Primer combination, kit and method for identifying boletus sinensis

Abstract

The invention provides a primer combination, a kit and a method for identifying boletus sinensis, and belongs to the technical field of boletus identification, the primer combination comprises a specific primer pair and an internal reference primer pair; the sequences of the specific primer pair are as shown in SEQ ID NO. 1 to SEQ ID NO. 2; the specific primer pair is used for amplifying a target sequence on a nuclear genome of boletus sinensis, and the target sequence is as shown in SEQ ID NO.3; the sequences of the internal reference primer pair are as shown in SEQ ID NO. 4 to SEQ ID NO. 5. The primer combination provided by the invention can be used for rapidly and accurately identifying the boletus sinensis.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of boletus identification, and particularly relates to a primer combination, a kit and a method for identifying Chinese saprobic boletus. BACKGROUND

[0002] Chinese saprobic boletus is an important group of Basidiomycota, widely distributed in forest ecosystems, with ecological functions of degrading lignocellulose and promoting material circulation. Some strains also have the potential for edible or medicinal development. Some strains have tender taste and rich nutrition, containing protein, polysaccharide, mineral substance and dietary fiber, which meet the needs of consumers for natural edible fungi and have the potential for edible development. The metabolic products of the strains contain polysaccharide, terpenes, phenols and other active substances, which may have potential medicinal value in antioxidant, immunomodulatory and antibacterial aspects, and provide new resources for natural medicine research and development.

[0003] The fruiting body morphology of Chinese saprobic boletus is easily affected by the growth environment and development stage, and there is morphological overlap with closely related species, so it is difficult to accurately distinguish them only by traditional characteristics such as cap color and tube structure, which may lead to misclassification. The common Chinese saprobic boletus products on the market are mostly dry slices or products, which cannot be accurately distinguished from morphology. The existing identification technology based on universal genes such as ITS has low resolution among species in the genus Suillus, and cannot achieve rapid and specific identification of Chinese saprobic boletus. SUMMARY

[0004] Therefore, the purpose of the present application is to provide a primer combination, a kit and a method for identifying Chinese saprobic boletus, which can quickly and accurately identify Chinese saprobic boletus.

[0005] The present application provides a primer combination for identifying Chinese saprobic boletus, which comprises a specific primer pair and an internal reference primer pair.

[0006] Preferably, the specific primer pair is used to amplify the target sequence on the nuclear genome of Chinese saprobic boletus, and the target sequence is shown as SEQ ID NO. 3.

[0007] Preferably, the sequence of the internal reference primer pair is shown as SEQ ID NO. 4-5.

[0008] The present application provides a kit for identifying Chinese saprobic boletus, which comprises the primer combination.

[0009] Preferably, it further comprises PCR amplification reagents.

[0010] The application provides a method for identifying Chinese porcini, comprising the following steps: 1) extracting genomic DNA of a sample to be tested; 2) using the specific primer pair and the internal reference primer pair in the primer combination as a template, amplifying the genomic DNA obtained in step 1) to obtain an amplification product; 3) determining whether the sample to be tested is Chinese porcini according to the amplification product: when the amplification product of the internal reference primer is only 443 bp, and the amplification product of the specific primer is only 300 bp, the sample to be tested is identified as Chinese porcini; when the amplification product of the internal reference primer is only 443 bp, and the amplification product of the specific primer is not 300 bp or the amplification product is not 300 bp, the sample to be tested is identified as other species of the genus porcini other than Chinese porcini.

[0011] Preferably, the amplification system in step 2) comprises the following components in 40 μl: 2×Taq MasterMix (Dye) 20 μl upstream primer 2 μl downstream primer 2 μl ddH2O 15 μl genomic DNA 1 μl.

[0012] Preferably, the amplification program in step 2) is as follows: 94 ℃ pre-denaturation for 3 min; 94 ℃ denaturation for 30 s, 58 ℃ annealing for 30 s, 72 ℃ extension for 30 s, 35 cycles; 72 ℃ extension for 5 min.

[0013] Compared with the prior art, the application has the following beneficial effects: the primer combination for identifying Chinese porcini provided by the application comprises a specific primer pair for amplifying a specific target sequence on the nuclear genome of Chinese porcini, and an internal reference primer pair for amplifying 18S rDNA of the genus porcini. The primer combination provided by the application can quickly and accurately identify Chinese porcini. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 It is a gel electrophoresis map of PCR products of different samples. DETAILED DESCRIPTION

[0015] The application provides a primer combination for identifying Chinese porcini, comprising a specific primer pair and an internal reference primer pair; the sequence of the specific primer pair is shown in SEQ ID NO. 1-2. The sequence of the internal reference primer pair is shown in SEQ ID NO. 4-5.

[0016] The specific primer pair is used for amplifying the target sequence on the nuclear genome of Chinese saprophytic boletus, and the target sequence is shown as SEQ ID NO. 3, and the specific sequence is as follows: CCCAATCCTTGCCAATCTACTCGCTCGCGATGGTGAAAACAGTCCCGACCAAGATGACCGGCTCGCACAGGAGAGTTTGAGGCAAGAACGCAATCAAACTCGCGCAAGCTCCCCACTGTCCGAACCACCGGAGAGTGAGGATGTGCCGGTCCAGTACTCGCTCCCTCACCGCGAGTCGACGGCTGGTCCAAGCACGGAAAGAGAATTCCGGTCTGGTGCTCACGCGGGTGCTTTGGGTCAAAACAAGGATGGCAGCGGTTCTAGTCGCCCGAGACCCAAGGTTGTCTATGGGAAGCAGAA.

[0017] The target sequence of the internal reference primer pair for amplifying the 18S rDNA of boletus is shown as SEQ ID NO. 6: AAATTAGAGTGTTCAAAGCAGGCGTTTCGCCCGAATACATTAGCATGGAATAATGAAATAGGACGTGCGGTTCTATTTTGTTGGTTTCTAGAGTCGCCGTAATGATTAATAGGGACAGTTGGGGGCATTAGTATTCAGTCGCTAGAGGTGAAATTCTTGGATTGACTGAAGACTAACTATTGCGAAAGCATTTGCCAAGGATGTTTTCATTAATCAAGAACGAAGGTTAGGGGATCGAAAACGATCAGATACCGTTGTAGTCTTAACAGTAAACTATGCCGACTAGGGATCGGGCGACCTCTTTTTGATGTGTCGCTCGGCACCTTACGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACCAGGAGTGGAGCCTGCGGCTTAATTTGACTCAACAC.

[0018] The present application provides a kit for identifying Chinese saprophytic boletus, comprising the primer combination.

[0019] In the present application, the kit preferably further comprises a PCR amplification reagent, which is preferably 2x Taq MasterMix (Dye).

[0020] The present application also provides a method for identifying A. sinuosa, comprising the following steps: 1) extracting genomic DNA of a sample to be tested; 2) using the specific primer pair and the internal reference primer pair in the primer combination as templates, respectively amplifying the genomic DNA obtained in step 1) to obtain an amplification product; 3) determining whether the sample to be tested is A. sinuosa according to the amplification product: when the amplification product of the internal reference primer is only one kind of 443 bp, and the amplification product of the specific primer is only one kind of 300 bp, it is identified as A. sinuosa; when the amplification product of the internal reference primer is only one kind of 443 bp, and the amplification product of the specific primer is not one kind of 300 bp or no amplification product, it is identified as other species of Amanita other than A. sinuosa.

[0021] In the present application, genomic DNA of a sample to be tested is first extracted. The method for extracting the genomic DNA is not particularly limited in the present application, and a conventional method for extracting genomic DNA in the art can be used, and preferably a genomic DNA extraction kit is used.

[0022] After obtaining the genomic DNA, the specific primer pair and the internal reference primer pair in the primer combination are used as templates to amplify the genomic DNA, respectively, to obtain an amplification product.

[0023] In the present application, the system for amplification preferably comprises the following components in 40 μl: 2x Taq MasterMix (Dye) 20 μl Upstream primer 2 μl Downstream primer 2 μl ddH2O 15 μl Genomic DNA 1 μl.

[0024] The concentrations of the upstream primer and the downstream primer are preferably 8-12 mol / L, more preferably 9-11 mol / L, and most preferably 10 μmol / L, respectively; and the concentration of the genomic DNA is preferably 80-120 ng / μL, more preferably 95-105 ng / μL, and most preferably 100 ng / μL.

[0025] The program for amplification is preferably as follows: pre-denaturation at 94℃ for 3 min; denaturation at 94℃ for 30 s, annealing at 58℃ for 30 s, extension at 72℃ for 30 s, 35 cycles; and extension at 72℃ for 5 min.

[0026] After the amplification is completed, electrophoresis is preferably performed. Based on the electrophoresis results of the amplification products, it is determined whether the sample to be tested is *Boletus saprophyticus*: when the amplification product of the internal reference primer is only 443 bp and the amplification product of the specific primer is only 300 bp, it is identified as *Boletus saprophyticus*; when the amplification product of the internal reference primer is only 443 bp and the specific primer has no amplification product or the amplification product is not 300 bp, it is identified as another species of *Boletus* that is not *Boletus saprophyticus*.

[0027] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0028] Example 1

[0029] Primer combination design

[0030] Based on high-throughput sequencing data analysis and assembly Buchwaldoboletus xylophilus Genome sequence, referenced from NCBI. Boletales 18S rDNA and nuclear genome sequences were used as the basis for PCR validation primer design using Primer 3 version 4.1.0 software. Internal control primers were designed on the 18S rDNA sequence, and specific primers were designed on... Buchwaldoboletus xylophilus On the nuclear genome sequence. Theoretically, the internal reference primer can only amplify... Boletales Specific primers can only amplify specific regions on the 18S rDNA sequence of the subordinate species. Buchwaldoboletus xylophilus Specific regions on the nuclear genome.

[0031] The PCR primer information is shown in Table 1.

[0032] Table 1 PCR Primer Information

[0033] Genomic DNA extraction

[0034] Genomic DNA was extracted from bacterial culture samples using the FastPure Microbiome DNA Isolation Kit (catalog number: DC502-01) from Nanjing Novizan Biotechnology Co., Ltd. The procedures were performed according to the instruction manual.

[0035] Table 2 Sample Information Table

[0036] PCR amplification

[0037] 1) Reagents and consumables

[0038] DNA polymerase: 2×Taq MasterMix (Dye) (purchased from Jiangsu Kangwei Century Technology Co., Ltd., item number: CW0682L); The primers are shown in Table 1; RNase-free water.

[0039] 2) PCR reaction system

[0040] Table 3 PCR reaction system

[0041] 3) PCR reaction conditions

[0042] Table 4 PCR reaction conditions

[0043] PCR product detection

[0044] Adhesive concentration: 1.1%, voltage 160V, time 30min.

[0045] Marker: M stands for DM2000, purchased from Jiangsu Kangwei Century Biotechnology Co., Ltd., item number CW0632M.

[0046] The DM2000 DNA Marker consists of six DNA fragments: 2,000 bp, 1,000 bp, 750 bp, 500 bp, 250 bp, and 100 bp. A 3 μl sample was directly electrophoresed. During electrophoresis, the 750 bp DNA fragment, approximately 90 ng in volume, showed a bright band, while the other bands contained approximately 30 ng of DNA.

[0047] The numbers in the figure represent the sample numbers from Table 2 and the primer pair numbers from Table 1. As shown in the figure, the PCR product size is as expected. Sample S1 is Buchwaldoboletus xylophilus The PCR products of the internal control primers and the specific primers both showed a single band; sample S2 was... Boletales Subspecies, and not Buchwaldoboletus xylophilus The PCR product of the internal reference primer has a single band, while the PCR product of the specific primer has no band.

[0048] Example 2

[0049] Primer design, genomic DNA extraction, PCR amplification, and PCR product detection were all performed using the same steps and parameters as in Example 1, except that the detection sample S1 from Example 1 was prepared using *Boletus sinensis* (…). Buchwaldoboletus xylophilus) replaced by the Yunnan wild boletus (the species is not accurately identified) which is extremely similar to Boletus sinensis, PCR product detection found that the detection sample S1 and the control sample S2 both showed that the PCR product of the internal reference primer had a single band, and the PCR product of the specific primer had no band. Further illustrate that the method can be used for accurately detecting Boletus sinensis Buchwaldoboletus xylophilus )。

[0050] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A primer combination for identifying *Boletus sinensis*, characterized in that, It includes specific primer pairs and internal reference primer pairs; the sequences of the specific primer pairs are shown in SEQ ID NO.1~SEQ ID NO.

2.

2. The primer combination according to claim 1, characterized in that, The specific primer pair is used to amplify the target sequence on the nuclear genome of Boletus sinensis, the target sequence being shown in SEQ ID NO.

3.

3. The primer combination according to claim 1 or 2, characterized in that, The sequences of the internal reference primer pairs are shown in SEQ ID NO.4~SEQ ID NO.

5.

4. A kit for identifying *Boletus sinensis*, characterized in that, Includes the primer combination described in any one of claims 1 to 3.

5. The reagent kit according to claim 4, characterized in that, It also includes PCR amplification reagents.

6. A method for identifying *Boletus sinensis*, characterized in that, Includes the following steps: 1) Extract genomic DNA from the sample to be tested; 2) Using the genomic DNA extracted in step 1) as a template, amplify the product by using the specific primer pair and the internal reference primer pair in the primer combination described in claim 1. 3) Determine whether the sample to be tested is *Boletus saprophyticus* based on the amplification products: When the amplification product of the internal reference primer is only 443 bp and the amplification product of the specific primer is only 300 bp, it is identified as *Boletus saprophyticus*; when the amplification product of the internal reference primer is only 443 bp and the specific primer has no amplification product or the amplification product is not 300 bp, it is identified as another species of *Boletus* that is not *Boletus saprophyticus*.

7. The method according to claim 6, characterized in that, Step 2) The amplification system comprises the following components per 40 μl: 2×Taq MasterMix (Dye) 20μl Upstream primer 2μl 2 μl of downstream primer ddH2O 15μl 1 μl of genomic DNA.

8. The method according to claim 7, characterized in that, Step 2) The amplification program is as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 5 min.

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