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Primer, probe and kit for absolute quantification of Rhizopus oryzae

A technology for detection kits and probes, applied in biochemical equipment and methods, measurement of color/spectral characteristics, measurement/inspection of microorganisms, etc., can solve the problem of the absence of Rhizopusoryzae specific sequences, high cost of equipment, and the inability to realize Rhizopusoryzae Quantitative detection and other issues

Pending Publication Date: 2021-08-06
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] A probe, kit and application for the absolute quantification of Rhizopus oryzae of the present invention solve at least one of the following technical problems: (1) there is no specific sequence of Rhizopus oryzae reported at present; (2) existing The method cannot achieve the total detection of all Rhizopus oryzae; (3) the existing quantitative method has low species resolution and / or insufficient detection accuracy; (4) the existing quantitative method requires high equipment and / or strict The operating environment is not suitable for timely detection after production sampling; (5) The existing quantitative methods are cumbersome to operate, etc.

Method used

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  • Primer, probe and kit for absolute quantification of Rhizopus oryzae
  • Primer, probe and kit for absolute quantification of Rhizopus oryzae
  • Primer, probe and kit for absolute quantification of Rhizopus oryzae

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1: Screening of Rhizopus oryzae-specific fragments based on conserved housekeeping genes

[0063] In order to develop specific fragments with high inter-species specificity and high intra-species coverage, the present invention first selects high-coverage marker gene sequences to screen specific fragments, including 26S, ITS, and 18S, and the results of homologous comparison are shown in the table As shown in 1, the housekeeping genes of Rhizopus oryzae and other species of the Rhizopus genus have a very high similarity (similarity higher than 99%), and the screening of specific fragments cannot be achieved.

[0064] Table 1 Interspecific similarity between housekeeping genes of Rhizopus oryzae and other species

[0065]

Embodiment 2

[0066] Example 2: Screening of Rhizopus oryzae-specific fragments based on comparative genomics

[0067] (1) Screening of specific genes

[0068] Collect 98 Rhizopus genomes collected by NCBI, including 5 species including Rhizopus oryzae. Through gene prediction and similarity homologous clustering, 10,608 Rhizopus oryzae specific genes were obtained.

[0069] (2) Screening of fragments of interspecies specificity and intraspecies coverage in specific genes

[0070] 10,102 pairs of specific primers were designed from 10,608 specific genes. The specificity and coverage of 10,102 pairs of specific primers were calculated and screened respectively. After a large number of screenings, the specific primers with the highest coverage in Rhizopus oryzae were finally obtained. As shown in Table 2, their specificity and coverage were 100%. The primer sequences (SEQ ID NO.5, SEQ ID NO. 6) It can be used as a specific fragment of Rhizopus oryzae for the construction of signaling prob...

Embodiment 3

[0073] Example 3: Rhizopus oryzae Quantitative Probe Combination Reagents

[0074] Probe combination reagent; containing independently packaged signal probe reagent and quenching probe reagent; wherein, the signal probe sequence is as shown in SEQ ID NO.1, and the quenching probe sequence is as shown in SEQ ID NO.2 .

[0075]Signal probe reagents and quenching probe reagents are in dry powder or liquid form; in dry powder form, they can be diluted to an appropriate concentration before the experiment, for example, diluted with sterile water or buffer solution to a concentration of 20 μM; in liquid form , the concentration can be 20-200μM, and the reagent can be diluted before use, or used directly.

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Abstract

The invention discloses a primer, a probe and a kit for absolute quantification of Rhizopus oryzae, and belongs to the field of biology, the field of foods and the field of detection. According to the invention, a specific fragment of the Rizopus oryzae is obtained by a lot of screening, and is used for construction of a signal probe reagent and a quenching probe, and a Rizopus oryzae quantitative probe and kit are designed. According to the invention, the total amount detection of the Rhizoplus Oryzae can be implemented, expensive instruments are not required during the detection and the Rhizoplus Oryzae quantification, and the quantification work can be rapidly completed within 2.5 h. Meanwhile, nucleic acid extraction does not need to be carried out on a sample used in the method. The probe and the detection kit disclosed by the invention are used for quantification of the Rhizopus oryzae, and have the characteristics of rapidness, convenience, cheapness and accuracy.

Description

technical field [0001] The invention relates to a primer, a probe and a kit for absolute quantification of Rhizopus oryzae, which belong to the fields of biology, food and detection. Background technique [0002] Rhizopus oryzae widely exists in food, especially traditional fermented food, and participates in the biological reaction process in the fermentation process, including liquor, Puer tea, etc. For example, during the fermentation process of liquor, Rhizopus oryzae can secrete a variety of amylases and proteases, which act on the hydrolysis of starch and protein, and are crucial to the metabolism of ethanol and flavor components. Therefore, it is necessary to track the biomass of Rhizopus oryzae in real time, which has important guiding significance for judging the stability of fermentation batches and regulating fermentation parameters. However, most of the traditional fermented food systems are multi-strain co-fermentation systems at present, and the content of Rhi...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/6816C12Q1/06G01N21/31
CPCC12Q1/6816G01N21/31C12Q2563/103
Inventor 吴群徐岩杜如冰
Owner JIANGNAN UNIV
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