CN104004846A - Method for identifying bacillus coagulans - Google Patents

Method for identifying bacillus coagulans Download PDF

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Publication number
CN104004846A
CN104004846A CN201410245441.8A CN201410245441A CN104004846A CN 104004846 A CN104004846 A CN 104004846A CN 201410245441 A CN201410245441 A CN 201410245441A CN 104004846 A CN104004846 A CN 104004846A
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bacillus coagulans
primer
hfq2
hfq1
pcr amplification
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CN104004846B (en
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符雷
郗洪生
施腾鑫
陆亚怡
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JIANGSU HENGFENGQIANG BIOTECHNOLOGY CO Ltd
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JIANGSU HENGFENGQIANG BIOTECHNOLOGY CO Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • 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 invention relates to a method for identifying bacillus coagulans, and particularly relates to a novel molecular biology method for identifying bacillus coagulans. By adopting the method, a specific primer HFQ1/HFQ2 is designed for a specific deoxyribonucleic acid (DNA) sequence of a bacillus coagulans gene, polymerase chain reaction (PCR) amplification detection is carried out on genome DNA of a to-be-detected sample by using the primer, and a bacillus coagulans strain can be identified to the level of seeds. A practical method which is more accurate and easier to operate is provided for identifying the bacillus coagulans, and classification and research work of the bacillus coagulans strain can be facilitated.

Description

A kind of method of identifying Bacillus coagulans
Technical field
The present invention relates to a kind of microorganism volume authentication method, be specifically related to a kind of method of identifying Bacillus coagulans, belong to molecular biological technical field.
Background technology
Bacillus coagulans (Bacillus coagulans) is shaft-like, the blunt circle in two ends, and gram-positive microorganism, hydrogen peroxide enzyme 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 homotype lactic acid fermenting bacteria.It is the bacillus class milk-acid bacteria that " generally believes safety " through the one of U.S. FDA approval.Bacillus coagulans (Bacillus coagulans) is the up-and-coming youngster in probiotic bacterium field 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 inhibition pathogen enterobacteria ability.
Be widely used in abroad the fields such as health care, medical treatment, food and herding, but just at the early-stage about the research and development of Bacillus coagulans in China.Bacillus coagulans is a kind of important microbe that causes tinned pre-flat cover to become sour.
In sum, utilize as probiotic bacterium no matter be, 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, thereby, be necessary to the research of Bacillus coagulans rapid detection.PCR method has advantages of 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 of identifying Bacillus coagulans is provided, to overcome the existing above-mentioned shortcoming and defect of prior art.The invention provides the rapid detection Bacillus coagulans that a kind of detection speed is fast, cost is low and arrive the PCR method of planting.
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 of identifying Bacillus coagulans, is characterized in that, described primer is a pair of Auele Specific Primer HFQ1/HFQ2, wherein, forward primer HFQ1 sequence is identical with SEQ ID NO.1, for: GTCACGGAAGAGCAAGCTTG; Reverse primer HFQ2 sequence is identical with SEQ ID NO.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, comprises the following steps:
(1) extract Bacillus coagulans (Bacillus coagulans), prepare control strain;
(2), taking the DNA of bacteria in step (1) as 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 SEQ ID NO.1, for: GTCACGGAAGAGCAAGCTTG; Reverse primer HFQ2 sequence is identical with SEQ ID NO.2, for: GTTTCTGAAATGTATGCACG.
In step (2), pcr amplification system is: 20 μ l are by 2 μ l10 × Bufferwith MgCl 2, 1 μ ldNTP2.5mmol/L, 1 μ l HFQ110mmol/L, 1 μ l HFQ210mmol/L, 0.1 μ l Taq DNAPolymerase5U/ μ l, 1 μ l DNA profiling, 13.9 μ l ddH 2o;
Pcr amplification condition is: 94 DEG C of 5min of denaturation, and 94 DEG C of 35s of sex change, the 50 DEG C of 40s that anneal, extend 72 DEG C of 40s, 30 circulations, 72 DEG C are extended 10min, 4 DEG C of preservations.
As a third aspect of the present invention, a kind of purposes of the Auele Specific Primer of identifying Bacillus coagulans, is characterized in that: for the Molecular Detection of Bacillus coagulans.
Beneficial effect of the present invention:
1, numerous and diverse step such as the 16sDNA amplification of more traditional qualification Bacillus coagulans, order-checking, comparison, physiological and biochemical test, whether method of the present invention can Direct Identification goes out is Bacillus coagulans, save largely experimental resources, 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 and can from all kinds of sample DNAs, directly amplify length and be approximately the Bacillus coagulans object fragment of 290bp.
Brief description of the drawings
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.
The amplification that No. 1 swimming lane is Bacillus coagulans.
No. 2 negative contrasts of swimming lane.
3-10 swimming lane is respectively and contains Bacillus subtillis (Bacillus subtilis), bacillus cereus (Bacillus cereus), bacillus megaterium (Bacillus megaterium), Bacillus licheniformis (Bacilluslicheniformis), intestinal bacteria type strain (Escherichia coli ATCC25922), streptococcus aureus (Staphylococcus aureus), the DNA of pneumococcus (Pneumococcus) and hemophilus influenzae (Haemophilusinfluenzae).
Fig. 2 is the gel electrophoresis figure of the different sample DNA amplified productions of HFQ1/HFQ2 primer pair in embodiment 2.
M swimming lane is standard DNA marker2000.
The amplification that No. 1 swimming lane is Bacillus coagulans.
No. 2 negative contrasts of swimming lane.
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 content of mentioning specially below, 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 (Bacillus coagulans), Bacillus subtillis (Bacillus subtilis), bacillus cereus (Bacillus cereus), bacillus megaterium (Bacillus megaterium), Bacillus licheniformis (Bacilluslicheniformis), intestinal bacteria type strain (Escherichia coli ATCC25922), streptococcus aureus (Staphylococcus aureus), the DNA of pneumococcus (Pneumococcus) and hemophilus influenzae (Haemophilusinfluenzae), above-mentioned bacterial classification is originated in table 1.
2,, taking the DNA of bacteria in step 1 as template, carry out pcr amplification with HFQ1/HFQ2 primer.
Pcr amplification system is: 20 μ l are by 2 μ l10 × Buffer (with MgCl 2), 1 μ l dNTP (2.5mmol/L), 1 μ l HFQ1 (10mmol/L), 1 μ l HFQ2 (10mmol/L), 0.1 μ l TaqDNA Polymerase (5U/ μ l), 1 μ l DNA profiling, 13.9 μ l ddH 2o.
Pcr amplification condition is: 94 DEG C of 5min of denaturation, and 94 DEG C of 35s of sex change, the 50 DEG C of 40s that anneal, extend 72 DEG C of 40s, 30 circulations, 72 DEG C are extended 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 and amplify nucleic acid fragment from other.No. 1 amplified fragments corresponding to swimming lane is checked order, through Blast compare of analysis, determine 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 that become sour rotten and 2 parts of Jiangsu Province Haimen City's pedotheque are carried out to the extraction of DNA, above-mentioned materials is originated in table 2.
2,, taking the DNA of bacteria in step 1 as template, carry out pcr amplification with HFQ1/HFQ2 primer.
Pcr amplification system is: 20 μ l are by 2 μ l10 × Buffer (with MgCl 2), 1 μ l dNTP (2.5mmol/L), 1 μ l HFQ1 (10mmol/L), 1 μ l HFQ2 (10mmol/L), 0.1 μ l TaqDNA Polymerase (5U/ μ l), 1 μ l DNA profiling, 13.9 μ l ddH 2o.
Pcr amplification condition is: 94 DEG C of 5min of denaturation, and 94 DEG C of 35s of sex change, the 50 DEG C of 40s that anneal, extend 72 DEG C of 40s, 30 circulations, 72 DEG C are extended 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 is placed 2 months
Tinned tomatoes B Plum forests board, after opening, normal temperature is placed 2 months
Experimental result
Detected result as shown in Figure 2, as can be seen from Figure 2 the target sequence fragment that present embodiment can amplify Bacillus coagulans accurately for the Auele Specific Primer HFQ1/HFQ2 of the Bacillus coagulans that increases can amplify Bacillus coagulans specific gene fragment from 2 parts of become sour tinned tomatoes sample DNA and 1 part of pedotheque DNA (No. 4 swimming lanes).5,6 and No. 4 amplified fragments corresponding to swimming lane are checked order, through Blast compare of analysis, determine that it is Bacillus coagulans special gene sequence.This is extensively present in soil and the tinned tomatoes that becomes sour consistent with Bacillus coagulans in bibliographical information.
Above the specific embodiment of the present invention is illustrated, but the present invention is as limit, only otherwise depart from aim of the present invention, the present invention can also have various variations.

Claims (5)

1. an Auele Specific Primer of identifying Bacillus coagulans, is characterized in that, described primer is a pair of Auele Specific Primer HFQ1/HFQ2, and wherein, forward primer HFQ1 sequence is identical with SEQ ID NO.1, for: GTCACGGAAGAGCAAGCTTG; Reverse primer HFQ2 sequence is identical with SEQ ID NO.2, for: GTTTCTGAAATGTATGCACG.
2. a method for Auele Specific Primer qualification Bacillus coagulans as claimed in claim 1, is characterized in that, comprises the following steps:
(1) extract Bacillus coagulans (Bacillus coagulans), prepare control strain;
(2), taking the DNA of bacteria in step (1) as template, carry out pcr amplification with HFQ1/HFQ2 primer; With
(3) with 1% agarose gel electrophoresis, above-mentioned pcr amplification result is verified.
3. the method for stating according to claim 2, is characterized in that: in step (2), forward primer HFQ1 sequence is identical with SEQ ID NO.1, for: GTCACGGAAGAGCAAGCTTG; Reverse primer HFQ2 sequence is identical with SEQ ID NO.2, for: GTTTCTGAAATGTATGCACG.
4. the method for stating according to claim 2, is characterized in that:
In step (2), pcr amplification system is: 20 μ l are by 2 μ l10 × Bufferwith MgCl 2, 1 μ ldNTP2.5mmol/L, 1 μ l HFQ110mmol/L, 1 μ l HFQ210mmol/L, 0.1 μ l Taq DNAPolymerase5U/ μ l, 1 μ l DNA profiling, 13.9 μ l ddH 2o;
Pcr amplification condition is: 94 DEG C of 5min of denaturation, and 94 DEG C of 35s of sex change, the 50 DEG C of 40s that anneal, extend 72 DEG C of 40s, 30 circulations, 72 DEG C are extended 10min, 4 DEG C of preservations.
5. 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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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105256054A (en) * 2015-11-13 2016-01-20 通威股份有限公司 Method for rapidly detecting bacillus coagulans and multiplex PCR reagent kit
WO2017058741A1 (en) 2015-10-01 2017-04-06 Muhammed Majeed Novel pcr primers and methods thereof for the identification of 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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Publication number Priority date Publication date Assignee Title
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

Patent Citations (6)

* Cited by examiner, † Cited by third party
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

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017058741A1 (en) 2015-10-01 2017-04-06 Muhammed Majeed Novel pcr primers and methods thereof for the identification of bacillus coagulans
CN105256054A (en) * 2015-11-13 2016-01-20 通威股份有限公司 Method for rapidly detecting bacillus coagulans and multiplex PCR reagent kit
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

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