CN113151538A

CN113151538A - Candidate DNA bar code, primer and method for identifying cordyceps fungus

Info

Publication number: CN113151538A
Application number: CN202110256392.8A
Authority: CN
Inventors: 文庭池; 张艳; 龙凤瑶
Original assignee: Guizhou University
Current assignee: Guizhou University
Priority date: 2021-03-09
Filing date: 2021-03-09
Publication date: 2021-07-23

Abstract

The invention discloses a candidate DNA bar code, a primer and a method for identifying cordyceps fungi, which are used for identifying the cordyceps fungi which partially cannot amplify 5.8S-ITS. Candidate DNA barcode primer sequences are SEQ ID N0.1 (5'-CAAATTACCCAATCCCG-3') and SEQ ID N0.2 (GGCCCCGTATGCTCTTACT). The identification method comprises the steps of using a DNA bar code primer as an amplification primer, using genome DNA of a sample to be identified as a template to carry out PCR reaction, carrying out electrophoresis detection on a PCR product, sending the sample with a 2700bp strip in an electrophoresis result to a sequencing company for sequencing, and carrying out classification identification on the cordyceps fungus by adopting molecular systematics after obtaining a sequencing result. The primer and the identification method designed by the invention can accurately identify the cordyceps fungus in the sample, and compared with the traditional method for identifying the cordyceps fungus and medicinal materials by naked eyes, the method has the advantages of simplicity, convenience, quickness and capability of identifying at any time.

Description

Candidate DNA bar code, primer and method for identifying cordyceps fungus

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to a candidate DNA bar code, a primer and a method for identifying cordyceps fungi.

Background

Cordyceps genus (Cordyceps) The fungus is parasitic on insects, the insect body is changed into stiff insects full of hypha, and a head-shaped or rod-shaped stroma with a handle is grown from the front end of the stiff insects.

Disclosure of Invention

The invention aims to provide a candidate DNA barcode primer pair and a method for identifying cordyceps fungi, the design can accurately identify which cordyceps fungi a sample is, and compared with the traditional method for identifying the cordyceps fungi and medicinal materials by naked eyes, the method has the advantages of simplicity, convenience and quickness, and can identify the cordyceps fungi and medicinal materials at any time, and the sequence of the primer pair is as follows:

a forward primer: 5'-CAAATTACCCAATCCCG-3', respectively;

reverse primer: 5'-GGCCCCGTATGCTCTTACT-3' are provided.

The invention relates to a cordyceps fungus identification method, which is characterized by comprising the following steps:

(1) extracting a genome of a cordyceps fungus sample to be detected;

(2) adding the extracted sample genome serving as a template into a reaction tube of a reaction system containing a reaction solution and polymerase, and performing PCR reaction on the sample genome in a sequence shown in SEQ ID N0.1: 5'-CAAATTACCCAATCCCG-3' and SEQ ID N0.2: 5'-GGCCCCGTATGCTCTTACT-3' as primers for PCR amplification;

(3) and (3) carrying out electrophoresis detection on the PCR amplification product, wherein if one band of 2700bp appears in the electrophoresis result, the detected sample is the target band.

The PCR reaction used is preferably: the PCR reaction system is a conventional 25 mu L universal system; the reaction procedure is a conventional PCR reaction procedure.

According to the technical scheme, the beneficial effects of the invention are as follows:

(1) the candidate DNA barcode primer can amplify a complete cordyceps fungus ITS-DNA fragment (including 18S, ITS1,5.8S, ITS2 and 28S), and has the advantages of simplicity and rapidness compared with the common primer;

(2) the invention identifies the cordyceps fungi on the basis of molecular biology, and provides a more scientific method for classifying the cordyceps fungi.

Drawings

FIG. 1 is an electrophoresis diagram of PCR amplification products of different Cordyceps samples (band 1 is Isaria farinosa, band 2 is Beauveria bassiana, band 3 is Cordyceps cicadae, band 4 is Cordyceps militaris, and M is DNA molecular weight standard of 5000 bp).

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Example 1

A candidate DNA bar code, a primer and a method for identifying cordyceps fungi, which take determined cordyceps fungi as a sample, and specifically comprise the following steps:

step 1: sample collection

The sample is selected from Cordyceps strains collected in wild county of Uchuan county of Guizhou province.

Step 2: extraction of genomic DNA

4mg of 2% CTAB extract (CTAB 20 g/L, NaCl 81.816 g/L, EDTA 9.306 g/L, Tris-HCl 12.114 g/L, pH 8.0.0) was added to a 10 ml centrifuge tube, then 2% beta-mercaptoethanol and 5% PVP-40 were added, and the mixture was incubated at 65 ℃. Adding Cordyceps fungus sample into the preheated extractive solution, sealing, keeping at 65 deg.C for 60 min, and shaking 1 time every 10 min.

The sample was centrifuged at 11000 rpm for 10 minutes and the supernatant was taken. Adding chloroform and isoamyl alcohol (24:1) 4 ml into the supernatant, shaking up, centrifuging at 11000 r/min for 10 min, taking the supernatant, and repeating the step for 1 time. 2 ml of 5 mol/l NaCl is added into the supernatant, then 2 ml of isopropanol refrigerated at 4 ℃ is added, the mixture is gently shaken and uniformly mixed, and the mixture is kept stand for more than 1 hour at minus 20 ℃. Centrifuging the frozen liquid at 11000 r/min for 10 min, pouring out the supernatant, adding 2 ml of absolute ethyl alcohol to wash twice, pouring the tube on toilet paper after washing, sucking dry, and drying in a vacuum concentrator for about 5 min. Adding 1 ml of 1 XTE, dissolving the precipitated DNA, namely the cordyceps fungus genome DNA, bouncing or shaking to fully dissolve, and putting into a refrigerator for later use. DNA concentration was determined by intensity comparison with 3000bp fragments of 5000bp DNA Ladder.

And step 3: PCR amplification

mu.mol/L of primers with known base sequences SEQ ID N0.1 and SEQ ID N0.2 was added to 25. mu.L of a reaction solution containing 10 XBuffer 1.5. mu. L, dNTPs 0.12.12 mmol/L and Taq DNA polymerase 0.10U/. mu. L, MgCl₂1.4mmol/L, 12ng of a genomic DNA template of a Cordyceps fungus, pre-denaturing at 94 ℃ for 3 minutes, completing 34 cycles of 94 ℃ for 30 seconds, 51 ℃ for 30 seconds and 72 ℃ for 1 minute, and finally, further extending at 72 ℃ for 5 minutes, thereby obtaining a PCR product.

Step 4 electrophoresis

Preparation of a composition containing Gold View^TM0.05. mu. mol/L of 1.2% agarose gel, 2. mu.L of bromophenol blue and 10. mu.L of the PCR product obtained in step 3 were mixed well, added to the well, and the well-spotted agarose gel was placed on an electrophoresis apparatus, added with 0.5 XTBE buffer, and subjected to electrophoresis at 140V for 30 minutes.

The electrophoresis result is shown in figure 1, and it can be seen from figure 1 that cordyceps fungus has bright and clear bands at 2700bp, thus indicating that the amplification rate of the primer pair DNA bands is high, and the cordyceps fungus species is identified by Blast comparison after the DNA bands are sequenced to obtain sequence fragments. The primers and the method designed by the invention can be used for identifying the types of the cordyceps fungi.

Sequence listing

Isaria farinosa (obtained by 2700bp band sequencing: (Isaria farinosa) ITS-DNA fragment sequence:

GGTCGATGATGCGCCGAAGCTCTCACCTGCGTTCACTTTCATTACGCGTAGGGGTTTGACACCCGAACACTCGCATACGAAGACGACTCCTTGGTCCGTGTTTCAAGACGGGTCGCTGATGACCATTACGCCAGCATCCTTGCGATGCGCGTACCTCAGTCCGGCGCAGGGTATTACGCAATGGGCTATAACACTCCCGAGGGAGCCACATTCCCAAAACCTTTTTCCCCCGCGCCAAACTGATGCTGGCCTGTGCCCGGCAAAGTGCACCAGCGAGAACGCTGGATGATTCACCGGGCCCAAGTCTGGTCATAGGCGCTTCCCTTTCAACAATTTCACGTACTTTTTAACCCTCTTTTCAAAGTGCTTTTCATCTTTCGATCACTCTACTTGTGCGCTATCGGTCTCTGGCCAATATTTAGCTTTAGAAGACATATACCTCCCATTTTGAGCAGCATTCCCAAACTACTCGACTCGTCGAAGGAGCTTTACAGAGGCTCGGTGTCCGACCAGACGGGGCTCTCACCCTCTATGGCGTCCCGTTCCAGGGAACTCGGAAGGCACCTCGCCAAAAGCATCCTCTACAAATTACAACTCGGACCCGNAGGCCAGATTTCAAATTTGAGCTGTTGCCGCTTCACTCGCCGTTACTGGGGCAATCCCTGTTGGTTTCTTTTCCTCCGCTTATTGATATGCTTAAGTTCAGCGGGTATTCCTACCTGATCCGAGGTCAACGTTCAGAAGTCGGGGGTTTTACGGCGTGGCCACGTCGGGGTTCCGGTGCGAGTTGGATTACTACGCAGAGGTCGCCGCGGACGGGCCGCCACTTCATTTCGGGGCCGGCGGTATACGGCCGGTCCCCAACGCCGATTTCCCCAAAGGGAAGTCGAGGGTTGAAATGACGCTCGAACAGGCATGCCCGCCAGAATGCTGGCGGGCGCAATGTGCGTTCAAAGATTCGATGATTCACTGAATTCTGCAATTCACATTACTTATCGCATTTCGCTGCGTTCTTCATCGATGCCAGAACCAAGAGATCCGTTGTTGAAAGTTTTGATTCATTTGTGTTGCCTTGCGGCGGATTCAGAAGATACTGAGAATACAGAGTTTGGGGGTCTCCGGCGGCCGCCTGGATCCAGGCCGCGGCCGGCGCGGGGCCGGCCGGACGCTGGGGCGAGTCCGCCGAAGCAACGATAGGTATGTTCACAGAAGGGTTTGGGAGTTGAAAACTCGGTAATGATCCCTCCGCTGGTTCACCAACGGAGACCTTGTTACGACTTTTACTTCCTCTAAATGACCGAGTTTGGAGAGCTTTCCGGCCCTGGGTGGTAGTTGCCCACCTCCCTGGGCCAGTCCGGACGCCTCACTGAGCCATTCAATCGGTAGTAGCGACGGGCGGTGTGTACAAAGGGCAGGGACGTAATCAACGCAAGCTGATGACTTGCGCTTACTAGGGATTCCTCGTTGAAGAGCAATAATTGCAATGCTCTATCCCCAGCACGACGGAGTTTAACAAGATTACCCGGGCCTTTCGGCCAAGGAAGTACTCGCTGGCTCCGTCAGTGTAGCGCGCGTGCGGCCCAGAACATCTAAGGGCATCACAGACCTGTTATTGCCTCAAACTTCCATCGGCTTGAGCCGATAGTCCCTCTAAGAAGCCGGCGTACTGCCAAAGCAATACGGGCTATTTAGCAGGTTAAGGTCTCGTTCGTTATCGCAATTAAGCAGACAAATCACTCCACCAACTAAGAACGGCCATGCACCACCACCCACAAAATCAAGAAAGAGCTCTCAATCTGTCAATCCTCATTGTGTCTGGACCTGGTGAGTTTCCCCGTGTTGAGTCAAATTAAGCCGCAGGCTCCACCCCTGGTGGTGCCCTTCCGTCAATTTCTTTAAGTTTCAGCCTTGCGACCATACTCCCCCTGGAGCCCAAGCACTTTGATTTCTCGTAAGGTGCCGAACGCGTCAAAAAATAACATCGTCCGATCCCTAGTCGGCATAGTTTATGGTTAAGACTACGACGGTATCTGATCGTCTTCGATCCCCTAACTTTCGTTCCTGATTAATGAAAACATCCTTGGCAAATGCTTTCGCAGTAGTTAGTCTTCAATAAATCCAAGAATTTCACCTCTGACAATTGAATACTGATGCCCCCGACTGTCCCTATTAATCATTACGGCGGTCCTAGAAACCAACAAAATAGAACCACACGTCCTATTTCATTATTCCATGCTAATGTATTCGAGCATAGGCCTGCCTGGAGCACTCTAATTTTTTCAAAGTAAAAGTCCTGTTTCCCCGCCACACCCAGTGAAGGGCATGAGGTTCCACAGAGGGAAAGGCCCGGCCGGACCAGTACACGCGGTGAGGCGGACCGGCCAGCCAGGCCCAAGGTTCAACTACGAGCTTTTTAACCACAACAACTTTAATATACGCTATTGGAGCTGGAATTACCGCGGCTGCTGGCACCAGACTTGCCCTCCAATTGTTCCTCGTTAAGGGATTTAAATTGTACTCATTCCAATTACAAGACCCAAAAGAGCCCTGTATCAG。

Claims

1. a candidate DNA barcode primer pair for identifying cordyceps fungi is characterized by comprising the following primers:

forward primer SEQ ID N0.1: 5'-CAAATTACCCAATCCCG-3'

Reverse primer SEQ ID N0.2: 5'-GGCCCCGTATGCTCTTACT-3' are provided.

2. The pair of candidate DNA barcodes for identifying cordyceps fungi of claim 1, wherein the pair of primers can amplify a complete ITS-DNA fragment comprising 18S, ITS1,5.8S, ITS2,28S as a candidate DNA barcode of cordyceps fungi for identifying cordyceps fungi which are partially incapable of amplifying 5.8S-ITS.

3. The candidate DNA barcode primer pair for identifying Cordyceps fungus as claimed in claim 1, wherein the size of the sequence band amplified by the primer pair is 2700 bp.

4. An application of a candidate DNA barcode primer pair for identifying cordyceps fungi is characterized in that the candidate DNA barcode primer pair is identified by Blast sequence comparison based on GenBank.

5. A PCR detection method for identifying cordyceps fungi is characterized by comprising the following operation steps:

(1) extracting a genome of a cordyceps fungus sample to be detected;

(2) adding the extracted sample genome as a template into a reaction tube of a reaction system containing a reaction solution and polymerase, and performing PCR reaction by using a PCR reaction solution shown in SEQ ID N0.1: 5'-CAAATTACCCAATCCCG-3' and SEQ ID N0.2: 5'-GGCCCCGTATGCTCTTACT-3' as primers for PCR amplification;

(3) carrying out agarose gel electrophoresis detection on the PCR amplification product, wherein if a 2700bp strip appears in an electrophoresis result, the detection sample is a target strip;

(4) sequencing to obtain Blast alignment identification of the sequence fragments.