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DNA (deoxyribonucleic acid) bar code and primer group for identifying source area of champignon luteo-virens and application of DNA bar code and primer group

A technology of origin and primer set, which is applied in the direction of recombinant DNA technology, DNA/RNA fragments, microbial determination/inspection, etc., can solve the problems of difficulty in popularization, poor reliability and repeatability of results, and high cost, and achieve the goal of overcoming inaccuracy Time-consuming and labor-intensive, stable and reliable results, and short detection cycle

Pending Publication Date: 2022-03-01
杨满军
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the existing DNA barcoding technology, ITS (ribosomal RNA internal transcriptional spacer) and the non-coding region or conserved gene sequence in mitochondria are mainly used for species identification; restriction fragment length polymorphism (restriction fragment length polymorphism, RFLP) operations are very complicated, the reliability and repeatability of the results are poor, random amplified polymorphic DNA (RAPD) is susceptible to interference, and requires a high level of operator skills. In assisted breeding work Difficult to promote; single nucleotide polymorphism (singlenucleotide polymorphism, SNP) requires high equipment and high cost
[0004] Therefore, in view of the shortcomings of traditional breeding methods that are not accurate enough and time-consuming and labor-intensive, it is necessary for those skilled in the art to provide a DNA barcode for accurate and fast identification of the place of origin of the yellow-green mushroom.

Method used

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  • DNA (deoxyribonucleic acid) bar code and primer group for identifying source area of champignon luteo-virens and application of DNA bar code and primer group
  • DNA (deoxyribonucleic acid) bar code and primer group for identifying source area of champignon luteo-virens and application of DNA bar code and primer group
  • DNA (deoxyribonucleic acid) bar code and primer group for identifying source area of champignon luteo-virens and application of DNA bar code and primer group

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] The construction of the DNA barcode of embodiment 1 yellow-green mushroom

[0067] Genome sequencing was carried out on the samples of Dangxiong County in Tibet Autonomous Region, Qilian County in Qinghai Province, and Shiqu County in Sichuan Province. The SSR loci in the genome sequences were analyzed using the MISA program.

[0068] Design primers for PCR amplification of these SSR sites, keep primers that can amplify the corresponding fragments, and discard invalid primers

[0069] The samples from the above three origins were respectively amplified using effective primers and detected by capillary electrophoresis. After analysis, the corresponding simple sequence repeat (SSR) locus was established to distinguish the origin. Finally, 4 pairs of primers were obtained (see Table 1), and the fragment polymorphism obtained by using these 4 pairs of primers to amplify the sample genome can assist in the identification of the origin of the yellow-green mushroom.

[0070]...

Embodiment 2

[0072] Embodiment 2 Identification of the place of origin of yellow-green mushroom

[0073] (1) Use the Sangon Bioengineering (Shanghai) Co., Ltd. Ezup Column Fungal Genomic DNA Extraction Kit (Cat. No. B518259) to extract the genome of the yellow-green mushroom sample, and dilute it to 20 ng / μL for fluorescent PCR amplification.

[0074] (2) Using the primers in Table 1 to amplify the SSR DNA barcode by fluorescent PCR, the origin of the yellow-green Pleurotus edodes can be identified by reading the information of the DNA barcode.

[0075] Fluorescent PCR amplification reaction system (10 μL): 2×Taq PCR Master Mix 5 μL, template (genome DNA) 1 μL, upstream primer 0.1 μL, downstream primer 0.4 μL (concentration of both upstream and downstream primers is 10 uM), fluorescent M13 primer (concentration 10uM) 0.4 μL, dilute to 10 μL with sterile deionized water;

[0076] Reaction conditions: pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 30 s, drop PCR annealing ...

Embodiment 3

[0122] Example 3 Yellow-green Pleurotus pubescens origin identification DNA barcode verification

[0123] Validation of DNA barcodes for identification of the origin of Pleurotus edulis by blind test.

[0124] In the first blind test, samples were taken from Qilian County of Qinghai Province (number of origin 1-16), Shiqu County of Sichuan Province (number of origin 17-32) and Damxung County of Tibet Autonomous Region (number of origin 33-48) 16 samples were blind tested;

[0125] The second step test utilizes primers (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 18, SEQ ID NO: 19) was amplified and subjected to capillary electrophoresis. Primer sets can be amplified using one or more pairs of combinations to distinguish blind samples and identify samples of origin with DNA barcode features;

[0126] The third step is unblinding. The results are shown in Table 3. All 16 samples from Qilian County in Qinghai Province, Shiqu...

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Abstract

The invention discloses a DNA bar code and a primer group for identifying the source area of champignon luteo-virens and application, and belongs to the technical field of edible mushroom germplasm resource screening. Compared with a traditional breeding method and other existing DNA bar code technologies, the method has the advantages of being time-saving, labor-saving, money-saving, accurate and efficient, plays a positive role in identification of the high-quality virens luteo-virens origin and genetic breeding, and also provides an effective method for identification and protection of germplasm resources.

Description

technical field [0001] The invention relates to the technical field of edible fungus germplasm resources screening, and more specifically relates to a DNA barcode, primer set and application for screening the origin of the yellow-green mushroom. Background technique [0002] Yellow-green mushroom, golden in color, also known as chanterelle and golden mushroom, is a high-quality edible fungus with unique flavor, which cannot be cultivated artificially at present. Wild yellow-green mushrooms are mainly distributed in the Qinghai-Tibet Plateau. The main production areas are Damxung County in the Tibet Autonomous Region, Qilian County in Qinghai Province, and Shiqu County in Sichuan Province. The quality of these three main production areas is also the best. Different origins of yellow-green mushrooms have different nutritional values, different flavors, different biological activities, and different market prices. In the past, the breeding of yellow-green mushrooms mainly used...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6858C12N15/11
CPCC12Q1/6895C12Q1/6858C12Q2600/156C12Q2531/113C12Q2563/107C12Q2565/125C12Q2563/185C12Q2525/151
Inventor 杨满军
Owner 杨满军
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