CN107513569A - The LAMP primer group and detection method of a kind of Listeria Monocytogenes - Google Patents
The LAMP primer group and detection method of a kind of Listeria Monocytogenes Download PDFInfo
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- CN107513569A CN107513569A CN201710845296.0A CN201710845296A CN107513569A CN 107513569 A CN107513569 A CN 107513569A CN 201710845296 A CN201710845296 A CN 201710845296A CN 107513569 A CN107513569 A CN 107513569A
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- listeria monocytogenes
- primer
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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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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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
Abstract
The present invention devises loop-mediated isothermal amplification (LAMP) primer according to the prfA genes of Listeria Monocytogenes, and it includes a pair of outer primers and a pair of inner primers, and establishes a kind of LAMP detection method of Listeria Monocytogenes.The present invention realizes single quick detection for increasing listeria spp from molecular level, has the characteristics of easy, sensitive, quick, high specificity, suitable for the quick detection of test for Listeria Monocytogenes in Foods.
Description
Technical field
The invention belongs to food-borne pathogens detection technique field, and in particular to a kind of Listeria Monocytogenes
Loop-mediated isothermal amplification technique quick detection primer and detection method.
Background technology
Listeria Monocytogenes (Listeria monocytogenes) are a kind of important Amphixenosises
Pathogen, and a kind of most commonly seen foodborne bacterial pathogenses, WHO are classified as four big pathogenic bacteria in food the 1990s
One of.Listeria Monocytogenes are widely present in meat products, dairy products, aquatic products and soil, 4 DEG C of environment
In still can growth and breeding, be one of the main pathogenic fungi that chilled food threatens human health.With the quickening pace of modern life, fast food
Food is gradually welcome by China people, and the harm of Listeria Monocytogenes is also following, therefore to Li Si
The detection work of special Salmonella is particularly important.
Mainly there are traditional isolation and identification method, immunological method and PCR method to the detection method of the bacterium at present.Three kinds of sides
Method respectively has advantage and disadvantage, wherein traditional isolation and identification method accuracy is higher, but step is more, process is complicated and the cycle of detection compared with
It is long, it is unfavorable for the monitoring of production procedure, the quality control of finished product and the management of government department；The immunology detection cycle is short,
But sensitivity is poor；PCR method detection time is short, sensitivity is high, but requires higher to instrument and equipment.Study newly quick, accurate
Really, it is single easily to operate to increase listeria spp detection method, have important practical significance.
Loop-mediated isothermal amplification technique (Loop-mediated isothermal amplification, LAMP) is 2000
A kind of new constant temperature nucleic acid amplification technology that year Notomi etc. grows up on round pcr, its amplification do not need thermal cycle
It can be carried out under a steady temperature, DNA can be detected in low-down test limit with respect to other method.The method is than tradition
PCR amplifications are highly efficient, and gene magnification and detection can be carried out simultaneously, have easy, quick, high sensitivity, high specificity etc.
Advantage, without using large-scale or high price instrument, detected suitable for laboratory rapid screening, and can promote and answer in laboratories
With.
The content of the invention
It is an object of the present invention to provide a kind of Listeria Monocytogenes loop-mediated isothermal amplification technique
Quick detection primer.Loop-mediated isothermal amplification (LAMP) primer, profit are designed according to the prfA genes of Listeria Monocytogenes
With the specific region of LAMP technology amplification gene, single quick detection for increasing listeria spp is realized from molecular level, is increased to be single
Listeria spp detection provides effective and quick screening method.
Another object of the present invention is to provide a kind of Listeria Monocytogenes ring mediated isothermal amplification
(LAMP) quick determination method, detection method of the invention have the characteristics of easy, sensitive, quick, high specificity, can use extensively
In food security field of fast detection.
To achieve these goals, the present invention uses following technical scheme：
A kind of LAMP primer group of Listeria Monocytogenes, including a pair of outer primers and a pair of inner primers, sequence
Row are respectively：
Inner primer 1：(sequence is shown in SEQ ID to TAGCCAACCGATGTTTCTGTATCAAACAATACTACAAAGGTGCTTTCG
No.1)；
Inner primer 2：(sequence is shown in SEQ ID to AGAAGTCATTAGCGAACAGGCTACGCGTAAGATTCTTGCTCAGTAG
No.2)；
Outer primer 1：GTGAGAACGGGACCATCA (sequence is shown in SEQ ID No.3)；
Outer primer 2：CTTGTTTTTGTAGGGTTTGGA (sequence is shown in SEQ ID No.4).
The present invention also provides a kind of Listeria Monocytogenes LAMP detection sides established based on above-mentioned primer sets
Method, comprise the following steps：
(1) processing of measuring samples, increasing bacterium and DNA extractions：According to National Standard of the People's Republic of China GB 4789.30-
2016《National food safety standard food microbiological examination Listeria Monocytogenes are examined》In the method specified
Measuring samples are handled and Zengjing Granule, DNA is extracted from measuring samples using water-boiling method；
(2) LAMP detects reaction system and condition：25 μ L reaction systems contain：Inner primer 1 (10 μm of ol/L) and inner primer 2
(10 μm of ol/L) each 1.25 μ L, outer primer 1 (10 μm of ol/L) and outer primer 2 (10 μm of ol/L) each 0.25 μ L, 10 × Buffer are anti-
Answer the μ of 2.5 μ L, 50mmol/L MgSO4 of buffer solution, 1.5 μ L, 25mmol/L dNTPs, 0.5 μ L, 8U/ μ L Bst archaeal dna polymerases 1
L, the μ L of DNA profiling 2.5, with ultra-pure water polishing to 25 μ L；Add appropriate sealing fluid；Yin and yang attribute is set to compare simultaneously；It will prepare
Reaction solution in 65 DEG C expand 60min；
(3) result judges：1~2 μ L 1000 × SYBR Green I are added in the reaction system after the completion of amplification, are mixed
Even, visual color reaction, under the premise of negative control is greeny in orange, positive control, measuring samples are sentenced in green
For the positive, that is, detect in sample and contain Listeria Monocytogenes；Measuring samples are judged to feminine gender in orange, that is, detect sample
Listeria Monocytogenes are not contained in product.
The Listeria Monocytogenes LAMP primer group and detection method that the present invention establishes, compared with prior art
With high sensitivity, specific good, simple operation and other advantages, suitable for food security quick detection.
Embodiment
The present invention is expanded on further with reference to specific embodiment.It should be understood that these embodiments are merely to illustrate this
Invention, and it is not limited to the scope of the present invention.
Embodiment 1：The design of LAMP primer
Using the prfA genes of Listeria Monocytogenes as target gene, using LAMP primer design software
Primer Explorer 4.0 design specific primer, and sequence is as follows：
Inner primer 1：(sequence is shown in SEQ ID to TAGCCAACCGATGTTTCTGTATCAAACAATACTACAAAGGTGCTTTCG
No.1)；
Inner primer 2：(sequence is shown in SEQ ID to AGAAGTCATTAGCGAACAGGCTACGCGTAAGATTCTTGCTCAGTAG
No.2)；
Outer primer 1：GTGAGAACGGGACCATCA (sequence is shown in SEQ ID No.3)；
Outer primer 2：CTTGTTTTTGTAGGGTTTGGA (sequence is shown in SEQ ID No.4).
Embodiment 2：The foundation of Listeria Monocytogenes LAMP detection method
1st, the processing of measuring samples, increasing bacterium
According to National Standard of the People's Republic of China GB 4789.30-2016《National food safety standard food microorganisms
Learn and examine Listeria Monocytogenes to examine》In the method specified measuring samples are handled and Zengjing Granule.
2nd, the preparation of template DNA
1) preparation of enrichment liquid template DNA：The measuring samples enrichment culture medium 1mL for taking step 1 to prepare is placed in 1.5mL centrifugations
Guan Zhong, 10000r/min centrifuge 2min, abandon supernatant；100 μ L DNA extract solutions are added, 100 DEG C are boiled 10min；10000r/min
2min is centrifuged, supernatant is template DNA.
2) preparation of suspicious bacterium colony template DNA：The suspicious bacterium colony being separated to for step 1, can the direct suspicious bacterium colony of picking,
It is added in 100 μ L DNA extract solutions, vibration is mixed, and 100 DEG C are boiled 10min；10000r/min centrifuges 2min, and supernatant is mould
Plate DNA.
Above-mentioned DNA extract solutions contain：0.5mol/L Tris-HCl, 0.05mol/L disodium ethylene diamine tetraacetates, 0.5%
TritonX-100。
3rd, the foundation of LAMP detection method
Listeria Monocytogenes LAMP reaction systems are shown in Table 1.
The Listeria Monocytogenes LAMP reaction systems of table 1
| Component | Working solution concentration | Sample-adding amount/μ L | Reaction system final concentration |
| Inner primer 1 | 10μmol/L | 1.25 | 0.5μmol/L |
| Inner primer 2 | 10μmol/L | 1.25 | 0.5μmol/L |
| Outer primer 1 | 10μmol/L | 0.25 | 0.1μmol/L |
| Outer primer 2 | 10μmol/L | 0.25 | 0.1μmol/L |
| Buffer reaction buffers | 10× | 2.5 | 1× |
| MgSO4 | 50mmol/L | 1.5 | 3mmol/L |
| dNTPs | 25mmol/L | 0.5 | 0.5mmol/L |
| Bst archaeal dna polymerases | 8U/μL | 1 | 0.32U/μL |
| DNA profiling | — | 2.5 | — |
| Ultra-pure water | — | 14 | — |
Note：Above-mentioned 10 × Buffer reaction buffers contain：200mmol/L Tris-HCl (25 DEG C of pH8.8@), 100mmol/
L KCl, 100mmol/L (NH4)2SO4, 20mmol/L MgSO4, 1%Triton X-100.
Appropriate sealing fluid isolation pollution is covered above the above-mentioned reaction solution prepared, and does yin and yang attribute control, wherein cloudy
Property control be DNA extract solutions, positive control is Listeria Monocytogenes reference culture DNA extracts, and sealing fluid is
Sterilized liquid paraffin.
After 25 μ L LAMP reaction systems are configured into reaction solution, 65 DEG C of amplification 60min.
4th, result judgement
1~2 μ L 1000 × SYBR Green I are added in the reaction system after the completion of step 3 amplification, are mixed, naked eyes
Color reaction is observed, testing result is judged.
Chromogenic reaction judges：Colour developing situation is visually observed under natural light, in negative control in orange, positive control in green
On the premise of color, measuring samples are judged to the positive in green, that is, detect in sample and contain Listeria Monocytogenes；It is to be checked
Sample is judged to feminine gender in orange, that is, detects in sample and do not contain Listeria Monocytogenes.If yin and yang attribute is compareed with before
The situation of stating is not inconsistent, then testing result is invalid, should detect again.
Embodiment 3：Listeria Monocytogenes LAMP detection method specific test
The LAMP detection method established using embodiment 2 is high to common food-borne pathogenic bacteria strain and homology respectively
Bacterial strain is detected, while it is positive control to set Listeria Monocytogenes DNA, and DNA extract solutions are negative control,
Each sample is repeated 2 times, and the results are shown in Table 2.As a result show, containing single display positive findings for increasing listeria spp, remaining is the moon
Property, show that the Listeria Monocytogenes LAMP detection method that the present invention establishes has good specificity.
The specificity experiments result of table 2
Note："+" represents the positive, and "-" represents feminine gender；16-20 is cross pollution sample.
Embodiment 4：Listeria Monocytogenes LAMP detection method sensitivity tests
Take after counting after 10 times of doubling dilutions of bacterium solution progress, the LAMP detection method established using embodiment 2 is respectively to it
Detected.As a result show, be 10 when singly increasing Listeria bacteria concentration2Positive reaction can be presented during CFU/mL, but due to mould
The reasons such as plate extraction, dilution error, the sensitivity order of magnitude may be 103, therefore, this method is to monocyte hyperplasia Listeria
The concentration limit of bacterium bacterium solution detection is 102~103CFU/mL。
Embodiment 5：The accordance of Listeria Monocytogenes LAMP detection method and national standard is tested
It is sample to select pork, duck, steamed buns stuffed with sweetened bean paste, the rice dumpling, uses the Listeria Monocytogenes pair of various concentrations
It is added, using the LAMP detection method that embodiment 2 is established and National Standard of the People's Republic of China GB 4789.30-
2016《National food safety standard food microbiological examination Listeria Monocytogenes are examined》In the method specified
Detected simultaneously, compare the testing result of two methods, each sample is repeated 3 times, while sets up yin and yang attribute control, is as a result seen
Table 3.As a result show, different sample LAMP method testing results are consistent with national standard method, and both coincidence rates are 100%.
The Listeria monocytogenes kit for detecting nucleic acid of table 3 and national standard method testing result
Sequence table
<110>Beijing Kwinbon Biotechnology Co., Ltd.
<120>The LAMP primer group and detection method of a kind of Listeria Monocytogenes
<141> 2017-09-19
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 48
<212> DNA
<213>Artificial sequence inner primer 1 (artificial sequence)
<400> 1
tagccaaccg atgtttctgt atcaaacaat actacaaagg tgctttcg 48
<210> 2
<211> 46
<212> DNA
<213>Artificial sequence inner primer 2 (artificial sequence)
<400> 2
agaagtcatt agcgaacagg ctacgcgtaa gattcttgct cagtag 46
<210> 3
<211> 18
<212> DNA
<213>Artificial sequence outer primer 1 (artificial sequence)
<400> 3
gtgagaacgg gaccatca 18
<210> 4
<211> 21
<212> DNA
<213>Artificial sequence outer primer 2 (artificial sequence)
<400> 4
cttgtttttg tagggtttgg a 21
Claims (2)
- A kind of 1. LAMP primer group of Listeria Monocytogenes, it is characterised in that：Including a pair of outer primers and a pair Inner primer, sequence are respectively：Inner primer 1：TAGCCAACCGATGTTTCTGTATCAAACAATACTACAAAGGTGCTTTCG；Inner primer 2：AGAAGTCATTAGCGAACAGGCTACGCGTAAGATTCTTGCTCAGTAG；Outer primer 1：GTGAGAACGGGACCATCA；Outer primer 2：CTTGTTTTTGTAGGGTTTGGA.
- A kind of 2. LAMP detection method of Listeria Monocytogenes, it is characterised in that：Comprise the following steps：(1) processing of measuring samples, increasing bacterium and DNA extractions；(2) LAMP reactions are carried out with the primer sets described in claim 1；(3) result judges.
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Citations (6)
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|---|---|---|---|---|
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| CN102676506A (en) * | 2012-05-30 | 2012-09-19 | 曹际娟 | Mononuclear cell proliferation listeria monocytogenes nucleic acid standard sample and establishing method and application thereof |
| CN103421904A (en) * | 2013-08-14 | 2013-12-04 | 华中农业大学 | Listeria monocytogenes LAMP (loop-medicated isothermal amplification) visualized detection method |
| CN105177127A (en) * | 2015-08-21 | 2015-12-23 | 上海市计量测试技术研究院 | Polynucleotide, method and kit for detecting listeria monocytogenes |
| CN106480209A (en) * | 2016-11-22 | 2017-03-08 | 无锡艾科瑞思产品设计与研究有限公司 | Listerial detection method in a kind of food |
| CN106520757A (en) * | 2016-11-02 | 2017-03-22 | 福建出入境检验检疫局检验检疫技术中心 | Preparation method of listeria monocytogenes nucleic acid standard substance |
-
2017
- 2017-09-19 CN CN201710845296.0A patent/CN107513569A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102382885A (en) * | 2011-11-04 | 2012-03-21 | 山东出入境检验检疫局检验检疫技术中心 | Rapid detection kit for Listeie monocytogenes (L.M) through strand displacement isothermal amplification (SDA) and detection method thereof |
| CN102676506A (en) * | 2012-05-30 | 2012-09-19 | 曹际娟 | Mononuclear cell proliferation listeria monocytogenes nucleic acid standard sample and establishing method and application thereof |
| CN103421904A (en) * | 2013-08-14 | 2013-12-04 | 华中农业大学 | Listeria monocytogenes LAMP (loop-medicated isothermal amplification) visualized detection method |
| CN105177127A (en) * | 2015-08-21 | 2015-12-23 | 上海市计量测试技术研究院 | Polynucleotide, method and kit for detecting listeria monocytogenes |
| CN106520757A (en) * | 2016-11-02 | 2017-03-22 | 福建出入境检验检疫局检验检疫技术中心 | Preparation method of listeria monocytogenes nucleic acid standard substance |
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