CN110527733A - The primer and kit of one group of Visual retrieval helicobacter pylori - Google Patents
The primer and kit of one group of Visual retrieval helicobacter pylori Download PDFInfo
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- 229960003531 phenolsulfonphthalein Drugs 0.000 claims description 5
- 238000011896 sensitive detection Methods 0.000 abstract description 3
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- 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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Abstract
The present invention relates to the primers and kit of one group of Visual retrieval helicobacter pylori, are related to field of molecular detection.The primer of Visual retrieval helicobacter pylori provided by the invention include outer primer to F3 and B3, inner primer to FIP and BIP and ring primer LB；The sequence of the F3 is as shown in SEQ ID NO.1, and the sequence of the B3 is as shown in SEQ ID NO.2；The sequence of the FIP is as shown in SEQ ID NO.3, and the sequence of the BIP is as shown in SEQ ID NO.4；Shown in the sequence SEQ ID NO.5 of the LB.Primer provided by the invention can be realized helicobacter pylori high specific, highly sensitive detection.
Description
Technical field
The present invention relates to field of molecular detection, and in particular to the primer of one group of Visual retrieval helicobacter pylori and
Kit.
Background technique
Helicobacter pylori (HelicobacterPylori, HP) is a kind of helical form gram-negative in human body gastric mucosa
Property bacterium.This bacterium is to lead to chronic gastritis, gastric ulcer and the duodenal ulcer even arch-criminal of gastric cancer.It is generally acknowledged that stomach
The clinical process of Helicobacter pylori infection is such that settle down infection, warp after stomach Helicobacter pylori orally reaches gastric mucosa
Several weeks or several months cause chronic, superficial gastritis, develop into duodenal ulcer, gastric ulcer, lymph after several years or many decades
Hypertrophic gastric lymphoma, atrophic gastritis etc., and the latter is the factor for causing gastric cancer most dangerous.Brainstrust thinks that stomach is deep and remote
Door helicobacter infection makes the danger to get a cancer of the stomach increase 2.7~12 times, if without Helicobacter pylori infection, at least 35
~89% gastric cancer will not occur.
Chinese patent CN102688505A discloses a kind of composition and reagent for detecting helicobacter pylori gastrin IR cells,
The composition and reagent are observed that distribution situation of the helicobacter pylori in stomach under gastroscope.But the patented method
It carries out in vivo, with certain blindness when requiring height to experimental implementation, and taking biopsy.
Chinese patent CN103293305A discloses a kind of oral cavity helicobacter infection detection method and the saliva for detection
Liquid test plate (panel), takes saliva to be placed in test glass, and buffer is added dropwise and mixes well, and draws the sample-adding window that mixed liquor instills sputum test plate
Mouthful, 5~15 minutes observation results.Sputum test plate used in detection method has sputum test item, and sputum test item has
Glass layer, saliva absorb paper layer, colloidal gold paper layer, glass layer, absorption paper layer and successively join end to end and be pasted together,
It is successively arranged control line, p-wire along absorption paper layer to colloidal gold paper layer direction on glass layer, it is wherein attached on control line
There is sheep polyclonal antibody, has urease antibody or flagellar antibody on p-wire, have urease antibody glue on colloidal gold paper layer
Body gold or flagellar antibody colloidal gold.Urease antibody or flagellar antibody are the ureases discharged respectively by toxic type pylori
Or the antibody that flagellar antigen is prepared.But the patented method raw material is complicated, it is at high cost.
Chinese patent CN105755105A discloses the promotion urea based on urease and decomposes and generate ammonia, and indicator is caused to become
The principle of color determines the method for testing result, is easy to be influenced and lose by external environmental condition using urease in this method
It is living, thus often there is false negative in testing result.
The detection of helicobacter pylori at present needs a kind of highly efficient method.
Summary of the invention
The purpose of the present invention is to provide the primers and kit of one group of Visual retrieval helicobacter pylori.The present invention mentions
The primer of confession can be realized helicobacter pylori high specific, highly sensitive detection.
The present invention provides the primer of one group of Visual retrieval helicobacter pylori, including outer primer to F3 and B3, interior draw
Object is to FIP and BIP and ring primer LB；The sequence of the F3 is as shown in SEQ ID NO.1, the sequence of the B3 such as SEQ ID
Shown in NO.2；The sequence of the FIP is as shown in SEQ ID NO.3, and the sequence of the BIP is as shown in SEQ ID NO.4；The LB
Sequence SEQ ID NO.5 shown in.
The present invention also provides a kind of kits of primer described based on the above-mentioned technical proposal, including above-mentioned technical proposal institute
State the reaction buffer of primer, dNTP, BstDNA polymerase and the indicator containing yin-yang.
Preferably, the yin-yang indicator includes phenolsulfonphthalein.
Preferably, the outer primer is respectively 0.2 μM using final concentration.
Preferably, the inner primer is respectively 1.6 μM using final concentration.
Preferably, the ring primer uses final concentration of 0.8 μM.
Preferably, every 25 μ L detection architecture includes primers F 3 and B3 described in above-mentioned technical proposal each 0.5 μ L, LF and LB each
Each 2.5 μ L of 1.5 μ L, dNTP of 1.5 μ L, FIP and BIP, 2 μ L, BstDNA polymerase of template DNA, 1 μ L and 2 × indicator containing yin-yang
12.5 μ L of reaction buffer.
The present invention provides the primers of one group of Visual retrieval helicobacter pylori.Primer provided by the invention can be realized
Helicobacter pylori high specific, highly sensitive detection.Test result shows detection of the present invention for helicobacter pylori
It is specific high, the helicobacter pylori DNA that sensitivity is reacted up to 1pg/.
Detailed description of the invention
Fig. 1 is the specific detection result figure that the embodiment of the present invention 1 provides；
Fig. 2 is the sensitivity technique result figure that the embodiment of the present invention 1 provides；
Fig. 3 is the helicobacter pylori positive patient that the embodiment of the present invention 2 provides and the signal of Healthy People saliva testing result
Figure.
Fig. 4 is the differential responses temperature detection result figure that the embodiment of the present invention 3 provides；
Fig. 5 is the differential responses time testing result figure that the embodiment of the present invention 4 provides.
Specific embodiment
The present invention provides the primer of one group of Visual retrieval helicobacter pylori, including outer primer to F3 and B3, interior draw
Object is to FIP and BIP and ring primer LB；The sequence of the F3 is as shown in SEQ ID NO.1, the sequence of the B3 such as SEQ ID
Shown in NO.2；The sequence of the FIP is as shown in SEQ ID NO.3, and the sequence of the BIP is as shown in SEQ ID NO.4；The LB
Sequence SEQ ID NO.5 shown in.In the present invention, the Visual retrieval is preferably based on loop-mediated isothermal amplification.This
Invent that the primer specificity is strong, the kit based on the primer detects high specificity, high sensitivity, and easy to operate.This
Invention does not have special restriction to the synthetic method of the primer, is synthesized using custom primer well known to those skilled in the art public
Department is synthesized.
In the present invention, the nucleotide sequence of the primer specifically:
F3:5'-AGAAGAAAGAGCGATTGAAGA-3'(SEQ ID NO.1)；
B3:5'-CTTATAAGCCGCGCCATT-3'(SEQ ID NO.2)；
FIP:
5'-CCTTTTAGCGTTGCCAACGCTTTTCTTTCACAATGAAGAATTGTTGC-3'(SEQ ID NO.3)；
BIP:
5'-GATGTCATAGGGCGCTATATCGCTTTTCTAACACGATCCTTAAACTTTG-3'(SEQ ID NO.4)；
LB:5’-TCT CGC CCACTT TATAGC TAGAA-3’(SEQ ID NO.5)。
The present invention also provides a kind of kits of primer described based on the above-mentioned technical proposal, including above-mentioned technical proposal institute
State the reaction buffer of primer, dNTP, BstDNA polymerase and the indicator containing yin-yang.In the present invention, the reaction buffer
Preferably LAMPbuffer.In the present invention, the yin-yang indicator includes that there is perception isothermal amplification system to change energy
The Phenolsulfonphthalein (PSP) of power.In the present invention, the PSP can be perceived in loop-mediated isothermal amplification
The variation of the quantity of cation, and make reaction system color change, concentration of the yin-yang indicator in reaction buffer is
0.1mM.In the present invention, the final concentration of the outer primer is respectively 0.2 μM.In the present invention, the final concentration of the inner primer is each
It is 1.6 μM.In the present invention, final concentration of 0.8 μM of the ring primer.Specifically, in order to preferably carry out Contrast on effect, this
The kit is invented it is also preferable to include positive control and negative control, the positive control is preferably the mixed of helicobacter pylori
Close liquid；The negative control is preferably non-helicobacter pylori, and the present invention does not have the type of the non-helicobacter pylori
Body limits.Kit of the present invention can be realized the Visual retrieval of helicobacter pylori by the combination of said components, inspection
It is obvious to survey result color difference, without other ancillary equipments, directly can observe amplification, colour-difference by naked eyes
Different high sensitivity.
In the present invention, every 25 μ L detection architecture includes each 0.5 μ L, LF and LB of primers F 3 and B3 described in above-mentioned technical proposal
The reaction buffering of each 1.5 μ L, FIP and BIP each 2.5 μ L, BstDNA polymerase of 1.5 μ L, dNTP, 1 μ L and the 2 × indicator containing yin-yang
12.5 μ L of liquid.In the present invention, the concentration of the dNTP is 10mM.In the present invention, the reaction system is in specific detection,
It is preferred that adding the template DNA of 2 μ L, water is for supplying.The present invention does not have special restriction to the source of the water, using this field
Standard PCR water known to technical staff, such as ddH2O。
Kit of the present invention when in use, carries out isothermal after preferably mixing each component with sample according to said ratio
Amplification.In the present invention, the sample to be tested is preferred from human saliva sample, extracts the DNA of saliva sample as template.Institute
Kit is stated in the detection for helicobacter pylori, the amplification program of the detection is preferably reacted at 58~64 DEG C of constant temperature
40~70min, more preferably 60 DEG C reaction 40min.In the present invention, the reaction carries out preferably in constant temperature water bath apparatus.
In the present invention, the condition for terminating reaction is preferably 80 DEG C of reaction 5min.
After obtaining amplified production, the direct naked-eye observation testing result of the present invention, when the color of testing result is yellow, inspection
Surveying result is the positive；When the color of testing result is red or pink, testing result is feminine gender.The present invention is not necessarily to produce reaction
Object is further processed, and testing result, and detection sensitivity of the present invention directly can be obtained by the color of observing response liquid
Height, for the helicobacter pylori DNA of 1pg/ reaction.Primer and kit provided by the present application in the detection process, are not required to valuableness
Equipment, do not need fluorescent dye and special installation, it is thus only necessary to metal bath or water-bath, naked eyes it may determine that as a result,
It has a clear superiority.
Primer to one group of Visual retrieval helicobacter pylori of the present invention and examination combined with specific embodiments below
Agent box is further described in detail, and technical solution of the present invention includes but is not limited to following embodiment.
Embodiment 1
Helicobacter pylori visible detection method
The kit of Visual retrieval helicobacter pylori provided by the invention includes following component in the detection process:
Final concentration containing primers F 3 and B3 in reaction tube is respectively 0.2 μM, final concentration of 0.8 μM of LF and LB, FIP and
The final concentration of BIP is respectively 1.6 μM, 2.5 μ L, BstDNA polymerase of dNTP, 1 μ L, and template DNA 2 μ L, 2 × 12.5 μ L (contain yin and yang attribute
As a result indicator), use ddH2O is supplemented to 25 μ L of total volume.The condition of reaction is 60 DEG C of reaction 50min.In specific test,
In order to carry out Contrast on effect, kit has positive control and negative control, and positive control is the mixed liquor of helicobacter pylori；Yin
Property control be non-helicobacter pylori.After the reaction was completed, reaction system is then the positive in yellow, and taking on a red color is for feminine gender.
The specific detection result of visible detection method is as shown in Figure 1, wherein Fig. 1 a is helicobacter pylori, Fig. 1 b
For mycobacterium tuberculosis, Fig. 1 c is staphylococcus aureus, and Fig. 1 d is ddH2O.It is found that having helicobacter pylori warp only in Fig. 1
It crosses after detection, reaction system shows yellow, and after other microorganisms are reacted, reaction system is displayed in red or pink, table
Whether the differentiation sample that the bright method for visualizing is capable of specificity is helicobacter pylori, and it is special well to show that this method has
It is anisotropic.
The sensitivity technique result of visible detection method is as shown in Figure 2, wherein H. pylori in Fig. 2-1 to Fig. 2-5
Bacterium genome concentration be followed successively by 100pg DNA/ reaction, 10pg DNA/ reaction, 1pg DNA/ reaction, 100fg DNA/ reaction and
10fg DNA/ response diagram 2 it is found that with helicobacter pylori genome concentration successively reduction, system color after reaction
Red or pink is become by yellow.When color is pink, show this method and cannot detect the pylorus in sample
Pylori.When helicobacter pylori genome concentration, which is more than or equal to 1pg DNA/, to react, system color is yellow after reaction,
Illustrate the sensitivity of the detection method for 1pg DNA/ reaction.
The Visual retrieval kit that this embodiment provides, high specificity, high sensitivity are easy to operate, can pass through meat
Eye observation amplification, it is easy, easily operated.
Embodiment 2
Helicobacter pylori Visual retrieval
For the present embodiment using kit provided by embodiment 1 during detection, specific detection method is as follows:
(1) the saliva 1.5mL that will make a definite diagnosis the patient of the helicobacter pylori positive is added in micro centrifugal pipe, in boiling water bath
5min；Then 3000rpm is centrifuged 5min, draws 1 μ L supernatant as template.
(2) it is added in reaction tube in kit amplification reaction solution and reaction tube is placed in 60 DEG C of constant water bath box (or PCR
Instrument), keep the temperature 40min；
(3) reaction tube of step (2) is placed in 5min in 80 DEG C of constant water bath box, terminates reaction.
After the reaction was completed, reaction system is then positive in yellow, take on a red color be for feminine gender, testing result as shown in figure 3, its
Middle Fig. 3-1 is helicobacter pylori positive patient saliva sample, and Fig. 3-2 is positive control, and Fig. 3-1 to Fig. 3-2 is yellow, is in
It is positive；Fig. 3-3 is helicobacter pylori feminine gender personnel first saliva sample, and Fig. 3-4 is helicobacter pylori feminine gender personnel second saliva
Sample, Fig. 3-5 are ddH2O, Fig. 3-3 are red to Fig. 3-5, are negative.
The detection method for being used to detect helicobacter pylori that this embodiment provides, process is simple, high sensitivity, detection
Process is convenient, and amplified reaction is fast, saves the time, and amplification can be completed in 50min, and amplification is observable, energy by naked eyes
Specific detection helicobacter pylori.
Embodiment 3
During detection, using kit provided by embodiment 1, different reaction temperatures (58 is arranged in the present embodiment
DEG C, 60 DEG C, 62 DEG C, 64 DEG C and 66 DEG C), the reaction time is 50min.Helicobacter pylori DNA concentration is 8pg/ μ L, detection knot
Fruit is as shown in figure 4, the testing result that wherein Fig. 4-1 is 58 DEG C, the testing result that Fig. 4-2 is 60 DEG C, the detection that Fig. 4-3 is 62 DEG C
As a result, the testing result that Fig. 4-4 is 64 DEG C, the testing result that Fig. 4-5 is 66 DEG C.Fig. 4 is it is found that as reaction temperature is risen by 58 DEG C
To 64 DEG C, electrophorogram and reaction system color after reaction show that reaction temperature is 66 DEG C, which stops, without big volume production
Object occurs, and reaction system color is red or pink.Wherein, when reaction temperature is 60 DEG C, electrophorogram shows that product assay is high
And reaction system color shows that optimal reaction temperature is 60 DEG C in yellow.
Embodiment 4
The present embodiment is using kit provided by embodiment 1 during detection, and reaction temperature is set as 60 DEG C, setting
The different reaction time (40min, 50min, 60min and 70min).8pg/ μ L helicobacter pylori DNA is detected, is detected
As a result as shown in figure 5, wherein Fig. 5-1 is the testing result for reacting 40min, Fig. 5-2 is the testing result for reacting 50min, Fig. 5-3
For the testing result for reacting 60min, Fig. 5-4 is the testing result for reacting 70min.The electrophorogram and visualization map table of Fig. 5
Bright, the reaction time does not have significant difference in 40~70min, and wherein the more brightness of the product band of 50min are big, use 50min for
The optimum detection time.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
<110>Shanghai Ao Naite biotechnology Co., Ltd
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Claims (7)
1. the primer of one group of Visual retrieval helicobacter pylori, which is characterized in that including outer primer to F3 and B3, inner primer pair
FIP and BIP and ring primer LB；The sequence of the F3 is as shown in SEQ ID NO.1, the sequence of the B3 such as SEQ ID NO.2 institute
Show；The sequence of the FIP is as shown in SEQ ID NO.3, and the sequence of the BIP is as shown in SEQ ID NO.4；The sequence of the LB
Shown in SEQ ID NO.5.
2. a kind of kit based on primer described in claim 1, which is characterized in that including primer described in claim 1,
The reaction buffer of dNTP, BstDNA polymerase and the indicator containing yin-yang.
3. kit according to claim 2, which is characterized in that the yin-yang indicator includes
phenolsulfonphthalein。
4. kit according to claim 2, which is characterized in that the outer primer is respectively 0.2 μM using final concentration.
5. kit according to claim 2, which is characterized in that the inner primer is respectively 1.6 μM using final concentration.
6. kit according to claim 2, which is characterized in that the ring primer uses final concentration of 0.8 μM.
7. according to kit described in claim 2~6 any one, which is characterized in that every 25 μ L detection architecture includes right
It is required that 1 primers F 3 and each 2.5 μ L of 1.5 μ L, dNTP of B3 each 0.5 μ L, LF and LB each 1.5 μ L, FIP and BIP, template DNA 2
The 12.5 μ L of reaction buffer of 1 μ L of μ L, BstDNA polymerase and the 2 × indicator containing yin-yang.
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|---|---|---|---|---|
| CN103333903A (en) * | 2013-06-19 | 2013-10-02 | 中国疾病预防控制中心传染病预防控制所 | Target sequence, primer and probe for detecting helicobacter pylori and kit thereof |
| CN105755105A (en) * | 2016-04-19 | 2016-07-13 | 夏少时 | Reagent and kit for detecting helicobacter pylori and preparation method of kit |
| CN107099610A (en) * | 2017-06-20 | 2017-08-29 | 嘉兴雅康博贝南生物科技有限公司 | A kind of qualitative detection helicobacter pylori and the kit for carrying out Genotyping |
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| CN103333903A (en) * | 2013-06-19 | 2013-10-02 | 中国疾病预防控制中心传染病预防控制所 | Target sequence, primer and probe for detecting helicobacter pylori and kit thereof |
| CN105755105A (en) * | 2016-04-19 | 2016-07-13 | 夏少时 | Reagent and kit for detecting helicobacter pylori and preparation method of kit |
| CN107099610A (en) * | 2017-06-20 | 2017-08-29 | 嘉兴雅康博贝南生物科技有限公司 | A kind of qualitative detection helicobacter pylori and the kit for carrying out Genotyping |
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