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Method for absolutely quantifying fungi in sample by constructing recombinant plasmids

A recombinant plasmid and absolute quantification technology, applied in the field of bioengineering, can solve the problems of underestimation and overestimation of relative abundance, and achieve the effect of improving the depth, making up for the lack of absolute content, and high accuracy

Pending Publication Date: 2021-11-12
KWEICHOW MOUTAI COMPANY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] However, there are few existing methods for the absolute quantification of fungi in liquor samples by constructing recombinant plasmids, and the phenomenon that the relative abundance of fungi in liquor samples is generally overestimated or underestimated compared with the absolute abundance

Method used

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  • Method for absolutely quantifying fungi in sample by constructing recombinant plasmids
  • Method for absolutely quantifying fungi in sample by constructing recombinant plasmids
  • Method for absolutely quantifying fungi in sample by constructing recombinant plasmids

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1: the construction of the method that can be used for detecting the absolute content of fungus in the sample

[0046] Specific steps are as follows:

[0047] The genomic DNA of the sample to be tested was extracted, and according to the upstream and downstream primers provided by the reference "Fungi Sailing the Arctic Ocean: Speciose Communities in North Atlantic Driftwood as Revealed by High-Throughput Amplicon Sequencing", the nucleotide sequence was as SEQ ID NO. The DNA fragment shown in 2 is used as the upstream primer, and the DNA fragment with the nucleotide sequence as shown in SEQ ID NO.3 is used as the downstream primer to carry out high-throughput sequencing on the mixture to obtain the type of fungus in the sample to be tested; the internal reference is added to In the sample to be tested, the concentration of the internal reference in the sample to be tested is 1×10 7 copies / mL to obtain the sample to be tested containing the internal referenc...

Embodiment 2

[0051] Embodiment 2: the application of the method that can be used for detecting the absolute content of fungus in the sample

[0052] The method of embodiment 1 is used to detect the absolute content of fungi in Daqu, and the specific steps are as follows:

[0053] Use the Plant Genomic DNA Kit Plant Genomic DNA Extraction Kit to extract the genomic DNA of Daqu to be tested, use the DNA fragment with the nucleotide sequence as shown in SEQ ID NO.2 as an upstream primer, and use the nucleotide sequence as shown in SEQ ID NO.3 The DNA fragments shown were used as downstream primers for high-throughput sequencing of the mixture (high-throughput sequencing was completed by Beijing Aoweisen Gene Technology Co., Ltd.), and according to the results of high-throughput sequencing, Pichiakudriavzevii was selected , Rhodotorula mucilaginosa, Wickerhamomyces anomalus, Saccharomycopsis fibuligera, Torulaspora delbrueckii, Rhizopus microsporus, Saccharomyces cerevisiae, Paecilomyces vari...

Embodiment 3

[0081] Example 3: Verification of the method that can be used to detect the absolute content of fungi in a sample

[0082] Saccharomyces cerevisiae has good ethanol production ability and is the core fungus of the liquor brewing system, so Saccharomyces cerevisiae was selected from the 8 quantified fungi for the verification of the quantitative method.

[0083] The specific implementation method is as follows: screen and obtain Saccharomyces cerevisiae from the sample, and adopt the hemocytometer method to check the number of cells to be 1.0 × 10 9 cells / g, dilute it to the corresponding cell number is 10 8 、10 7 、10 6 、10 5 、10 4 After cells / g, use the Plant Genomic Kit Plant Genomic DNA Extraction Kit to extract the genomic DNA of Saccharomyces cerevisiae, and design Saccharomyces cerevisiae specific primers ScAIRIF-CACAATGGGGCAAAGGCTTC, ScAIRIR-GCCAGAACTGAAGCACAAGC, and then the extracted genome was passed through the StepOnePlus instrument (purchased from Thermo Fly) ...

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Abstract

The invention belongs to the field of bioengineering, and particularly relates to a method for absolutely quantifying fungi in a sample by constructing recombinant plasmids. The method comprises the following steps: firstly obtaining the types of fungi in the sample to be detected, then respectively constructing the recombinant plasmids by ITS2 genes of the fungi, finally mixing the recombinant plasmids, and directly calculating the concentrations of the fungi in the sample to be detected by taking the reads number of the recombinant plasmids in the mixed plasmids as a basis. The step of counting microorganisms is avoided, and the method is simpler and more convenient to operate, and has stronger purposiveness and higher accuracy when being used for absolutely quantifying the fungi in the sample.

Description

technical field [0001] The application belongs to the field of bioengineering, and in particular relates to a method for constructing recombinant plasmids for absolute quantification of fungi in samples. Background technique [0002] Chinese baijiu has a long history of brewing, and its unique taste is deeply loved by Chinese people. Historically, the special wine culture related to drinking has even become a part of Chinese traditional culture. Today, liquor has become a daily consumer product, and its sales are increasing year by year. Daqu is an essential part of traditional Chinese brewing, and plays an important role in saccharification, liquefaction and providing flavor substances in the production of liquor. In the brewing process of liquor, Daqu is also an important indicator of its product control. The ancestors of brewing came to the conclusion from the practice of brewing: "Qu is the bone of wine", "There must be good music in good wine". Commonly used Daqu ra...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6869C12Q1/06
CPCC12Q1/6895C12Q1/6869C12Q2600/166C12Q2535/122C12Q2545/101C12Q2537/165
Inventor 王莉杜海吕锡斌郝飞陈良强杨帆王和玉徐岩周天慈
Owner KWEICHOW MOUTAI COMPANY