为单17-7-1的条带大小为2222bp。
泳道9为15JGS2的条带大小估算为无。
泳道10为15JGS4的条带大小估算为无。
泳道11为15JGS11的条带大小估算为无。
泳道12为15JGS35的条带大小估算为无。
泳道13为15JGS38的条带大小估算为无。
图21为引物S11-2-F4和S11-2-R4对应的SCAR条带图谱
泳道1Marker，泳道2为单17-7-1的PCR结果，泳道3为15FY8的PCR结果，泳道4为15FY13的PCR结果，泳道5为15FY14的PCR结果，泳道6为14DBS50的PCR结果，泳道7为15LQ4的PCR结果，泳道8为阴性对照，其中泳道1Marker条带按照分子量大小从上往下顺序依次为：10000bp，5000bp，3000bp，2000bp，1500bp，1000bp，750bp，500bp，250bp，100bp。
泳道2为单17-7-1的条带大小为2222bp。
泳道3为15FY8的条带大小估算为无。
泳道4为15FY13的条带大小估算为无。
泳道5为15FY14的条带大小估算为无。
泳道6为14DBS50的条带大小估算为无。
泳道7为15LQ4的条带大小估算为无。
泳道8为阴性对照，无条带。
具体实施方式
以下实施例所用蝉花单17-7-1菌株于2009年11月18日在中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC)注册保藏，保藏号为CGMCC No.3453；其他不同地域蝉花分别采集自安溪(编号分别为：15AX22，15AX27，15AX30，15AX33，15AX34，15AX36，15AX38，15AX39)、九江(编号分别为：15JJ5，15JJ7，15JJ8，15JJ10，15JJ14，15JJ17，15JJ18)、井冈山(编号分别为：15JGS2，15JGS4，15JGS11，15JGS35，15JGS38)、富阳(编号分别为：15FY8，15FY13，15FY14)、大别山(编号为：14DBS50)、乐清(编号为：15LQ4)。上菌株均为蝉棒束孢。
实施例1
基于SCAR分子标记技术，先人工合成多个随机多态核苷酸序列作为引物，所述引物序列见表1。
表1引物序列
编号 引物名称 序列(5'到3')
SEQ ID NO：1 S11-2-F3 GTAGACCCGTGTTGTATGACAAACT
SEQ ID NO：2 S11-2-R3 GTAGACCCGTAAGGAGACGGAGGAT
SEQ ID NO：3 S11-2-F4 CCTCCCGTCGCTCATTGTT
SEQ ID NO：4 S11-2-R4 TCGGGCAAATTTCAAGCGCCA
SEQ ID NO：5 S11-2-F1 GCCCACAAGCCTCCTCAAGAT
SEQ ID NO：6 S11-2-R1 GGACACCCATACTGGCACGA
SEQ ID NO：7 S11-2-F2 CCCACAAGCCTCCTCAAGAT
SEQ ID NO：8 S11-2-R2 TCGCCAGAGGGTCATACATTTT
SEQ ID NO：9 S11-7-F1 GGCTCAGAACAACTCGGGAATG
SEQ ID NO：10 S11-7-R1 TACGAGACGACGGAAGAAGGAT
SEQ ID NO：11 S11-10-F1 CATAGGTGAAACCTGCGGACTGT
SEQ ID NO：12 S11-10-R1 TGGAACATTGGGAGAAGGGCTA
SEQ ID NO：13 S11-10-F2 GTCAACTGGCTCACCAATGTCA
SEQ ID NO：14 S11-10-R2 AATTCAACAGCCTCATACGATGTCT
SEQ ID NO：15 S11-11-F1 GAAGACGAAATCGACGAACAGT
SEQ ID NO：16 S11-11-R1 GTAAATGCGGCTCAGAACAACT
SEQ ID NO：17 S11-7-F2 GTAGACCCGTTGCAGAATACTAATG
SEQ ID NO：18 S11-7-R2 GTAGACCCGTCGATATTGATGAAGAT
SEQ ID NO：19 S11-10-F3 CTGGGGGTCAATTCGGCTCAT
SEQ ID NO：20 S11-10-R3 GTAGACCCGTTGTTAAGTTGGAGATT
SEQ ID NO：21 S11-11-F2 GCTCGGACGCTCAAGGTATC
SEQ ID NO：22 S11-11-R2 GTAGACCCGTTGCAGAATACTAATG
采用CTAB法提取蝉花单17-7-1和不同地域蝉花DNA，采用上述引物进行聚合酶链式反应扩增，然后进行琼脂糖电泳，检测电泳条带。
PCR反应条件如下：
Figure PCTCN2018082481-appb-000002
PCR反应程序：95℃ 5min；95℃ 30sec，55℃ 30sec，72℃ 2min 30sec，共循环35次；72℃ 10min，4℃保存。
1.2％琼脂糖凝胶电泳，电压100V 40min；凝胶成像仪(Alphamager)拍摄。
检测结果见图1-9。由电泳图可以看出，除引物S11-2-F3和S11-2-R3(图8)、S11-2-F4和S11-2-R4外(图9)，其他引物的电泳图中不同地域蝉花与单17-7-1菌株都具有一条或者多条相同的电泳条带，其中S11-7-F1和S11-7-R1、S11-11-F1和S11-11-R1PCR结果无条带；只有引物S11-2-F3和S11-2-R3、S11-2-F4和S11-2-R4的电泳图中，蝉花单17-7-1具有一条特异的条带，其片段长度分别为2414bp和2222bp，而其他不同地域的蝉花并不具有，可以明显的区分蝉花单17-7-1与其他不同地域蝉花。
实施例2
以实施例1筛选得到的引物S11-2-F3和S11-2-R3、S11-2-F4和S11-2-R4对蝉花单17-7-1建立对蝉花单17-7-1的鉴定方法。
SCAR分子标记稳定性实验1：
试验操作：
1.CTAB法提取同一批次蝉花单17-7-1和不同地域蝉花DNA，采用引物S11-2-F3和S11-2-R3、S11-2-F4和S11-2-R4进行聚合酶链式反应扩增，然后进行琼脂糖电泳，检测电泳条带。
2.PCR反应条件如下：
Figure PCTCN2018082481-appb-000003
PCR反应程序：95℃ 5min；95℃ 30sec，55℃ 30sec，72℃ 2min 30sec，共循环35次；72℃ 10min，4℃保存。
3. 1.2％琼脂糖凝胶电泳，电压100V 40min。
4.凝胶成像仪(Alphamager)拍摄。
实验结果：
以S11-2-F3和S11-2-R3为引物，对同一批次提取蝉花单17-7-1和不同地域蝉花的DNA为模板的PCR而获得的电泳图谱(图10、图11、图12所示)。
以S11-2-F4和S11-2-R4为引物，对同一批次提取蝉花单17-7-1和不同地域蝉花的DNA为模板的PCR而获得的电泳图谱(图13、图14、图15所示)。
从电泳图谱上看，以同一批次抽提的DNA为模板，并重复3次，得到主要的扩增片段是一致的。
从以上试验结果可以得出，SCAR标记检测同一批次的DNA多态性是一种稳定的、可靠的方法。
SCAR分子标记稳定性实验2：
试验操作：
1.CTAB法提取不同批次蝉花单17-7-1和不同地域蝉花DNA，采用引物S11-2-F3和S11-2-R3、S11-2-F4和S11-2-R4进行聚合酶链式反应扩增，然后进行琼脂糖电泳，检测电泳条带。
2.PCR反应条件如下：
Figure PCTCN2018082481-appb-000004
PCR反应程序：95℃ 5min；95℃ 30sec，55℃ 30sec，72℃ 2min 30sec，共循环35次；72℃ 10min，4℃保存。
3. 1.2％琼脂糖凝胶电泳，电压100V 40min。
4.凝胶成像仪(Alphamager)拍摄。
实验结果：
以S11-2-F3和S11-2-R3为引物，对不同批次提取蝉花单17-7-1和不同地域蝉花的DNA为模板的PCR而获得的电泳图谱(图16、图17、图18所示)。
以S11-2-F4和S11-2-R4为引物，对不同批次提取蝉花单17-7-1和不同地域蝉花的DNA为模板的PCR而获得的电泳图谱(图19、图20、图21所示)。
从电泳图谱上看，以不同批次抽提的DNA为模板，并重复3次，得到主要的扩增片段基本是一致的。
从以上试验结果可以得出，SCAR标记检测不同样本DNA多态性是一种稳定的、可靠的方法。
从中可见采用引物S11-2-F3和S11-2-R3、S11-2-F4和S11-2-R4，可以明显的区分蝉花单17-7-1与其他不同地域蝉花的不同，因此利用SCAR分子标记技术并以引物S11作为鉴定蝉花单17-7-1的方法非常可行。

Claims (5)

  1. 一种蝉花单17-7-1菌株的鉴定方法，其特征在于，所述方法包括如下步骤：
    步骤1，提取待检测菌株的DNA；
    步骤2，对步骤1获得的待检测菌株DNA采用下列引物进行PCR扩增：
    SEQ ID NO：1：GTAGACCCGTGTTGTATGACAAACT；
    SEQ ID NO：2：GTAGACCCGTAAGGAGACGGAGGAT；
    步骤3，将步骤2获得的扩增产物经琼脂糖凝胶电泳后获得待检测菌株的电泳条带图谱；
    步骤4，将步骤3获得的待检测菌株图谱进行分析获得判定结果。
  2. 如权利要求1所述的鉴定方法，其特征在于，步骤2的引物为：
    SEQ ID NO：3：CCTCCCGTCGCTCATTGTT；
    SEQ ID NO：4：TCGGGCAAATTTCAAGCGCCA。
  3. 如权利要求1或2所述的鉴定方法，其特征在于，所述步骤2所采用的PCR扩增反应体系为：
    Figure PCTCN2018082481-appb-100001
  4. 如权利要求1或2所述的鉴定方法，其特征在于，所述步骤2所采用的PCR扩增反应条件如下：先95℃5分钟；然后依次95℃30秒，55℃30秒，72℃2.5分钟，共循环35次；然后72℃延伸10分钟，最后降温至4℃保存。
  5. 如权利要求1或2所述的鉴定方法，其特征在于，所述步骤4中，将步骤3获得的SCAR条带图谱进行分析，待检测菌株在3000bp～2000bp之间具有一条条带，鉴定待检测菌株为蝉花菌株单17-7-1菌株。
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