CN104004846B - A kind of method identifying Bacillus coagulans - Google Patents
A kind of method identifying Bacillus coagulans Download PDFInfo
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- CN104004846B CN104004846B CN201410245441.8A CN201410245441A CN104004846B CN 104004846 B CN104004846 B CN 104004846B CN 201410245441 A CN201410245441 A CN 201410245441A CN 104004846 B CN104004846 B CN 104004846B
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The present invention relates to a kind of method identifying Bacillus coagulans, be specifically related to a kind of molecular biology method of qualification Bacillus coagulans newly.The method, by DNA sequence dna design Auele Specific Primer: the HFQ1/HFQ2 to Bacillus coagulans gene specific, is carried out pcr amplification detection with the genomic dna of this primer pair testing sample, coagulating bacillus strain can be identified the level of planting.The present invention by provide for Bacillus coagulans qualification a kind of more accurately, be easier to the practical approach that operates, be conducive to classification and the research work of coagulating bacillus strain.
Description
Technical field
The present invention relates to a kind of microorganism volume authentication method, be specifically related to a kind of method identifying Bacillus coagulans, belong to molecular biological technical field.
Background technology
Bacillus coagulans (Bacilluscoagulans) is in shaft-like, and the blunt circle in two ends, gram-positive microorganism, catalase positive, brood cell holds life, atrichia.Optimum growth temperature is 45 ~ 50 DEG C, and optimum pH is 6.6 ~ 7.0.It can decompose carbohydrate and generate Pfansteihl, is homofermentative lactic bacterium.It is the bacillus class milk-acid bacteria that the one ratified through U.S. FDA " generally believes safety ".Bacillus coagulans (Bacilluscoagulans) is the up-and-coming youngster of field of probiotic bacteria in recent years, it is except having the health-care effect such as milk-acid bacteria and bifidus bacillus, also there is the high resistance to cold and diseases such as high temperature resistant, acidproof and bile tolerance, and there is stronger suppression pathogen enterobacteria ability.
Be widely used in the fields such as health care, medical treatment, food and herding abroad, but just at the early-stage about the research and development of Bacillus coagulans in China.Bacillus coagulans is a kind of important microbe causing tinned pre-flat cover to become sour.
In sum, no matter be utilize as probiotic bacterium, or as the arch-criminal of becoming sour, all need one authentication method fast, and traditional biochemical identification complex operation, detection time are long, can not meet the demand of every field, thus, be necessary to the research of Bacillus coagulans rapid detection.PCR method has the advantage that detection speed is fast, highly sensitive, cost is low, is one method fast and effectively in current Bacteria Identification.
Summary of the invention
The object of the invention is to, a kind of method identifying Bacillus coagulans is provided, to overcome the above-mentioned shortcoming and defect existing for prior art.The invention provides a kind of detection speed is fast, cost is low rapid detection Bacillus coagulans to kind PCR method.
The technical problem that will solve required for the present invention, can be achieved through the following technical solutions:
As a first aspect of the present invention, a kind of Auele Specific Primer identifying Bacillus coagulans, is characterized in that, described primer is a pair Auele Specific Primer HFQ1/HFQ2, wherein, forward primer HFQ1 sequence is identical with SEQIDNO.1, for: GTCACGGAAGAGCAAGCTTG; Reverse primer HFQ2 sequence is identical with SEQIDNO.2, for: GTTTCTGAAATGTATGCACG.
As a second aspect of the present invention, the method for a species-specific primer qualification Bacillus coagulans, is characterized in that, comprise the following steps:
(1) extract Bacillus coagulans (Bacilluscoagulans), prepare control strain;
(2) with the DNA of bacteria in step (1) for template, carry out pcr amplification with HFQ1/HFQ2 primer; With
(3) with 1% agarose gel electrophoresis, above-mentioned pcr amplification result is verified.
In step (2), forward primer HFQ1 sequence is identical with SEQIDNO.1, for: GTCACGGAAGAGCAAGCTTG; Reverse primer HFQ2 sequence is identical with SEQIDNO.2, for: GTTTCTGAAATGTATGCACG.
In step (2), PCR amplification system is: 20 μ l are by 2 μ l10 × BufferwithMgCl
2, 1 μ ldNTP2.5mmol/L, 1 μ lHFQ110mmol/L, 1 μ lHFQ210mmol/L, 0.1 μ lTaqDNAPolymerase5U/ μ l, 1 μ lDNA template, 13.9 μ lddH
2o;
Pcr amplification condition is: denaturation 94 DEG C of 5min, sex change 94 DEG C of 35s, and anneal 50 DEG C of 40s, extends 72 DEG C of 40s, 30 circulations, and 72 DEG C extend 10min, 4 DEG C of preservations.
As a third aspect of the present invention, a kind of purposes identifying the Auele Specific Primer of Bacillus coagulans, is characterized in that: for the Molecular Detection of Bacillus coagulans.
Beneficial effect of the present invention:
1, increase compared with the 16sDNA of conventional identification Bacillus coagulans, order-checking, comparison, numerous and diverse step such as physiological and biochemical test, method of the present invention can Direct Identification whether go out be Bacillus coagulans, save experimental resources largely, can be used for the qualification Bacillus coagulans of extensive sample.
2, primer of the present invention designs according to the special conservative gene of Bacillus coagulans, applies primer HFQ1/HFQ2 of the present invention from all kinds of sample DNA, directly can amplify the Bacillus coagulans object fragment that length is approximately 290bp.
Accompanying drawing explanation
Fig. 1 is the gel electrophoresis figure of HFQ1/HFQ2 primer pair kind DNA of bacteria amplified production in embodiment 1.
M swimming lane is standard DNA marker2000.
No. 1 swimming lane is the amplification of Bacillus coagulans.
No. 2 swimming lanes are negative control.
3-10 swimming lane is respectively containing Bacillus subtillis (Bacillussubtilis), bacillus cereus (Bacilluscereus), bacillus megaterium (Bacillusmegaterium), Bacillus licheniformis (Bacilluslicheniformis), intestinal bacteria type strain (EscherichiacoliATCC25922), streptococcus aureus (Staphylococcusaureus), the DNA of pneumococcus (Pneumococcus) and hemophilus influenzae (Haemophilusinfluenzae).
Fig. 2 is the gel electrophoresis figure of the different sample DNA amplified production of HFQ1/HFQ2 primer pair in embodiment 2.
M swimming lane is standard DNA marker2000.
No. 1 swimming lane is the amplification of Bacillus coagulans.
No. 2 swimming lanes are negative control.
No. 3 swimming lanes are soil A sample DNA.
No. 4 swimming lanes are soil B sample DNA.
5-swimming lane is tinned tomatoes A sample DNA.
No. 6 swimming lanes are tinned tomatoes B sample DNA.
Embodiment
In conjunction with following specific embodiments and the drawings, the present invention is described in further detail.Implement process of the present invention, condition, reagent, experimental technique etc., except the following content mentioned specially, be universal knowledege and the common practise of this area, the present invention is not particularly limited content.
Embodiment 1
Experimental procedure
1, extract Bacillus coagulans (Bacilluscoagulans), Bacillus subtillis (Bacillussubtilis), bacillus cereus (Bacilluscereus), bacillus megaterium (Bacillusmegaterium), Bacillus licheniformis (Bacilluslicheniformis), intestinal bacteria type strain (EscherichiacoliATCC25922), streptococcus aureus (Staphylococcusaureus), the DNA of pneumococcus (Pneumococcus) and hemophilus influenzae (Haemophilusinfluenzae), above-mentioned bacterial classification source is in table 1.
2, with the DNA of bacteria in step 1 for template, carry out pcr amplification with HFQ1/HFQ2 primer.
PCR amplification system is: 20 μ l are by 2 μ l10 × Buffer (withMgCl
2), 1 μ ldNTP (2.5mmol/L), 1 μ lHFQ1 (10mmol/L), 1 μ lHFQ2 (10mmol/L), 0.1 μ lTaqDNAPolymerase (5U/ μ l), 1 μ lDNA template, 13.9 μ lddH
2o.
Pcr amplification condition is: denaturation 94 DEG C of 5min, sex change 94 DEG C of 35s, and anneal 50 DEG C of 40s, extends 72 DEG C of 40s, 30 circulations, and 72 DEG C extend 10min, 4 DEG C of preservations.
3, with 1% agarose gel electrophoresis, above-mentioned pcr amplification result is verified.
Experimental result
Detected result as shown in Figure 1, as can be seen from Figure 1 present embodiment can amplify the target sequence fragment of Bacillus coagulans accurately for the Auele Specific Primer HFQ1/HFQ2 of the Bacillus coagulans that increases, and fails to belong to DNA of bacteria and blank from other to amplify nucleic acid fragment.Amplified fragments corresponding for No. 1 swimming lane is checked order, through Blast compare of analysis, determines that it is Bacillus coagulans special gene sequence.
Table 1 bacterial classification source table
Embodiment 2
Experimental procedure
1,2 parts of tinned tomatoes samples becoming sour rotten and 2 parts of Jiangsu Province Haimen City's pedotheque are carried out to the extraction of DNA, above-mentioned materials source is in table 2.
2, with the DNA of bacteria in step 1 for template, carry out pcr amplification with HFQ1/HFQ2 primer.
PCR amplification system is: 20 μ l are by 2 μ l10 × Buffer (withMgCl
2), 1 μ ldNTP (2.5mmol/L), 1 μ lHFQ1 (10mmol/L), 1 μ lHFQ2 (10mmol/L), 0.1 μ lTaqDNAPolymerase (5U/ μ l), 1 μ lDNA template, 13.9 μ lddH
2o.
Pcr amplification condition is: denaturation 94 DEG C of 5min, sex change 94 DEG C of 35s, and anneal 50 DEG C of 40s, extends 72 DEG C of 40s, 30 circulations, and 72 DEG C extend 10min, 4 DEG C of preservations.
3, with 1% agarose gel electrophoresis, above-mentioned pcr amplification result is verified.
Table 2 material source registry form
| Title material | Source |
| Soil A | San Chang town, Haimen City of Jiangsu Province Bao Jiaqiao |
| Soil B | Community Service Center of Green Dragon port village, San Chang town, Haimen City of Jiangsu Province |
| Tinned tomatoes A | Benincasa board, after opening, normal temperature places 2 months |
| Tinned tomatoes B | Plum forests board, after opening, normal temperature places 2 months |
Experimental result
Detected result as shown in Figure 2, as can be seen from Figure 2 the present embodiment target sequence fragment that can amplify Bacillus coagulans accurately for the Auele Specific Primer HFQ1/HFQ2 of the Bacillus coagulans that increases, can amplify Bacillus coagulans gene-specific fragments from 2 parts of become sour tinned tomatoes sample DNA and 1 part of pedotheque DNA (No. 4 swimming lanes).Amplified fragments corresponding for 5,6 and No. 4 swimming lanes is checked order, through Blast compare of analysis, determines that it is Bacillus coagulans special gene sequence.This and Bacillus coagulans in bibliographical information are extensively present in soil and the tinned tomatoes that becomes sour consistent.
Be illustrated the specific embodiment of the present invention above, but the present invention is not as limit, only otherwise depart from aim of the present invention, the present invention can also have various change.
Claims (3)
1. identify an Auele Specific Primer for Bacillus coagulans, it is characterized in that, described primer is a pair Auele Specific Primer HFQ1/HFQ2, and wherein, forward primer HFQ1 sequence is identical with SEQIDNO.1, for: GTCACGGAAGAGCAAGCTTG; Reverse primer HFQ2 sequence is identical with SEQIDNO.2, for: GTTTCTGAAATGTATGCACG.
2. a method for Auele Specific Primer qualification Bacillus coagulans as claimed in claim 1, is characterized in that, comprise the following steps:
(1) extract Bacillus coagulans (Bacilluscoagulans), prepare control strain;
(2) with the DNA of bacteria in step (1) for template, carry out pcr amplification with HFQ1/HFQ2 primer; Forward primer HFQ1 sequence is identical with SEQIDNO.1, for: GTCACGGAAGAGCAAGCTTG; Reverse primer HFQ2 sequence is identical with SEQIDNO.2, for: GTTTCTGAAATGTATGCACG; PCR amplification system is: 20 μ l are by 2 μ l10 × BufferwithMgCl
2, 1 μ ldNTP2.5mmol/L, 1 μ lHFQ110mmol/L, 1 μ lHFQ210mmol/L, 0.1 μ lTaqDNAPolymerase5U/ μ l, 1 μ lDNA template, 13.9 μ lddH
2o;
Pcr amplification condition is: denaturation 94 DEG C of 5min, sex change 94 DEG C of 35s, and anneal 50 DEG C of 40s, extends 72 DEG C of 40s, 30 circulations, and 72 DEG C extend 10min, 4 DEG C of preservations;
(3) with 1% agarose gel electrophoresis, above-mentioned pcr amplification result is verified.
3. a purposes for the Auele Specific Primer of qualification Bacillus coagulans as claimed in claim 1, is characterized in that: for the Molecular Detection of Bacillus coagulans.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2017058741A1 (en) | 2015-10-01 | 2017-04-06 | Muhammed Majeed | Novel pcr primers and methods thereof for the identification of bacillus coagulans |
| CN105256054B (en) * | 2015-11-13 | 2019-02-15 | 通威股份有限公司 | A kind of method and multiple PCR reagent kit of quick detection bacillus coagulans |
| CN107418920A (en) * | 2017-08-31 | 2017-12-01 | 江苏微康生物科技有限公司 | A kind of screening technique of bacillus coagulans |
Citations (6)
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|---|---|---|---|---|
| JP2010004855A (en) * | 2008-06-30 | 2010-01-14 | Q P Corp | Oligonucleotide and method for detecting bacillus coagulans using the same as primer |
| JP2011000008A (en) * | 2009-06-16 | 2011-01-06 | Toyo Seikan Kaisha Ltd | Method for detecting bacillus coagulans in test sample specifically, in a short period of time and with high sensitivity |
| CN102041298A (en) * | 2009-10-09 | 2011-05-04 | 中粮新疆屯河股份有限公司 | PCR (Polymerase Chain Reaction) rapid detection method for Bacillus coagulans |
| CN102304559A (en) * | 2010-02-05 | 2012-01-04 | 山东出入境检验检疫局检验检疫技术中心 | Fluorescence quantitative polymerase chain reaction (PCR) method for detecting bacillus coagulans quickly |
| JP4888622B2 (en) * | 2001-06-04 | 2012-02-29 | キユーピー株式会社 | Oligonucleotide and method for detecting batylscore glance using the same as primer |
| CN102533743A (en) * | 2011-12-23 | 2012-07-04 | 南京农业大学 | Specific primer for amplifying bacillus coagulans and application thereof |
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- 2014-06-04 CN CN201410245441.8A patent/CN104004846B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4888622B2 (en) * | 2001-06-04 | 2012-02-29 | キユーピー株式会社 | Oligonucleotide and method for detecting batylscore glance using the same as primer |
| JP2010004855A (en) * | 2008-06-30 | 2010-01-14 | Q P Corp | Oligonucleotide and method for detecting bacillus coagulans using the same as primer |
| JP2011000008A (en) * | 2009-06-16 | 2011-01-06 | Toyo Seikan Kaisha Ltd | Method for detecting bacillus coagulans in test sample specifically, in a short period of time and with high sensitivity |
| CN102041298A (en) * | 2009-10-09 | 2011-05-04 | 中粮新疆屯河股份有限公司 | PCR (Polymerase Chain Reaction) rapid detection method for Bacillus coagulans |
| CN102304559A (en) * | 2010-02-05 | 2012-01-04 | 山东出入境检验检疫局检验检疫技术中心 | Fluorescence quantitative polymerase chain reaction (PCR) method for detecting bacillus coagulans quickly |
| CN102533743A (en) * | 2011-12-23 | 2012-07-04 | 南京农业大学 | Specific primer for amplifying bacillus coagulans and application thereof |
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