Primer and method for identifying leuconostoc mesenteroides subsp.mesenteroides and application of leuconostoc mesenteroides subsp.mesenteroides

A technology for identification of Leuconostoc enteritiformis and its identification method, which is applied in the field of primers for identifying Leuconostoc enteritidis subspecies, which can solve the time-consuming, cumbersome identification of Leuconostoc enteritidis subspecies, and the impossibility of subspecies identification. Level identification and other issues to achieve the effect of accelerated application and simple identification

Active Publication Date: 2017-01-11
BRIGHT DAIRY & FOOD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The purpose of the present invention is to provide a new primer for identifying Leuconostoc enteritidis subspecies and its method and application in view of the tedious and time-consuming identification of Leuconostoc enteritidis subspecies at present.
The identification method of the present invention is simpler and more effective than the traditional biochemical identification, and at the same time solves the problem that the currently used targets (such as 16S rDNA and gyrB, etc.) A highly efficient molecular biology approach is developed to facilitate the screening of Leuconostoc enterolis strains with superior properties and their application in food

Method used

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  • Primer and method for identifying leuconostoc mesenteroides subsp.mesenteroides and application of leuconostoc mesenteroides subsp.mesenteroides
  • Primer and method for identifying leuconostoc mesenteroides subsp.mesenteroides and application of leuconostoc mesenteroides subsp.mesenteroides
  • Primer and method for identifying leuconostoc mesenteroides subsp.mesenteroides and application of leuconostoc mesenteroides subsp.mesenteroides

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Embodiment 1

[0029] Primer design and validation for the identification of Leuconostoc enteritidis subspecies:

[0030] By comparing and analyzing the existing genome sequences of Leuconostoc enterococci (see attached table 1 for each strain and its genome sequence extraction code), a ubiquitous GH70 glycosyltransferase coding gene was identified and named gtf1. The gtf1 gene contains two highly conserved base sequences encoding the GH70 domain. Between these two highly conserved sequences, there is a highly variable sequence, which shows a high degree of polymorphism in different strains ( polymorphism).

[0031] Table 1 The complete genome information of Leuconostoc enterolis

[0032]

[0033] The schematic diagram of the gtf1 gene and its encoded product is attached figure 1 As shown, we designed primers in the highly conserved GH70 coding region to amplify the central highly variable region, and identified the strain by sequencing the amplified product.

[0034] According to the ...

Embodiment 2

[0039] A method for identifying Leuconostoc enteritidis isolated from traditional foods at the level of Enteromenis subspecies, the method comprising the following steps:

[0040] (1) Taking Leuconostoc enterococci BD1710 isolated from traditional food in our laboratory as the test bacteria, the genomic DNA of the test strains was extracted, and the obtained DNA was used as a template for PCR amplification.

[0041] (2) Using the genomic DNA to be tested extracted from the above strains as a template, the PCR amplification reaction system is as follows: 2 μl of 2ng / μl DNA template, 1 μl of 0.5 μmol / L primer, 2 μl of 0.2mmol / L dNTP, 1.5mmol / L MgSO4 Add 1 μl, 2 μl of 10×PCR reaction buffer, 0.5 μl of 5U / μl rTaq DNA polymerase to 20 μl.

[0042] (3) Reaction parameters of PCR amplification: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 30 s, annealing at 48°C for 30 s, extension at 72°C for 2 min, and 35 cycles; extension at 72°C for 15 min, and storage at 4°C.

...

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Abstract

The invention provides a primer and method for identifying leuconostoc mesenteroides subsp.mesenteroides and application of the leuconostoc mesenteroides subsp.mesenteroides. The primer for identifying the leuconostoc mesenteroides subsp.mesenteroides is designed by utilizing a high-conservation GH70 structural domain coding design in a GH70 glycosyltransferase coding gene; the primer is used for amplifying on a high-variation region; and a segment subjected to amplification is subjected to sequencing, and subsequent sequence alignment analysis is carried out to realize identification of leuconostoc mesenteroides in a subsp.mesenteroides level. The primer disclosed by the invention can be used for realizing the identification of the leuconostoc mesenteroides in the subspecies level; and compared with a traditional biochemical identification method, the identification method of the primer is more efficient and can be used for rapidly, simply and conveniently identifying potential fermentation agent strains in a food industry, so that evidences are provided for application of novel strains in foods, and application of novel strain resources in the field of the foods is accelerated.

Description

technical field [0001] The invention belongs to the technical field of food biology, and in particular relates to a primer for identifying Leuconostoc enteritidis subspecies and its method and application. Background technique [0002] Currently, Leuconostoc mesensteroides obtained by isolation and screening is a plant epiphytic bacterium commonly found in traditional fermented dairy products and pickles. Because of its ability to ferment to produce lactic acid and diacetyl substances, it is considered to have an important contribution to the flavor of these traditional foods. In 2012, the Ministry of Health included Leuconostoc mesensteroides subsp.mesenteroides in the catalog of strains that can be used in food (Ministry of Health Announcement No. 8 in 2012), about the fermentation characteristics of Leuconostoc mesensteroides subsp.mesenteroides And the research on its special physiological activity is gradually in-depth. Studies have confirmed that Leuconostoc enteroco...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/6869C12Q1/689C12Q2537/165C12Q2531/113
Inventor 鄢明辉吴正钧刘振民高彩霞韩瑨
Owner BRIGHT DAIRY & FOOD
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