Quantitative polymerase chain reaction (qPCR) detection method for residual viable bacteria and spores of production strain in vitamin B2 as well as primers and probes used in qPCR detection method

A technology for producing strains and detection methods, applied in the field of primers and probes, and can solve problems such as cumbersome operations

Inactive Publication Date: 2019-12-20
HUBEI GUANGJI PHARMA
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Problems solved by technology

The 16S rRNA gene sequence analysis method is commonly used to identify bacteria, but this method is cumbersome and has limitations in the identification of transgenic strains, and the 16S rRNA gene similarity of Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus licheniformis reaches more than 99%. This method cannot be used to distinguish, so we use the unique sequence of the GMB.subtilis strain to design qPCR primers and probes, which can specifically identify the residues of the production strain

Method used

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  • Quantitative polymerase chain reaction (qPCR) detection method for residual viable bacteria and spores of production strain in vitamin B2 as well as primers and probes used in qPCR detection method
  • Quantitative polymerase chain reaction (qPCR) detection method for residual viable bacteria and spores of production strain in vitamin B2 as well as primers and probes used in qPCR detection method
  • Quantitative polymerase chain reaction (qPCR) detection method for residual viable bacteria and spores of production strain in vitamin B2 as well as primers and probes used in qPCR detection method

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[0023] For Vitamin B 2 Detection of residual viable bacteria and spores of Bacillus subtilis KCCM-10445 (referred to as GMB.subtilis) production strain in the product, and vitamin B was designed according to its chloramphenicol resistance gene 2 The qPCR detection primers for the residual live bacteria and spores of Bacillus subtilis GM B.subtilis production strains, the nucleotide sequence of the primers are: the forward primer is 5'-CGA GCT TTT GCG CGT ATA-3', and the reverse primer is 5'-GCC ATT CCAATA CAAAAC CAC ATA-3'. and vitamin B 2 The qPCR detection probe of the residual DNA of the production strain of Bacillus subtilis GM B. subtilis, the probe sequence is FAM-CGG ATC TAA CGC ATG CTC CGC A-BBQ. As shown in Table 1:

[0024]

[0025] Table 1 Primer and probe sequences

[0026] For Vitamin B 2Detection of residual DNA from production strains of Bacillus subtilis GM B.subtilis in products designed for vitamin B 2 A qPCR detection kit for the residual DNA of str...

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Abstract

The invention relates to the technical field of biology, in particular to a quantitative polymerase chain reaction (qPCR) detection method for residual viable bacteria and spores of a production strain in vitamin B2 as well as primers and probes used in the qPCR detection method. The qPCR detection primers for the residual viable bacteria and spores of the production strain in vitamin B2 are designed. The production strain is B.subtilius KCCM-10445. The nucleotide sequences of the primers are as follows: the forward primer is 5'-CGA GCT TTT GCG CGT ATA-3', and the reverse primer is 5'-GCC ATTCCA ATA CAA AAC CAC ATA-3'. The probes, a kit and the detection method are also designed. The residual viable bacteria and spores of the production strain B.subtilius KCCM-10445 in a vitamin B2 product can be accurately detected, and the primers, the probes and the detection method related to detection are developed.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to vitamin B 2 The qPCR detection method and the primers and probes used for the residual viable bacteria and spores of the production strains in medium. Background technique [0002] Vitamin B 2 Also known as riboflavin, it is slightly soluble in water and is stable when heated in neutral or acidic solution. It is an integral part of the prosthetic group of yellow enzymes in the body (yellow enzyme plays the role of hydrogen delivery in biological redox). When it is lacking, it will affect the biological oxidation of the body, cause metabolic disorders, and lead to various diseases. It is indispensable for life activities. vitamins. For humans and animals, vitamin B must be obtained from food 2 . The human body needs about 0.3-1.8mg vitamin B per day 2 , while animal feed must contain 1-4mg / kg vitamin B 2 In order to meet its growth needs and improve nutrient utilization. [00...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/04C12Q1/686C12N15/11C12R1/125
CPCC12Q1/686C12Q1/689
Inventor 卢正东肖其磊陈情尹露露付强雷华
Owner HUBEI GUANGJI PHARMA
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