CN102433385B

CN102433385B - Primers for identifying leuconostoc mesenteroides and application thereof

Info

Publication number: CN102433385B
Application number: CN201110429471.0A
Authority: CN
Inventors: 刘婷; 刘滢; 胡婷; 杜建涛; 王安如
Original assignee: BEIJING DBN AGRICULTURAL FEED Co Ltd; HARBIN DABEI FARMING AND ANIMAL HUSBANDRY TECHNOLOGY Co Ltd; Beijing Dabeinong Technology Group Co Ltd
Current assignee: Nanning Dabeinong Feed Technology Co., Ltd.; Beijing Dabeinong Technology Group Co Ltd
Priority date: 2011-12-20
Filing date: 2011-12-20
Publication date: 2014-10-15
Anticipated expiration: 2031-12-20
Also published as: CN102433385A

Abstract

The invention belongs to the filed of molecular biology, and particularly relates to primers for identifying leuconostoc mesenteroides and an application thereof. The primers for identifying leuconostoc mesenteroides provided by the invention can specifically amplify partial sequences of leuconostoc mesenteroides 16S rDNA (recombinant deoxyribonucleic acid) with high amplification specificity, and can accurately and rapidly identify leuconostoc mesenteroides. The process of identifying leuconostoc mesenteroides by the primers is simple, and the identifying method is stable and efficient, thus not only shortening detection time but also lowing detection cost. According to the invention, a detection method for identifying germ plasm resources of leuconostoc mesenteroides is provided, thus establishing foundation for screening excellent strains of leuconostoc mesenteroides.

Description

Primer and application thereof for the identification of Leuconostoc mesenteroides

Technical field

The invention belongs to biology field, be specifically related to primer and application thereof for the identification of Leuconostoc mesenteroides.

Background technology

Leuconostoc mesenteroides (Leuconostoc mesenteroides) is the important bacterial classification of the leuconos toc in milk-acid bacteria, is generally present in plant surface, is usually used in cultured milk prod, ensiling, pickles and fruit wine.Its glucomannan of can degrading is oligosaccharides, fermenting carbohydrate produces multiple acid and alcohol, there is the characteristics such as high acid ability, resistance of oxidation and antagonism pathogenic bacterium, be widely used in the industries such as medicine, food and biochemical preparation, also will be expected to become novel probiotics.

Current domestic isolation identification Leuconostoc mesenteroides adopts traditional methods of inspection such as plane culture method, physiological and biochemical test more, and aforesaid method exists the shortcomings such as long, level of automation is low, poor accuracy of the cycle that detects.Along with molecular biological development, 16S rDNA sequential analysis is used to the isolation identification of milk-acid bacteria gradually.Before order-checking, pcr amplification is that to take the DNA of the method extractings such as alkaline lysis be template, and success ratio is high, yet seems comparatively loaded down with trivial details.From a large amount of samples, during certain bacterium of directed screening, 16S rDNA sequence sequencing analysis cost is also higher.

Summary of the invention

The object of this invention is to provide a kind of primer for the identification of Leuconostoc mesenteroides, the partial sequence that this primer can specific amplification Leuconostoc mesenteroides 16S rDNA, specific amplification is high, and the time is short, can be accurately, Rapid identification Leuconostoc mesenteroides.

Another object of the present invention is to provide the application of this primer in identifying Leuconostoc mesenteroides, and this primer is simple for the identification of Leuconostoc mesenteroides process, and authentication method is stable, efficient.Meanwhile, adopt colony polymerase chain reaction (PCR) method, sample is skipped to this step of DNA extracting, and directly take the DNA exposing after thalline pyrolysis, as template, carry out pcr amplification, not only shortened detection time, also reduced testing cost.

The object of the invention is to be achieved through the following technical solutions:

The invention provides a kind of primer for the identification of Leuconostoc mesenteroides (Leuconostoc mesenteroides), it is characterized in that: forward primer has the base sequence described in SEQ ID No:1 in sequence table, reverse primer has the base sequence described in SEQ ID No:2 in sequence table.

Primer sequence of the present invention can be used for identifying Leuconostoc mesenteroides, and qualification process is as follows:

(1) use described primer pair bacterial strain to be measured to carry out colony PCR amplification;

(2) utilize electrophoresis detection PCR product, at 430bp place, produce the bacterial strain of specific amplification band, be judged as Leuconostoc mesenteroides.

Wherein, prepare the process of template of bacterial strain to be measured as follows: single bacterium colony of getting micro-bacterial strain to be measured with toothpick or the rifle choicest of sterilizing is to the centrifuge tube of 0.5mL, and during 94 ℃ of sex change of pcr amplification, the DNA exposing after thalline pyrolysis is template.

Wherein, colony PCR amplification program is as follows: 95 ℃ of denaturation 5min, and 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 30s, carry out 30 circulations, and last 72 ℃ are extended 10min.

Wherein, described sepharose is that mass volume ratio is 2% agarose gel electrophoresis, and uses ethidium bromide staining.

Wherein, it is only experiment material that the present invention prepares the Leuconostoc mesenteroides relating in the process of bacterial strain template to be measured, the present invention does not limit specific bacterial strain, can be any strain Leuconostoc mesenteroides that openly maybe can buy now both at home and abroad, can be arbitrarily parting material voluntarily, no matter any strain Leuconostoc mesenteroides does not all affect enforcement of the present invention yet.

Primer of the present invention can be any strain Leuconostoc mesenteroides now and identifies, compared with prior art, technical scheme tool of the present invention has the following advantages: the partial sequence that the primer sequence for the identification of Leuconostoc mesenteroides of the present invention can specific amplification Leuconostoc mesenteroides 16S rDNA, specific amplification is high, can be accurately, Rapid identification Leuconostoc mesenteroides.This primer is simple for the identification of the bacterium colony PCR process of Leuconostoc mesenteroides, and authentication method is stable, efficient, has not only shortened detection time, and has reduced testing cost (table 1).The present invention identifies detection method is provided for Leuconostoc mesenteroides germ plasm resource, and then lays a good foundation for screening the strain excellent of Leuconostoc mesenteroides.

The comparison of table 1 detection method of the present invention and traditional detection method

	Detection time	Detect cost
			Traditional method	One week left and right	25 yuan
The inventive method	2 hours	3 yuan

Accompanying drawing explanation

Fig. 1 is the gel electrophoresis spectrum of each bacterial strain amplified production in PCR specific amplification.

Wherein, M is nucleic acid molecular weight standard; 1. Leuconostoc mesenteroides strain (Leuconostoc mesenteroides) FLSJ, 2. Leuconostoc mesenteroides strain, 3. Leuconostoc mesenteroides strain, 4. lactobacillus salivarius, 5. Lactobacillus johnsonii, 6. lactobacillus rhamnosus, 7. digestion Bacterium lacticum, 8. cibarium Wei Si Salmonella, 9. lactobacillus sake, 10. lactobacillus curvatus, 11. plant lactobacilluss, 12. thermophilus streptococcuses and 13. faeciums, CK is water contrast.

Fig. 2 is the gel electrophoresis spectrum of the amplified production of bacterium in pickles sample.

By the following example, by more specific description the present invention, it should be understood that described embodiment is only for the present invention is described, rather than limit the scope of the invention by any way.

Embodiment

The design of embodiment 1 Leuconostoc mesenteroides Auele Specific Primer is with synthetic

The Genebank of NCBI database ( http://www.ncbi.nlm.nih.gov/genome/) in obtain the Lactobacillus johnsonii (Lactobacillus johnsonii) of the lactobacillus in Leuconostoc mesenteroides and source nearly with it, short lactobacillus (Lactobacillus brevis), lactobacillus rhamnosus (Lactobacillus rhamnosus), lactobacillus salivarius (Lactobacillus saliarius), plant lactobacillus (Lactobacillus plantarum), lactobacillus sake (Lactobacillus sakei), lactobacterium casei (Lactobacillus casei) etc., the faecium of enterococcus spp (Enterococcus faecium), the subtilis of Sporolactobacillus (Bacillu subtilis), the Pediococcus pentosaceus of Pediococcus (Pediococcus pentosaceus), and the 16S rDNA sequence of the bacterial strain such as staphylococcus epidermidis (Staphylococcus epidermidis).Utilize the 16S rDNA sequence of these bacterial strains of Multiple Sequence Alignment software DNAMAN compare of analysis, search in Leuconostoc mesenteroides kind and guard and the not conservative specific regions of all the other Bacterium lacticum, design primer also screens and optimizes specific sequence, has finally obtained preferably primer of a pair of parameter.In the concrete sequence of above-mentioned primer and sequence table, corresponding relation is as shown in table 2 below:

Table 2

Primer title	Sequence	Sequence number
			Primer 1	5’-TTAAAAGGCGCTTCGGCG-3’	SEQ ID NO：1
Primer 2	5’-CATTCCGGAGTTGAGCTCCG-3’	SEQ ID NO：2

Above-mentioned sequence can be synthesized by ordinary method, also can entrust professional biological reagent company synthetic, and primer of the present invention is synthetic by the English Weihe River prompt base (Shanghai) trade Co., Ltd.

The checking of embodiment 2 Auele Specific Primers

The thalline of lactobacillus salivarius, Lactobacillus johnsonii, lactobacillus rhamnosus, digestion Bacterium lacticum, cibarium Wei Si Salmonella, lactobacillus sake, lactobacillus curvatus, plant lactobacillus, thermophilus streptococcus and faecium of Leuconostoc mesenteroides and nearly edge thereof of take is respectively template, with water belongs with yin contrast, carry out bacterium colony PCR.Wherein, the bacterial strains such as the lactobacillus salivarius relating in this experiment, Lactobacillus johnsonii, lactobacillus rhamnosus, digestion Bacterium lacticum, cibarium Wei Si Salmonella, lactobacillus sake, lactobacillus curvatus, plant lactobacillus, thermophilus streptococcus and faecium are control strain experiment material, must not be defined as certain strain bacterial strain, can be as required or available experiment material selected, on enforcements of the present invention without affecting.

On the culture dish of picking trace, single bacterium colony of each bacterial strain is to 0.5mL sterilizing centrifuge tube, and the synthetic base sequence of take in embodiment 1 carries out bacterium colony PCR reaction as primer, and reaction system is:

To divide the centrifuge tube of the system that installs to put into PCR instrument, response procedures be:

After reaction finishes, pcr amplification product is through 2% agarose gel electrophoresis result of determination.That at 430bp place, finds specific amplification band is judged to be positive findings.

Experimental result shows: only have the pcr amplification product of Leuconostoc mesenteroides to occur positive amplification, and amplified signal does not all appear in other nearly edge milk-acid bacteria and negative control, the results are shown in Figure 1.As can be seen here, the specificity of this primer pair Leuconostoc mesenteroides is very high, can be used in rapid detection and identifies the Leuconostoc mesenteroides in sample.

The isolation identification of Leuconostoc mesenteroides in embodiment 3 pickles samples

By the line separation on MRS substratum respectively of 6 parts of pickles samples collecting, respectively select 5 colonies typicals and continue line separated 2 times.MRS substratum be milk-acid bacteria partly select substratum, milk-acid bacteria is had to certain selective action.

Step 1 Auele Specific Primer is identified

On the single bacterium colony growing from flat board, picking trace thalline, joins in pcr amplification system (20 μ L), and PCR primer is Leuconostoc mesenteroides Auele Specific Primer, carries out bacterium colony PCR.Then carry out agarose gel electrophoresis, amplify 430bp fragment person for Leuconostoc mesenteroides.

As shown in Figure 2, bacterial strain 8,13,14 and 15 has amplified 430bp fragment to result.This 4 strain bacterium is initially identified as to Leuconostoc mesenteroides.

Step 2 order-checking is identified

PCR system is expanded as to 50 μ L, by 16S universal primer colony PCR amplification step 1, detect the 16S rDNA sequence fragment of bacterial strain.Re-use glue recovery technology purifying amplified fragments, then send the order-checking of the English Weihe River prompt base (Shanghai) trade Co., Ltd.Utilize the BLASTn instrument comparison sequencing result of NCBI website.According to sequencing result, identification of strains to be measured is arrived and planted.

16S order-checking is identified and is shown: the bacterial strain that amplifies 430bp fragment is Leuconostoc mesenteroides.This specificity of primer pair Leuconostoc mesenteroides that shows design is very high.In conjunction with MRS, partly select the Leuconostoc mesenteroides of substratum in can Rapid Detection pickles.

Step 3 Physiology and biochemistry is identified

The bacterial strain that step 1 is detected carries out gramstaining and examines under a microscope (* 1000).According to the biochemical characteristic of Leuconostoc mesenteroides, to detecting bacterial strain, carry out respectively following biochemical test: glucose fermentation test, catalase test, gelatin liquification test, hydrogen sulfide produce test and indoles produces test.

Result shows: Auele Specific Primer is accredited as the bacterial strain of Leuconostoc mesenteroides, Gram-positive, and thalline is spherical, its biochemical test result all meets the feature of Leuconostoc mesenteroides, that is: glucose fermentation produces acid, and liquefy gelatin, does not produce catalase, hydrogen sulfide and indoles.

Claims

For the identification of Leuconostoc mesenteroides ( leuconostoc mesenteroides) primer, it is characterized in that, the base sequence of forward primer is as shown in SEQ ID No:1 in sequence table, the base sequence of reverse primer is as shown in SEQ ID No:2 in sequence table.
2. the application of primer claimed in claim 1 in identifying Leuconostoc mesenteroides, is characterized in that, qualification process is as follows:

(1) right to use requires the primer pair bacterial strain to be measured described in 1 to carry out colony PCR amplification;

(2) utilize electrophoresis detection PCR product, at 430bp place, produce the bacterial strain of specific amplification band, be judged as Leuconostoc mesenteroides.
3. the application of primer as claimed in claim 2 in identifying Leuconostoc mesenteroides, it is characterized in that, the preparation process of the template of bacterium colony PCR is as follows: single bacterium colony of getting micro-bacterial strain to be measured with toothpick or the rifle choicest of sterilizing is to the centrifuge tube of 0.5mL, during 94 ℃ of sex change of pcr amplification, the DNA exposing after thalline pyrolysis is template.
4. the application of primer as claimed in claim 2 in identifying Leuconostoc mesenteroides, is characterized in that, colony PCR amplification program is as follows: 95 ℃ of denaturation 5min, 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 30s, carry out 30 circulations, last 72 ℃ are extended 10min.
5. the application of primer as claimed in claim 2 in identifying Leuconostoc mesenteroides, is characterized in that, described electrophoresis is that mass volume ratio is 2% agarose gel electrophoresis, and uses ethidium bromide staining.