CN107365851A - A kind of helicobacter hepaticus LAMP quick determination methods - Google Patents
A kind of helicobacter hepaticus LAMP quick determination methods Download PDFInfo
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- CN107365851A CN107365851A CN201710691336.0A CN201710691336A CN107365851A CN 107365851 A CN107365851 A CN 107365851A CN 201710691336 A CN201710691336 A CN 201710691336A CN 107365851 A CN107365851 A CN 107365851A
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Abstract
The present invention provides a kind of helicobacter hepaticus LAMP detection primer group, including two outer primers, two inner primers and a ring primer, the primer have SEQ ID NO.1~NO 5 nucleotide sequence.Present invention also offers helicobacter hepaticus LAMP PCR detection architectures, including following components：Primer sets：Two outer primers, two inner primers and a ring primer；2 × reaction buffer, archaeal dna polymerase, fluorescent dye, DNA profiling and sterile purified water.The present invention discloses a kind of LAMP primer and amplification method of specific detection helicobacter hepaticus, is not limited, possessed simple to operate by condition of culture, specific good, high sensitivity, is as a result easy to observe, low advantage, available for the quick detection of helicobacter hepaticus, suitable popularization and application are required to instrument and equipment.
Description
Technical field
The invention belongs to biochemistry detection field, and in particular to a kind of helicobacter hepaticus LAMP quick determination methods.
Background technology
Helicobacter hepaticus (Helicobacterhepaticus) is a kind of important Amphixenosis's cause of disease, can be caused chronic
A variety of diseases such as active hepatitis, liver cancer, breast cancer, typhlitis, colitis；Helicobacter hepaticus is microaerobion, and nutritional requirement is high,
Biochemical identification test operation is cumbersome, time and effort consuming, generally needs could obtain testing result within 2-3 weeks, and sensitivity is very low.Generally
Missing inspection is caused, therefore cultivation is unsuitable for quick detection.Serological method is easy to operate, and a large amount of samples can be detected within the same time
Product, but due to the presence of cross reaction between different helicobacters, cause the possibility of false positive so that helicobacter hepaticus species specificity is reflected
It is not difficult to.PCR method possesses quick, special, sensitive advantage, has been widely used, but detection process needs costliness
The equipment such as amplification instrument are supported and the problems such as pollution causes false positive easily occur, and limiting popularization of this method at line scene should
With.
The content of the invention
In view of this, outside it is an object of the invention to provide a kind of helicobacter hepaticus LAMP detection primer group, including two
Primer, two inner primers and a ring primer, the primer have following nucleotide sequence：F3 forward direction outer primers：5’-
GCAGCTTTAACATTAACTTGTGTA-3’(SEQ ID NO.1)；The reverse outer primers of B3：5’-AATGCGAATGTCGCTCAA-
3’(SEQ ID NO.2)；
FIP forward direction inner primers：
5’-GCCCAGCTTGATAAACTCCGTATAGTTGAAACAAGTTGAATCTGTG-3’(SEQ ID NO.3)；
The reverse inner primers of BIP：
5’-TCTGCCATATCCATAACTACCATTGATCTCCATTTTGATGGTATCG-3’；(SEQ ID NO.4)
The reverse ring primers of LB：5’-CCTTTAAGGCTTGTAACCCCA-3’(SEQ ID NO.5).
Preferably, in helicobacter hepaticus LAMP of the present invention PCR detection architectures, including following components：Primer sets：Two
Bar outer primer, two inner primers and a ring primer；2 × reaction buffer, archaeal dna polymerase, fluorescent dye, DNA profiling are with going out
Bacterium pure water.
Preferably, in helicobacter hepaticus LAMP of the present invention PCR detection architectures, the primer has following nucleotides
Sequence：
F3 forward direction outer primers：5’-GCAGCTTTAACATTAACTTGTGTA-3’(SEQ ID NO.1)；B3 is reversely outer to be drawn
Thing：5’-AATGCGAATGTCGCTCAA-3’(SEQ ID NO.2)；
FIP forward direction inner primers：
5’-GCCCAGCTTGATAAACTCCGTATAGTTGAAACAAGTTGAATCTGTG-3’(SEQ ID NO.3)；
The reverse inner primers of BIP：
5’-TCTGCCATATCCATAACTACCATTGATCTCCATTTTGATGGTATCG-3’；(SEQ ID NO.4).
Preferably, in helicobacter hepaticus LAMP of the present invention PCR detection architectures, the PCR detection architectures include：With
25 μ L LAMP PCR reaction system meters, 5pM each 1 μ L of outer primer F3 and B3；40pM each 1 μ L of inner primer FIP and BIP；
The 20pM reverse μ L of ring primer LB 1；The μ L of 2 × reaction buffer 12.5；The μ L of Bst archaeal dna polymerases 1；The μ L of fluorescent dye 1；DNA moulds
The μ L of plate 2, insufficient section are supplied with sterile purified water.
Another object of the present invention is to provide a kind of helicobacter hepaticus LAMP quick determination method, comprise the following steps：
1) helicobacter DNA is extracted from sample；
2) using helicobacter DNA as template, it is put into primer sets as described above in isothermal duplication instrument, enters performing PCR amplification；
3) amplification is estimated or tested and analyzed through 2.0% agarose gel electrophoresis.
Preferably, in helicobacter hepaticus LAMP of the present invention quick determination method, the PCR system includes primer
Group；And 2 × reaction buffer, archaeal dna polymerase, fluorescent dye, DNA profiling and sterile purified water.
Preferably, in helicobacter hepaticus LAMP of the present invention quick determination method, the PCR detection architectures include：
In terms of 25 μ L LAMP PCR reaction systems, 5pM each 1 μ L of outer primer F3 and B3；40pM each 1 μ L of inner primer FIP and BIP；
The 20pM reverse μ L of ring primer LB 1；The μ L of 2 × reaction buffer 12.5；The μ L of Bst archaeal dna polymerases 1；The μ L of fluorescent dye 1；DNA moulds
The μ L of plate 2, insufficient section are supplied with sterile purified water.
Preferably, in helicobacter hepaticus LAMP of the present invention quick determination method, the visual method is in LAMP
After reaction terminates, colour developing result observes that green fluorescence is then judged as the positive, orange, is judged as feminine gender.
Preferably, in helicobacter hepaticus LAMP of the present invention quick determination method, the agarose gel electrophoresis method：
Take 2 μ L amplified productions to be detected through 2% agarose gel electrophoresis, be judged as the positive if the trapezoid belt for LAMP characteristics occur, not
There is amplified band and be then judged as feminine gender.
It is still another object of the present invention to provide a kind of helicobacter hepaticus LAMP quick detection kit, including it is as described above
Primer sets or detection PCR system.
The present invention discloses a kind of LAMP primer and amplification method of specific detection helicobacter hepaticus, is not limited by condition of culture
System, it is only necessary to be loaded by the system of foundation, you can the detection of helicobacter hepaticus is completed in 60min, possesses simple to operate, specificity
Good, as a result high sensitivity is easy to observe, and low advantage is required to instrument and equipment, available for the quick detection of helicobacter hepaticus, suitably
Popularization and application.
Figure of description
Fig. 1 is specific test LAMP amplification figures in one embodiment of the present of invention；
Fig. 2 is specific test visual test result figure in one embodiment of the present of invention；
Fig. 3 is specific test electrophoresis detection result figure in one embodiment of the present of invention；
Fig. 4 is sensitivity tests visual test result figure in one embodiment of the present of invention；
Fig. 5 is sensitivity tests electrophoresis detection result figure in one embodiment of the present of invention.
Embodiment
The bacterial strain information that the present invention uses is as follows：It is helicobacter hepaticus ATCC 51449, courage type helicobacter ATCC51630, golden yellow
Color staphylococcus A TCC 6538, Pseudomonas aeruginosa ATCC 27853, Pasteurella pneumotropica ATCC35149, grinding tooth citric acid bacillus
ATCC 51116, Corynebacterium bovis ATCC 7715 are purchased from Unite States Standard biology product collecting center.Klebsiella pneumoniae CMCC
46117th, salmonella typhimurium CMCC50115, Shigella flexneri CMCC 51572, Bacillus foecalis alkaligenes CMCC 40001 are purchased from
Chinese medicine Microbiological Culture Collection administrative center.
DNA amplification kit (ring mediated isothermal amplification method) (is purchased from Rong Yan Bioisystech Co., Ltd), carefully
Bacterium genome DNA extracting reagent kit (is purchased from Tiangeng biochemical technology Co., Ltd).
Below in conjunction with the embodiment in the present invention, the technical scheme in the present invention is clearly and completely described.It is aobvious
So, described embodiment is only part of the embodiment of the present invention, rather than whole embodiments.Based on the reality in the present invention
Example is applied, the every other embodiment that those of ordinary skill in the art are obtained under the premise of creative work is not made, is all belonged to
In the scope of protection of the invention.
The helicobacter hepaticus LAMP detection primer of embodiment 1 designs
F3 forward direction outer primers：5’-GCAGCTTTAACATTAACTTGTGTA-3’(SEQ ID NO.1)
The reverse outer primers of B3：5’-AATGCGAATGTCGCTCAA-3’(SEQ ID NO.2)
FIP forward direction inner primers：5’-GCCCAGCTTGATAAACTCCGTATAGTTGAAACAAGTTGAATCTGTG-3’
(SEQ ID NO.3)
The reverse inner primers of BIP：5’-TCTGCCATATCCATAACTACCATTGATCTCCATTTTGATGGTATCG-3’
(SEQ ID NO.4)
The reverse ring primers of LB：5’-CCTTTAAGGCTTGTAACCCCA-3’(SEQ ID NO.5)
The extracting genome DNA of embodiment 2, LAMP amplification systems are established and result
1st, helicobacter hepaticus genomic DNA, ultraviolet spectrometry light are extracted using TIANGEN bacterial genomes DNA extraction agents box
OD260/280 is in the range of 1.6-2.0 for degree meter measure, and -20 DEG C save backup.
LAMP reaction systems are as follows：
The LAMP amplification systems of foundation are put into isothermal duplication instrument, 63 DEG C of reaction 45min.Amplification estimated or
Tested and analyzed through 2.0% agarose gel electrophoresis.
Respectively with helicobacter hepaticus, courage type helicobacter, staphylococcus aureus, Pseudomonas aeruginosa, Pasteurella pneumotropica, grinding tooth
Citric acid bacillus, Corynebacterium bovis, klebsiella pneumoniae, salmonella typhimurium, Shigella flexneri, Bacillus foecalis alkaligenes, water
LAMP amplifications are carried out for template, observe amplification, as a result as shown in Figures 1 to 3, only there is specific amplification in helicobacter hepaticus, its
He does not expand template；Fig. 2 show only helicobacter hepaticus occur green fluorescence (in Fig. 2 labels, test tube 1~12 be respectively 1
Helicobacter hepaticus；2：Courage type helicobacter；3：Staphylococcus aureus；4：Pseudomonas aeruginosa；5：Pasteurella pneumotropica；6：Grinding tooth lemon
Acidfast bacilli；7：Corynebacterium bovis；8：Klebsiella pneumoniae；9：Salmonella typhimurium；10：Shigella flexneri；11：Excrement produces alkali
Bacillus；12：Water)；Fig. 3 shows that only specific band occurs in helicobacter hepaticus, shows that the LAMP detection method that the present invention establishes is special
Property good (swimming lane label is respectively M in Fig. 3：DL 2000DNAMarker；1：Helicobacter hepaticus；2：Courage type helicobacter；3：It is golden yellow
Staphylococcus；4：Pseudomonas aeruginosa；5：Pasteurella pneumotropica；6：Grinding tooth citric acid bacillus；7：Corynebacterium bovis；8：Kerekou pneumonia
Primary bacillus；9：Salmonella typhimurium；10：Shigella flexneri；11：Bacillus foecalis alkaligenes；12：Water).
Embodiment 3：LAMP sensitivity analyses
The helicobacter hepaticus genomic DNA extracted to embodiment 2 carries out 10 multiple proportions gradient dilutions, with by gradient dilution
DNA is template, and the LAMP detection method established using the present invention is expanded, with the quick of the checking LAMP systems of the invention established
Perception.
Visual results are as shown in figure 4,1~test tube of test tube 9 shows green fluorescence, and test tube 10, test tube 11 does not occur green
Color fluorescence, wherein, concentration is respectively 1 in each test tube：4.3×109Copy/uL；2：4.3×108Copy/uL；3：4.3×107
Copy/uL；4：4.3×106Copy/uL；5：4.3×105Copy/uL；6：4.3×104Copy/uL；7：4.3×103Copy/
uL；8：4.3×102Copy/uL；9：4.3×101Copy/uL；10：4.3×100Copy/uL；11：4.3×10-1Copy/uL.
2% agarose electrophoresis is as shown in figure 5, wherein 1~swimming lane of swimming lane 9 shows specific band, swimming lane 10, swimming lane 11
Do not occur specific band, wherein each swimming lane is respectively M：DL 2000DNAMarker；1：4.3×109Copy/uL；2：4.3
×108Copy/uL；3：4.3×107Copy/uL；4：4.3×106Copy/uL；5：4.3×105Copy/uL；6：4.3×104Copy
Shellfish/uL；7：4.3×103Copy/uL；8：4.3×102Copy/uL；9：4.3×101Copy/uL；10：4.3×100Copy/
uL；11：4.3×10-1Copy/uL.
Fig. 4-5 results show that the lowest detection for the LAMP detection method that the present invention is established is limited to 43 copies/uL.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Sequence table
<110>Shanghai Laboratory Animal Research Institute
<120>A kind of helicobacter hepaticus LAMP quick determination methods
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gcagctttaa cattaacttg tgta 24
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aatgcgaatg tcgctcaa 18
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gcccagcttg ataaactccg tatagttgaa acaagttgaa tctgtg 46
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tctgccatat ccataactac cattgatctc cattttgatg gtatcg 46
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Claims (10)
1. a kind of helicobacter hepaticus LAMP detection primer group, including two outer primers, two inner primers and a ring primer, described
Primer has following nucleotide sequence：
F3 forward direction outer primers：5’-GCAGCTTTAACATTAACTTGTGTA-3’(SEQ ID NO.1)；
The reverse outer primers of B3：5’-AATGCGAATGTCGCTCAA-3’(SEQ ID NO.2)；
FIP forward direction inner primers：
5’-GCCCAGCTTGATAAACTCCGTATAGTTGAAACAAGTTGAATCTGTG-3’(SEQ ID NO.3)；
The reverse inner primers of BIP：
5’-TCTGCCATATCCATAACTACCATTGATCTCCATTTTGATGGTATCG-3’；(SEQ ID NO.4)
The reverse ring primers of LB：5’-CCTTTAAGGCTTGTAACCCCA-3’(SEQ ID NO.5).
2. a kind of helicobacter hepaticus LAMP PCR detection architectures, including following components：Primer sets：Two outer primers, two inner primers
With a ring primer；2 × reaction buffer, archaeal dna polymerase, fluorescent dye, DNA profiling and sterile purified water.
3. helicobacter hepaticus LAMP according to claim 2 PCR detection architectures, it is characterised in that the primer has such as
Lower nucleotide sequence：
F3 forward direction outer primers：5’-GCAGCTTTAACATTAACTTGTGTA-3’(SEQ ID NO.1)；
The reverse outer primers of B3：5’-AATGCGAATGTCGCTCAA-3’(SEQ ID NO.2)；
FIP forward direction inner primers：
5’-GCCCAGCTTGATAAACTCCGTATAGTTGAAACAAGTTGAATCTGTG-3’(SEQ ID NO.3)；
The reverse inner primers of BIP：
5’-TCTGCCATATCCATAACTACCATTGATCTCCATTTTGATGGTATCG-3’；(SEQ ID NO.4).
4. helicobacter hepaticus LAMP according to claim 2 PCR detection architectures, it is characterised in that the PCR detection architectures
Including：In terms of 25 μ LLAMP PCR reaction systems, 5pM each 1 μ L of outer primer F3 and B3；40pM inner primer FIP and BIP each 1
μL；The 20pM reverse μ L of ring primer LB 1；The μ L of 2 × reaction buffer 12.5；The μ L of Bst archaeal dna polymerases 1；The μ L of fluorescent dye 1；
The μ L of DNA profiling 2, insufficient section are supplied with sterile purified water.
5. a kind of helicobacter hepaticus LAMP quick determination method, it is characterised in that comprise the following steps：
1) helicobacter DNA is extracted from sample；
2) using helicobacter DNA as template, it is put into the primer sets described in claim 1 in isothermal duplication instrument, enters performing PCR amplification；
3) amplification is estimated or tested and analyzed through 2.0% agarose gel electrophoresis.
6. helicobacter hepaticus LAMP according to claim 5 quick determination method, it is characterised in that the PCR system bag
Include primer sets；
And 2 × reaction buffer, archaeal dna polymerase, fluorescent dye, DNA profiling and sterile purified water.
7. helicobacter hepaticus LAMP according to claim 5 quick determination method, it is characterised in that the PCR detects body
System includes：In terms of 25 μ LLAMP PCR reaction systems, 5pM each 1 μ L of outer primer F3 and B3；40pM inner primer FIP and BIP
Each 1 μ L；The 20pM reverse μ L of ring primer LB 1；The μ L of 2 × reaction buffer 12.5；The μ L of Bst archaeal dna polymerases 1；The μ of fluorescent dye 1
L；The μ L of DNA profiling 2, insufficient section are supplied with sterile purified water.
8. helicobacter hepaticus LAMP according to claim 5 quick determination method, it is characterised in that the visual method is
After LAMP reactions terminate, colour developing result observes that green fluorescence is then judged as the positive, orange, is judged as feminine gender.
9. helicobacter hepaticus LAMP according to claim 5 quick determination method, it is characterised in that the Ago-Gel
Electrophoresis：Take 2 μ L amplified productions to be detected through 2% agarose gel electrophoresis, be judged as if the trapezoid belt for LAMP characteristics occur
The positive, do not occur amplified band and be then judged as feminine gender.
10. a kind of helicobacter hepaticus LAMP quick detection kit, it is characterised in that including primer as claimed in claim 1
The detection PCR system of group or claim 2~4.
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| CN201710691336.0A CN107365851B (en) | 2017-08-14 | 2017-08-14 | LAMP (loop-mediated isothermal amplification) rapid detection method for helicobacter pylori |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108085402A (en) * | 2018-02-10 | 2018-05-29 | 上海实验动物研究中心 | The primer and its kit of one group of detection grinding tooth citric acid bacillus |
| CN108531557A (en) * | 2018-06-01 | 2018-09-14 | 厦门蓝特生物科技有限公司 | The LAMP primer group of detection helicobacter pylori cytotoxin GAP-associated protein GAP cagA a kind of and its application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2010130931A (en) * | 2008-12-03 | 2010-06-17 | Toshiba Corp | Primer set for specifically amplifying nucleic acid derived from bacterium belonging to genus helicobacter, and method for sensing and/or classifying the bacterium |
-
2017
- 2017-08-14 CN CN201710691336.0A patent/CN107365851B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2010130931A (en) * | 2008-12-03 | 2010-06-17 | Toshiba Corp | Primer set for specifically amplifying nucleic acid derived from bacterium belonging to genus helicobacter, and method for sensing and/or classifying the bacterium |
Non-Patent Citations (2)
| Title |
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| MASAAKI MINAMI ET AL.: "Use of a Combination of Brushing Technique and the Loop-Mediated", 《JOURNAL OF CLINICAL MICROBIOLOGY》 * |
| 孙颖杰: "《出入境动物检验检疫技术研究》", 31 July 2009 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108085402A (en) * | 2018-02-10 | 2018-05-29 | 上海实验动物研究中心 | The primer and its kit of one group of detection grinding tooth citric acid bacillus |
| CN108531557A (en) * | 2018-06-01 | 2018-09-14 | 厦门蓝特生物科技有限公司 | The LAMP primer group of detection helicobacter pylori cytotoxin GAP-associated protein GAP cagA a kind of and its application |
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| CN107365851B (en) | 2020-05-15 |
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