CN106119372A - A kind of method detecting meriones unguiculatus infection helicobacter pylori - Google Patents
A kind of method detecting meriones unguiculatus infection helicobacter pylori Download PDFInfo
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- CN106119372A CN106119372A CN201610522202.1A CN201610522202A CN106119372A CN 106119372 A CN106119372 A CN 106119372A CN 201610522202 A CN201610522202 A CN 201610522202A CN 106119372 A CN106119372 A CN 106119372A
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Abstract
The present invention relates to detect the method that meriones unguiculatus infects helicobacter pylori.It comprises the following steps: step one: meriones unguiculatus fasting be can't help water to empty the digest in its stomach；Step 2: fill normal saline or water in the stomach of described meriones unguiculatus；Step 3: extract liquid from the stomach of described meriones unguiculatus；Step 4: with described liquid for sample extraction genome；Step 5: carry out nest-type PRC: use pair of primers to carry out first round PCR with described genome for template and obtain the first PCR primer；Use second primer carries out second with described first PCR primer for template to take turns PCR and obtain the second PCR primer；Step 6: detect described second PCR primer, when detect the size of described second PCR primer with its expected from size identical, or the sequence of described second PCR primer with its expected from sequence identical time, then confirm that described meriones unguiculatus infects described helicobacter pylori.
Description
Technical field
The present invention relates to detect the method that meriones unguiculatus infects helicobacter pylori.
Background technology
Helicobacter pylori (Helicobacter pylori) morphological characteristic is similar with Campylobacter spp, and Grain-negative, in spiral shell
Shape, S type or sea-gull spread the wings shape, often arrange in shoal of fish sample in gastric mucus layer, and living after Secondary Culture can under the conditions of adverse environment
Become shaft-like or spherical.Can there be many flagellums thalline one or both ends.Helicobacter pylori is microaerophilic bacteria, the most suitable growth temperature
Degree is for 35-37 DEG C.Nutritional requirement is high, needs blood or serum, also needs to certain humidity during growth.The antibacterial of spherical change, generally locates
In resting state, it is difficult to Secondary Culture in vitro, but Spiral shaped bacteria when environmental condition is suitable in vivo, can be changed into.
This type of bacterium is mainly present in meriones unguiculatus (Latin name: Meriones with inapparent infection form
Unguiculatus；English name: Mongolian gerbil) gastric antrum portion, can cause host's pathological reaction in various degree from
And cause chronic gastritis, gastric ulcer, the intestinal metaplasia even generation of gastric cancer.When animal pattern infects Helicobacter pylori, induction one can be caused
A little complications, have a strong impact on Quality of Experimental Animals and the reliability of experimental data are produced potential interference.In addition pylorus spiral shell
Swing arm bacterium can mutually be propagated between people and meriones unguiculatus, and the meriones unguiculatus carrying helicobacter pylori can pass through contact transmission
To keeper and experimenter, personnel are caused to infect.
At present, the detection method of meriones unguiculatus Helicobacter pylori infection mainly has nosetiology, serology, urease test
And molecular biology method:
(1) bacteria distribution is cultivated.Taking gastric antrum stomach function regulating soma, under special culture medium and micro-oxygen environment, culture of isolated should
Bacterium.This method detection needs that animal is carried out execution and draws materials, and relatively costly experimental period is long, needs to cultivate 3-6d, biochemical identification
Test operation is loaded down with trivial details, time and effort consuming.Although can make a definite diagnosis after turning out antibacterial, but its false negative rate is higher.
(2) Serologic detection.Use ELISA method to detect helicobacter pylori resistant specificity in serum or feces to resist
Body.Immunological method is easy and simple to handle, can detect a large amount of sample, but meriones unguiculatus is being tested as one dynamic within the same time
The animal of materialization, does not has good antibody to be applicable to ELISA detection.
(3) PCR detection.Directly detect the DNA of this thalline in feces, intestinal contents and gastric tissue.But the method needs mostly
Animal tissue, intestinal contents and In vivo detection feces will be after death taken at animal.Desert is mainly lived in due to meriones unguiculatus
Area, so its feces water content is relatively low, is unfavorable for bacteria live, thus the false negative rate of feces detection helicobacter pylori also than
Higher.
Meriones unguiculatus is to use in laboratory animal widely and animal that quantity is a lot of, according to the literature, and meriones unguiculatus
Being the important laboratory animal as helicobacter pylori Study on Pathogenicity, under conditions of Helicobacter pylori infection, long pawl is husky
Mus there will be gastritis, gastric ulcer, intestinal metaplasia and gastric cancer even occurs.The infection of helicobacter pylori can be to keeper and experiment people
Member damages and the reliability of experimental data can be produced serious interference.
Based on current various detection methods, in addition it is also necessary to a kind of operation is relatively simple, reliable results and inspection with low cost
The method surveying experiment meriones unguiculatus live infection helicobacter pylori.
Summary of the invention
It is an object of the invention to provide and a kind of detect experiment meriones unguiculatus live body easier, more reliable and with low cost
The method infecting helicobacter pylori.
Therefore, the invention provides a kind of detection meriones unguiculatus (Latin name: Meriones Unguiculatus；English
Literary fame: Mongolian gerbil) infect helicobacter pylori (Helicobacter pylori) method, it includes following
Step: step one: described meriones unguiculatus fasting be can't help water to empty the digest in its stomach；Step 2: husky to described long pawl
The stomach of Mus fills normal saline or water；Step 3: extract liquid from the stomach of described meriones unguiculatus；Step 4: with described liquid
For sample extraction genome；Step 5: carry out nest-type PRC: use pair of primers to carry out first with described genome for template
Wheel PCR obtains the first PCR primer；Use second primer carries out second with described first PCR primer for template to take turns PCR and obtain the
Two PCR primer；Step 6: detect described second PCR primer, when detect the size of described second PCR primer with its expected from
Size is identical, or the sequence of described second PCR primer with its expected from sequence identical time, then confirm that described meriones unguiculatus infects
Described helicobacter pylori.When designing the fragment of the amplification of nest-type PRC of the present invention, in general, the first PCR primer is big
Little for 300-800bp, preferably 400-600bp；The size of the second PCR primer is 80-150bp, preferably 80-120bp.
In the present invention, described pair of primers is SEQ ID No.1 and SEQ ID No.2；Described second pair of primer is
SEQ ID No.3 and SEQ ID No.4.
In the present invention, it is 18-36 hour that the time of water is can't help in described meriones unguiculatus fasting, preferably 20-28 hour.
In the present invention, in described step 2, gastric perfusion needle is used to fill described physiology salt in the stomach of described meriones unguiculatus
Water or water 0.3-0.6ml.
In the present invention, in described step 3, the volume 70-120 μ L of extraction liquid.
In the present invention, the response procedures at first round PCR is as follows: 92-98 DEG C of denaturation 0-10min；92-98 DEG C of degeneration
0.5-2min, 65-78 DEG C of extension 0.5-2min of 0.5-2min, 40-55 DEG C of annealing, circulates 20-45 time；65-78 DEG C extends 0-eventually
15min；The preferably response procedures of first round PCR is as follows: 94-96 DEG C of denaturation 2-5min；94-96 DEG C of degeneration 0.5-1min, 42-
0.5-1min, 70-73 DEG C of extension 0.5-1min of 47 DEG C of annealing, circulates 25-35 time；70-73 DEG C extends 2-10min eventually.
In the present invention, the response procedures of PCR is taken turns second as follows: 92-98 DEG C of denaturation 0-7min；92-98 DEG C of degeneration
15-60sec, 65-78 DEG C of extension 15-60sec of 15-60sec, 43-58 DEG C of annealing, circulates 20-45 time；65-78 DEG C extends 0-eventually
15min；Preferably second to take turns the response procedures of PCR as follows: 94-96 DEG C of denaturation 30-180sec；94-96 DEG C of degeneration 20-40sec,
46-51 DEG C of annealing 20-40sec, 72 DEG C of extension 20-40sec, circulate 25-35 time；70-73 DEG C extends 1-5min eventually.
In the present invention, in the system of first round PCR, the proportionate relationship of each component is as follows:
Template: SEQ ID No.1:SEQ ID No.2:10 times PCR buffer: archaeal dna polymerase: water=10-
100ng:0.5-1.5 μm ol:0.5-1.5 μm ol:4-6 μ L:0.5-3U:32-48 μ L；Preferred template: SEQ ID No.1:SEQ
The PCR buffer of ID No.2:10 times: archaeal dna polymerase: water=20-40ng:0.5-1 μm ol:0.5-1 μm ol:4-6 μ L:0.5-
1U:38-42 μ L；More preferably template: SEQ ID No.1:SEQ ID No.2:2 × PCR Master Mix or 2 × PreMix
RTaq: water=20-40ng:0.5-1 μm ol:0.5-1 μm ol:23-28 μ L:17-25 μ L.Wherein 2 × PCR Master Mix purchases
From Thermo company；2 × PreMix rTaq is purchased from Thermo company.And 2 × PCR Master Mix or 2 × PreMix
Component in rTaq is to be as the criterion in description.
In the present invention, in the second system taking turns PCR, the proportionate relationship of each component is as follows:
Template: SEQ ID No.3:SEQ ID No.4:10 times PCR buffer: archaeal dna polymerase: water=10-
100ng:0.5-1.5 μm ol:0.5-1.5 μm ol:4-6 μ L:0.5-3U:32-48 μ L；Preferred template: SEQ ID No.3:SEQ
The PCR buffer of ID No.4:10 times: archaeal dna polymerase: water=20-40ng:0.5-1 μm ol:0.5-1 μm ol:4-6 μ L:0.5-
1U:38-42 μ L；More preferably template: SEQ ID No.3:SEQ ID No.4:2 × PCR Master Mix or 2 × PreMix
RTaq: water=20-40ng:0.5-1 μm ol:0.5-1 μm ol:23-28 μ L:17-25 μ L.Wherein 2 × PCR Master Mix purchases
From Thermo company；2 × PreMix rTaq is purchased from Thermo company.When carrying out second and taking turns PCR, for convenience's sake, it is possible to
In the system of first round PCR, appropriate archaeal dna polymerase (archaeal dna polymerase of such as 0.5-3U or 0.5-1U) is added on directly
With the above-mentioned appropriate SEQ ID No.3 and SEQ ID No.4 taking turns PCR for second.
Present invention also offers the primer of a kind of nested PCR amplification helicobacter pylori, wherein pair of primers is SEQ
ID No.1 and SEQ ID No.2, second pair of primer is SEQ ID No.3 and SEQ ID No.4.
Advantages of the present invention:
(1) animal survival: relative to traditional helicobacter pylori detection method, it is examined by the method by gastric juice
Surveying, less to animal injury, it is possible to ensure the survival of animal, animal can continue to be used as other experiments.This is for zoopery
Particularly relevant to helicobacter pylori experiment is most important.
(2) simple to operate, premix enzyme for example with 2 × PCR Master Mix, need the most available pylorus spiral of 2 steps PCR
Bacillus infection situation.
(3) detection speed is fast: relative to traditional approach, the method can reflect helicobacter pylori the most intuitively
Infection conditions.
(4) experimental result identification is relatively simple, objective: the interpretation of the method result can be according to agarose gel electrophoresis figure
Presence or absence or the direct Sequencing of middle band purpose band complete, and the interpretation making testing result is simpler, objective.
(5) with low cost, this experiment has only to micro-example DNA extraction kit and common PCR primers and DNA polymerization
Enzyme.Relative to the modes such as antibacterial In vitro culture, advantage of lower cost.
Accompanying drawing explanation
Fig. 1 is that the DNA that the meriones unguiculatus gastric juice to ten artificial infection helicobacter pylori extracts only carries out first round PCR
After PCR primer electrophoretogram.Wherein, swimming lane 1-10 is followed successively by the PCR primer of ten meriones unguiculatus Gastric juice sample, each swimming lane
Loading volume is identical.
Fig. 2 be use second primer to the 5th pair primer carried out second take turns PCR after the electrophoretogram that carries out.Wherein, each
The loading volume of swimming lane is identical.Swimming lane 1-5 is to use the second PCR primer to primer amplification；Swimming lane 6-10 is right for use the 3rd
The PCR primer of primer amplification；Swimming lane 11-15 is the PCR primer using the 4th pair of primer amplification；Swimming lane 16-20 is for using the 5th
PCR primer to primer amplification；The PCR annealing temperature of swimming lane 1, swimming lane 6, swimming lane 11 and swimming lane 16 is 44.8 DEG C；Swimming lane 2, swimming
The PCR annealing temperature of road 7, swimming lane 12 and swimming lane 17 is 46.1 DEG C；The PCR annealing temperature of swimming lane 3, swimming lane 8, swimming lane 13 and swimming lane 18
Degree be the PCR annealing temperature of 48.3 DEG C, swimming lane 4, swimming lane 9, swimming lane 14 and swimming lane 19 be 51.3 DEG C, swimming lane 5, swimming lane 10, swimming lane
15 and the PCR annealing temperature of swimming lane 20 be 55.3 DEG C.
Fig. 3 is the electrophoretogram detecting helicobacter pylori detection threshold value in gastric juice.Wherein swimming lane 1-8 be respectively 2 ×
108CFU/ml、2×107CFU/ml、2×106CFU/ml、2×105CFU/ml、2×104CFU/ml、 2×103CFU/ml、2×
102The helicobacter pylori bacterium solution of CFU/ml and 2 × 10CFU/ml is extracted postgenome and is carried out the PCR of nested amplification as template
Product, the loading volume of each swimming lane is identical.
Fig. 4 is the electrophoretogram that helicobacter pylori meriones unguiculatus gastric juice detection helicobacter pylori is infected in detection ten.Its
In, swimming lane 1-10 is followed successively by the PCR primer of ten meriones unguiculatus Gastric juice sample, and the loading volume of each swimming lane is identical.
Detailed description of the invention
Below in conjunction with embodiment, further illustrate the present invention.
Laboratory animal: male regular grade meriones unguiculatus 10,8 week old, body weight 50-60g.All meriones unguiculatuss are all from head
All medical university's laboratory animal portions, raise in the conventional environment controlling humiture [SYXK (capital) 2013-0005].
Helicobacter pylori: reference culture ATCC43504 is ground by National Institute for Food and Drugs Control's Laboratory Animal Resource
Study carefully and provided.
Reagent and instrument: columbia blood agar base culture medium (OXOID company, lot number CM0331)；Aseptic de-fiber
Sanguis caprae seu ovis (Beijing road and bridge Technology Co., Ltd.)；The micro-aerobic aerogenesis bag of MGC cultivates box (Mitsubishi Gas Chemical Co., Ltd) with sealing,
Helicobacter pylori diagnostic kit (YZB/ Fujian 0455-2009, Fujian Sanqiang Biochemical Co., Ltd.), micro-example gene
Group DNA extraction kit (TIANGEN Biotech (Beijing) Co., Ltd., lot number: DP316), PCR Master Mix (2 ×) is pre-
Mixed enzyme (K0171, Thermo Scientific), low melting-point agarose (A8350, Amresco), PCR instrument (Applied
Biosystems company), electrophresis apparatus (BIO-RAD company), gel imaging system (BIO-RAD company), pressure steam sterilizer
(Triumph Products), superclean bench, sterilizing gastric perfusion needle, sterilizing operating theater instruments (eye scissors, ophthalmic tweezers etc.).
The genome extracted in the following embodiments can be carried out quantitatively with ultraviolet spectrophotometer.
Embodiment 1
Use the method In vivo detection helicobacter pylori only carrying out a PCR
Aseptic de-fiber Sanguis caprae seu ovis is added in the Colombia's agar based media after autoclaving, be poured into culture dish
In prepare flat board, make containing the 5% Colombia's agar plate taking off fiber Sanguis caprae seu ovis.Helicobacter pylori is inoculated in Colombia
On blood agar plate, cultivate in box as sealing, after carrying out 36 DEG C of micro-aerobic cultivation 48h of routine, by lawn sterile physiological
Bacteria containing amount made by saline is 109The bacterium solution of CFU/mL concentration, is distributed into aliquot, standby to-80 DEG C of refrigerators, is finished in 2 weeks.
Take 10 healthy meriones unguiculatuss respectively with 0.5mL bacterium solution gavage, continuous gavage 3 times, every minor tick 24h.All
Before meriones unguiculatus gavage, water 24h is can't help in fasting.Gavage terminates rear conventional environment and raises 10 weeks.
The method extracting the meriones unguiculatus gastric juice of above-mentioned artificial infection helicobacter pylori: first infect pylorus spiral shell to artificial
Water 24h is can't help in the meriones unguiculatus fasting of swing arm bacterium, in order to empty digest in its stomach.Use gastric perfusion needle to meriones unguiculatus gavage
0.5ml normal saline.Wait after about 1min, then stretch into by gastric perfusion needle and meriones unguiculatus stomach extracts liquid in its stomach put into centrifugal
In pipe (about 100 μ L).
The extraction of helicobacter pylori DNA: use the micro-example base of TIANGEN Biotech's sky root
Because group DNA extraction kit (DP316) extracts meriones unguiculatus gastric juice helicobacter pylori genome.
Devise pair of primers for first round PCR, forward primer F1 as shown in SEQ ID No.1；Downstream primer R1
As shown in SEQ ID No.2.
First round PCR reaction system sample-adding is as follows:
The helicobacter pylori genome 20ng extracted
Forward primer F1 (SEQ ID No.1) the 1 μ L of 10 μm ol；
Downstream primer R1 (SEQ ID No.2) the 1 μ L of 10 μm ol；
2×PCR Master Mix 10μL；
ddH2O to 20 μ L.
First round PCR response procedures: 95 DEG C of denaturations 5min；95 DEG C of degeneration 30sec, 45 DEG C of annealing 30sec, 72 DEG C of extensions
30sec, circulates 35 times；72 DEG C extend 5min eventually.
After completing first round PCR, taking PCR primer and carry out the agarose gel electrophoresis of 2%, amplification purpose band is 499bp
(see Fig. 1).Finding that only carrying out taking turns PCR can not amplify purpose band well according to electrophoretogram, testing result occurs false cloudy
Property rate is higher.
Embodiment 2
In nest-type PRC second takes turns the screening of the primer pair of PCR
Devise 4 to taking turns the primer of PCR for second and detecting its accuracy and specificity by agarose gel electrophoresis.
Wherein, the forward primer F2 in second pair of primer as shown in SEQ ID No.3, downstream primer R2 such as SEQ ID
Shown in No.4；Forward primer F3 in 3rd pair of primer as shown in SEQ ID No.5, downstream primer R3 such as SEQ ID No.6 institute
Show；Forward primer F4 in 4th pair of primer is as shown in SEQ ID No.7, and downstream primer R4 is as shown in SEQ ID No.8；The
Forward primer F5 in five pairs of primers is as shown in SEQ ID No.9, and downstream primer R5 is as shown in SEQ ID No.10.
Experimental technique is the micro-example extracting genome DNA examination using TIANGEN Biotech's sky root
Agent box (DP316) extracting directly helicobacter pylori genome, H. pylori bacteria concentration is 2 × 109CFU/ml.Use pylorus
Pylori genome is masterplate, and the primer sequence of embodiment 1 carries out first round PCR；Then with the product of first round PCR as mould
Plate, carries out second take turns PCR with second pair of primer to the 5th pair primer, different annealing temperature, to filter out suitable primer and to move back
Fire temperature.
First round PCR reaction system sample-adding and reaction condition such as embodiment 1.
Second takes turns PCR reaction system is loaded as follows:
First round PCR primer 20ng；
The forward primer 1 μ L of 10 μm ol；
The downstream primer 1 μ L of 10 μm ol；
2×PCR Master Mix 10μL；
ddH2O to 20 μ L.
Second takes turns PCR response procedures: 95 DEG C of denaturations 5min；95 DEG C of degeneration 30sec, respectively 44.8 DEG C, 46.1 DEG C,
48.3 DEG C, 51.3 DEG C, 55.3 DEG C of annealing 30sec, 72 DEG C of extension 30sec, circulate 35 times；72 DEG C extend 5min eventually.
Taking the second of 4 pairs of primers respectively to take turns PCR primer and carry out the agarose gel electrophoresis of 2%, purpose band is 100bp
(such as Fig. 2), and described PCR primer is checked order, after sequencing result and Pubmed data are compared, 100% is correct.
Can obtain according to electrophoretogram, to primer, (i.e. forward primer is SEQ ID No.3, and downstream primer is SEQ in use second
The primer of ID No.4 to) the purpose band that amplifies is single, has higher accuracy and specificity.Therefore use second to drawing
Thing is as the second primer taking turns PCR.
Embodiment 3
In vivo detection helicobacter pylori detection threshold value
Before detection, water 24h is can't help in healthy meriones unguiculatus (the most not infecting the meriones unguiculatus of helicobacter pylori) fasting,
To empty digest in its stomach.Use gastric perfusion needle to meriones unguiculatus gavage 0.5ml normal saline.After about 1min, then use
Gastric perfusion needle stretches into and extracts liquid in its stomach in meriones unguiculatus stomach and put in centrifuge tube.
The gastric juice of 10 meriones unguiculatuss is mixed, then take out from the gastric juice of mixing 180 μ L add concentration be 2 ×
109The helicobacter pylori 20 μ L of CFU/ml concentration, after mixing, doubling dilution is concentration 2 × 108CFU/ml、2×107CFU/
ml、2×106CFU/ml、2×105CFU/ml、2×104CFU/ml、2×103CFU/ml、2×102CFU/ml and 2 × 10CFU/
The mixed liquor of ml.
It is that day root micro-example genome DNA extracting reagent kit (DP316) extracts in variable concentrations mixed liquor with test kit
Helicobacter pylori genome.
First round PCR reaction system sample-adding is as follows:
The helicobacter pylori genome 40ng extracted；
Forward primer F1 (SEQ ID No.1) the 1 μ L of 10 μm ol；
Downstream primer R1 (SEQ ID No.2) the 1 μ L of 10 μm ol；
2×PCR Master Mix 10μL；
ddH2O to 20 μ L.
First round PCR response procedures: 95 DEG C of denaturations 5min；95 DEG C of degeneration 30sec, 45 DEG C of annealing 30sec, 72 DEG C of extensions
30sec, circulates 35 times；72 DEG C extend 5min eventually.
Second takes turns PCR reaction system is loaded as follows:
First round PCR primer 40ng；
Forward primer F2 (SEQ ID No.3) the 1 μ L of 10 μm ol；
Downstream primer R2 (SEQ ID No.4) the 1 μ L of 10 μm ol；
2×PCR Master Mix 10μL；
ddH2O to 20 μ L.
Second takes turns PCR response procedures: 95 DEG C of denaturations 5min；95 DEG C of degeneration 30sec, 51.3 DEG C of annealing 30sec, 72 DEG C prolong
Stretch 30sec, circulate 35 times；72 DEG C extend 5min eventually.
The genome of the helicobacter pylori mixed liquor extraction taking variable concentrations carries out the product after nested PCR amplification (i.e.
Second PCR primer taken turns) carry out the agarose gel electrophoresis (see Fig. 3) of 2% and check order and Pubmed data base BLAST knot
Really.
From analysis result it can be seen that the detection of In vivo detection helicobacter pylori is limited to 2 × 102CFU/ml, this detection
The method detection of the limit explanation present invention has the feature more highly sensitive than prior art, is fully able to meet follow-up experiment need
Want.
Embodiment 4
In vivo detection artificially infects helicobacter pylori meriones unguiculatus
Aseptic de-fiber Sanguis caprae seu ovis is added in the Colombia's agar based media after autoclaving, be poured into culture dish
In prepare flat board, make containing the 5% Colombia's agar plate taking off fiber Sanguis caprae seu ovis.Helicobacter pylori is inoculated in Colombia
On blood agar plate, cultivate in box as sealing, after carrying out 36 DEG C of micro-aerobic cultivation 48h of routine, by lawn sterile physiological
Bacteria containing amount made by saline is 109The bacterium solution of CFU/mL concentration, is distributed into aliquot, standby to-80 DEG C of refrigerators, is finished in 2 weeks.Take 10
The most healthy meriones unguiculatus is respectively with 0.5mL bacterium solution gavage, continuous gavage 3 times, every minor tick 24h.All meriones unguiculatus gavages
Water 24h is can't help in front fasting.Gavage terminates rear conventional environment and raises 10 weeks.
The method extracting the meriones unguiculatus gastric juice of above-mentioned artificial infection helicobacter pylori: first infect pylorus spiral shell to artificial
Water 24h is can't help in the meriones unguiculatus fasting of swing arm bacterium, in order to empty digest in its stomach.Use gastric perfusion needle to meriones unguiculatus gavage
0.5ml normal saline.Wait after about 1min, then stretch into by gastric perfusion needle and meriones unguiculatus stomach extracts liquid in its stomach put into centrifugal
In pipe (about 100 μ L).
The extraction of helicobacter pylori DNA: use the micro-example base of TIANGEN Biotech's sky root
Because group DNA extraction kit (DP316) extracts meriones unguiculatus gastric juice helicobacter pylori genome.
PCR reacts amplification:
PCR system is specifically loaded: first round PCR: with the genome of said extracted be template 30ng, 2 × PreMix rTaq
10 μ L, add forward primer F1 (SEQ ID No.1) (10 μMs) the 1 μ L and downstream primer R1 (SEQ ID of pair of primers
No.2) (10 μMs) 1 μ L, then with ddH2O benefit carries out the reaction of first round PCR to 20 μ L, first with F1 and R1 as amplimer
Wheel PCR response procedures: 94 DEG C of denaturations 5min；94 DEG C of degeneration 30sec, 45 DEG C of annealing 30sec, 72 DEG C of extension 30sec, circulate 35
Secondary；72 DEG C extend 5min eventually.Second takes turns PCR: using first round PCR primer as template, then with F2 (SEQ ID No.3) and R2
(SEQ ID No.4) is that the second of amplimer takes turns PCR amplification, and reaction system is: template 30ng, 2 × PreMix rTaq 10 μ
L, the forward primer F2 (SEQ ID No.3) (10 μMs) 1 μ L and downstream primer R2 (SEQ ID No.4) (10 μMs) of second pair of primer
1 μ L, then with ddH2O benefit carries out second to 20 μ L and takes turns PCR reaction, and second with F2 and R2 as amplimer takes turns PCR reaction interval
Sequence: 95 DEG C of denaturations 5min；95 DEG C of degeneration 30sec, 51.3 DEG C of annealing 30sec, 72 DEG C of extension 30sec, circulate 35 times；72 DEG C of ends
Extend 5min.
Agarose gel electrophoresis:
Product (the i.e. second PCR primer taken turns) after nested PCR amplification carries out the agarose gel electrophoresis of 2%, expands mesh
Band be 100bp (see Fig. 4).
Embodiment 5
To with embodiment 4 same procedure artificially infect the gastric juice of 10 meriones unguiculatuss of helicobacter pylori, gastric tissue, ten
It is (i.e. husky to the long pawl of same artificial infection helicobacter pylori that two duodenum 12 contents and colonic faecal carry out DNA extraction respectively
Mus extracts its gastric juice, gastric tissue, duodenum content and the DNA of colonic faecal respectively) after, then to obtain DNA respectively
Template, (includes that the pair of primers and second of first round primer takes turns the second couple of primer with nest-type PRC as identical in embodiment 4
Primer is identical) detect, and agarose gel electrophoresis, it is considered as sun for amplifying purpose band (in conjunction with sequencing result)
Property.
RUT detection method: (Fujian three strongest ones biochemical industry is limited to use stomach Helicobacter pylori diagnostic kit
Company), remove the lid of substrate enzyme mark strip before using, every hole adds enzymatic reaction solution 2 (or 100 μ L), treats that medicine film is completely dissolved
After, the gastric mucosa extracted after killing infected animal with clean tweezers is put into the liquid, hatches under the conditions of 10 DEG C-30 DEG C
5 minutes, observed result.Ocular estimate, lining blank sheet of paper is used to observe gastric tissue mucosal tissue edge medicinal liquid in each hole under available light
Color changes.Helicobacter pylori feminine gender is yellow, and the helicobacter pylori positive is that light red is to rose.
Result: detect 10 meriones unguiculatuss in Gastric juice sample stomach function regulating tissue sample and all infect helicobacter pylori, recall rate
100%；Detecting 9 meriones unguiculatuss in duodenum contents samples and infect helicobacter pylori, recall rate is 90%；Colon excrement
Just detecting 1 meriones unguiculatus in sample and infect helicobacter pylori, recall rate is 10%；Rapid urease test detection 10 is long
Pawl gerbil jird infects helicobacter pylori, recall rate 100%.But use gastric tissue detection that meriones unguiculatus is had infringement, unfavorable
In using meriones unguiculatus to proceed follow-up experiment, the most just lose the meaning of In vivo detection, therefore, integrate and see, stomach
The Detection results of liquid sample is optimum, and the damage for meriones unguiculatus is little, can be similar to meriones unguiculatus as long term monitoring
Non-invasive detection methods etc. laboratory animal Helicobacter pylori infection.Add up above-mentioned experiment acquired results, as shown in table 1.Result
Represent with detection words and deeds success ratio, be described separately the Positive rate of various method.
Table 1
"+" is positive, "-be negative ".
Claims (10)
1. detection meriones unguiculatus (Meriones Unguiculatus) infects helicobacter pylori (Helicobacter
Pylori) method, it comprises the following steps:
Step one: described meriones unguiculatus fasting be can't help water to empty the digest in its stomach；
Step 2: fill normal saline or water in the stomach of described meriones unguiculatus；
Step 3: extract liquid from the stomach of described meriones unguiculatus；
Step 4: with described liquid for sample extraction genome；
Step 5: carry out nest-type PRC: use pair of primers to carry out first round PCR with described genome for template and obtain first
PCR primer；Use second primer carries out second with described first PCR primer for template to take turns PCR and obtain the second PCR primer；
Step 6: detect described second PCR primer, when detect the size of described second PCR primer with its expected from size phase
With, or the sequence of described second PCR primer with its expected from sequence identical time, then confirm described meriones unguiculatus infect described imprison
Door pylori.
Method the most according to claim 1, it is characterised in that described pair of primers is SEQ ID No.1 and SEQ ID
No.2；Described second pair of primer is SEQ ID No.3 and SEQ ID No.4.
Method the most according to claim 1 and 2, it is characterised in that it is 18-that the time of water is can't help in described meriones unguiculatus fasting
36 hours, preferably 20-28 hour.
4., according to the method described in any one in claim 1-3, it is characterised in that in described step 2, use gavage
Pin fills described normal saline or water 0.3-0.6ml in the stomach of described meriones unguiculatus.
5., according to the method described in any one in claim 1-4, it is characterised in that in described step 3, extract liquid
Volume 70-120 μ L.
6. according to the method described in any one in claim 1-5, it is characterised in that first round PCR response procedures such as
Under: 92-98 DEG C of denaturation 0-10min；0.5-2min, 65-78 DEG C of extension of 92-98 DEG C degeneration 0.5-2min, 40-55 DEG C annealing
0.5-2min, circulates 20-45 time；65-78 DEG C extends 0-15min eventually；
The preferably response procedures of first round PCR is as follows: 94-96 DEG C of denaturation 2-5min；94-96 DEG C of degeneration 0.5-1min, 42-47
DEG C annealing 0.5-1min, 70-73 DEG C extend 0.5-1min, circulate 25-35 time；70-73 DEG C extends 2-10min eventually.
7. according to the method described in any one in claim 1-6, it is characterised in that take turns the response procedures of PCR such as second
Under: 92-98 DEG C of denaturation 0-7min；15-60sec, 65-78 DEG C of extension 15-of 92-98 DEG C degeneration 15-60sec, 43-58 DEG C annealing
60sec, circulates 20-45 time；65-78 DEG C extends 0-15min eventually；
Preferably second to take turns the response procedures of PCR as follows: 94-96 DEG C of denaturation 30-180sec；94-96 DEG C of degeneration 20-40sec,
46-51 DEG C of annealing 20-40sec, 72 DEG C of extension 20-40sec, circulate 25-35 time；70-73 DEG C extends 1-5min eventually.
8. according to the method described in any one in claim 1-7, it is characterised in that each component in the system of first round PCR
Proportionate relationship as follows: template: SEQ ID No.1:SEQ ID No.2:10 times PCR buffer: archaeal dna polymerase: water=
10-100ng:0.5-1.5 μm ol:0.5-1.5 μm ol:4-6 μ L:0.5-3U:32-48 μ L；Preferred template: SEQ ID No.1:
The PCR buffer of SEQ ID No.2:10 times: archaeal dna polymerase: water=20-40ng:0.5-1 μm ol:0.5-1 μm ol:4-6 μ L:
0.5-1U:38-42 μ L；More preferably template: SEQ ID No.1:SEQ ID No.2:2 × PCR Master Mix or 2 ×
PreMix rTaq: water=20-40ng:0.5-1 μm ol:0.5-1 μm ol:23-28 μ L:17-25 μ L.
9. according to the method described in any one in claim 1-8, it is characterised in that take turns the system of PCR second as follows: mould
Plate: SEQ ID No.3:SEQ ID No.4:10 times PCR buffer: archaeal dna polymerase: water=10-100ng:0.5-1.5 μ
Mol:0.5-1.5 μm ol:4-6 μ L:0.5-3U:32-48 μ L；Preferred template: SEQ ID No.3:SEQ ID's No.4:10 times
PCR buffer: archaeal dna polymerase: water=20-40ng:0.5-1 μm ol:0.5-1 μm ol:4-6 μ L:0.5-1U:38-42 μ L；More excellent
Modeling plate: SEQ ID No.3:SEQ ID No.4:2 × PCR Master Mix or 2 × PreMix rTaq: water=20-40ng:
0.5-1 μm ol:0.5-1 μm ol:23-28 μ L:17-25 μ L.
10. the primer of nested PCR amplification helicobacter pylori, wherein pair of primers is SEQ ID No.1 and SEQ ID
No.2, second pair of primer is SEQ ID No.3 and SEQ ID No.4.
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| CN104919058A (en) * | 2012-12-05 | 2015-09-16 | 安普利戴格公司 | Method for detecting helicobacter pylori dna in a stool sample |
| CN104458725A (en) * | 2014-12-08 | 2015-03-25 | 本溪泰斯特捷生物科技有限公司 | Helicobacter pylori urease detection kit and preparation method thereof |
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