CN105950728A - LRRK2 gene 1628 polymorphism detection kit - Google Patents
LRRK2 gene 1628 polymorphism detection kit Download PDFInfo
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- CN105950728A CN105950728A CN201610332890.5A CN201610332890A CN105950728A CN 105950728 A CN105950728 A CN 105950728A CN 201610332890 A CN201610332890 A CN 201610332890A CN 105950728 A CN105950728 A CN 105950728A
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Abstract
The invention relates to an LRRK2 gene 1628 polymorphism detection kit, relating to gene polymorphism detection. The LRRK2 gene 1628 polymorphism detection kit comprises primers, fluorescent probes and reagents, wherein the primers are used for carrying out specific amplification on a DNA fragment containing the 1628 site of the LRRK2 gene, and the base sequences of the primers are shown as SEQ ID NO:1 and SEQ ID NO:2; the fluorescent probes are used for recognizing the polymorphism of the LRRK2 gene 1628, the 5' end of each fluorescent probe marks the fluorophore, the 3' end of each fluorescent probe marks the cancellation group; two nucleic acid probes used for marking two different fluorophore marks are adopted for simultaneously indicating two polymorphism conditions in one reaction tube, and the base sequences are shown as SEQ ID NO:3 and SEQ ID NO:4; the reagents comprise MgCl2, warm start Taq polymerase, Uracil-N-Glycosylase, dATP, dCTP, dGTP, dTTP, dUTP and PCR reaction buffer.
Description
Technical field
The present invention relates to genetic polymorphism detection, especially relate to a kind of LRRK2 gene 1628 polymorphic detection test kit.
Background technology
Parkinson disease (PD) are in addition to the second common neurodegenerative diseases beyond Alzheimer (AD).At me
PD prevalence in state over-65s crowd is 1.17%, similar to European and American developed countries, this ratio people more than 75 years old
Rising to 3% in Qun, therefore parkinson disease have become one of major disease of modern society of China serious harm human health.
The mean age that parkinson disease are made a definite diagnosis is 70 years old, but its symptom but occurs earlier than clinical definite, therefore finds handkerchief in early days
The gloomy sick Biomarkers of gold and carry out gene diagnosis, prevents particularly important for its risk profile and later stage.Parkinson disease from
Make a definite diagnosis death and typically can continue 15 years, some can live 20 years the most long.The current pathogenic factor about it is with definite
Pathogenesis be not the most fully aware of, but generally believe that this disease is the complexity caused by heredity, environment and aging interaction
Disease.Parkinsonian morbidity is closely related with genetic sudden change, studies by the analysis to familial Parkinson's disease case,
Have been found that at least 13 sites are relevant to familial Parkinson's disease on chromosome.Wherein LRRK2 gene in Chinese population
Suddenly change most commonly seen.
Up to the present having been found that nearly 50 different LRRK2 gene mutation, wherein great majority are missense mutation.LRRK2
Genovariation has the specificity of race, such as, study the most universal G2019R sudden change in the crowd of Europe, 1%～4%
Patients with Parkinson disease in find this gene mutation, in Portuguese, Jew and north African Arab patients with Parkinson disease,
The mutation probability of G2019R accounts for 6%, 15% and 40% (Tan E K, Shen H, Tan L C, et al.The G2019S LRRK2 respectively
mutation is uncommon in an Asian cohort of Parkinson′s disease patients.Neurosci Let,2005,384
(3):327-329).But in Chinese population, do not find the sudden change in this site, but be found that the specific site of two East Asia crowds
R1628P (rs33949390), G2385R (rs34778348) can increase risk, and the two is suddenlyd change in Asia parkinson disease
Crowd accounts for 5～9% (relative to the 3% of normal person), and the two sudden change both increase twice risk (Wang X,
Zhang X,Xue L,et al.The association between the LRRK2R1628P variant and the risk of
Parkinson's disease in Asian:a meta-analysis.Neurosci Lett.2016；623:22-27.).Have document report (Yu L,
Hu F,Zou X,Jiang Y,et al.LRRK2R1628P contributes to Parkinson's disease susceptibility in
Chinese Han populations from mainland China.Brain Res.2009；1296:113-6.), China's Mainland Chinese Han Population
R1628P carrier parkinson disease risk increases by 2.7 times, and TaiWan, China and Singapore R1628P carrier parkinson disease
Risk increases by 2.1 and 2.5 times respectively.
Therefore, detecting 1628 polymorphisms of PD risk genes LRRK2 clinically, whether detection testee carries phase
Correlation gene suddenlys change, and can assist a physician diagnosis parkinson disease, in order to direction of medication usage more in time, significantly alleviates patient and family members
Burden.Parkinson risk genes is detected contribute to the testee that risk genes carries do sth. in advance parkinson disease are carried out prevention and
Treatment, plays the effect of early detection early prevention.
The technology being applied to the detection of parkinson relative risk gene at present is mainly polymerase chain reaction-Restriction fragment length
Polymorphism (PCR-RFLP) method.The method a kind of restriction that has been the different choice according to LRRK2 gene 1628 pleomorphism site
Property restriction endonuclease, this kind of restriction endonuclease only has the effect of cutting to a kind of polymorphism therein, and will not produce another kind of polymorphism
Cutting.This method first passes through primer and LRRK2 gene 1628 pleomorphism site is carried out PCR reaction, then to PCR
Product uses restricted enzyme to carry out endonuclease reaction, then digestion products is carried out electrophoretic analysis, according to obtained segment size
LRRK2 gene R1628P pleomorphism site is analyzed.There is many deficiencies in the method.First, the method relies on
The activity of restricted enzyme, easily produces false positive and false-negative result during enzyme action；Secondly, the method for electrophoresis is held
Easily cause fatal PCR primer to pollute, cause the erroneous judgement of result；Again, the method time-consuming (about 5～6h), work consuming, extremely
Including PCR amplification, enzyme action, electrophoresis, four steps of gel imaging analysis less, need a large amount of manual operation, automaticity is low,
Personal error is big.
Real-time fluorescence PCR has feature quick, accurate, free of contamination, it has also become the conventional instrument platform of molecular diagnostic laboratories.
At present, there is not yet the report that LRRK2 gene 1628 polymorphism is studied by the method utilizing real-time fluorescence PCR.
Summary of the invention
It is an object of the invention to provide and can quickly, accurately, with high throughput LRRK2 gene 1628 polymorphism be detected
A kind of LRRK2 gene 1628 polymorphic detection test kit.
The present invention includes primer, fluorescent probe and reagent；
Described primer comprises the DNA fragmentation in LRRK2 gene 1628 site, its base sequence such as SEQ for specific amplification
Described in IDNO:1 and SEQ IDNO:2, primer sequence can be replaced by its complementary base sequences thereof；
Described fluorescent probe is used for identifying LRRK2 gene 1628 polymorphism, described fluorescent probe 5 ' end mark fluorescent group, 3 '
End labelling quenching group.The nucleic probe using two kinds of different fluorophor labellings of two labellings indicates in a reaction tube simultaneously
The situation of two kinds of polymorphisms, its base sequence is as described in SEQ IDNO:3 and SEQ IDNO:4, and probe sequence can be by its alkali
Base complementary series is replaced.
Two kinds of different fluorophors include the group arbitrarily launching the different two kinds of fluorophors (fluorophor and quenching group) of wavelength
Close.Conventional fluorophor including, but not limited to existing various fluorescent markers, as ALEX-350, FAM, HEX, VIC,
TET, JOE, ROX, TEXAS RED, CY3, CY5, CY5.5 etc..Conventional quenching group including, but not limited to
Current various quencher, such as DABCYL, BHQ1, BHQ2, BHQ3, TAMRA, ECLIPE etc..
Described fluorescent probe is including, but not limited to TaqMan probe, TaqMan-MGB probe, molecular beacon, improvement molecule
At least one in beacon, double-strand fluorescence displacement probe, LightCycler probe, dicyclo probe etc..
Described reagent includes MgCl2, hot start Taq polymerase, UNG enzyme, dATP, dCTP, dGTP, dTTP, dUTP and
PCR reaction buffer.
Described MgCl2Molar concentration can be 1～5mmol/L.
The molar concentration of described dATP, dCTP, dGTP, dTTP can be all 50～400 μm ol/L, the molar concentration of dUTP
Can be 10～1000 μm ol/L.
The unit of described hot start Taq polymerase can be 1～3U/ reaction, and the consumption of described UNG enzyme can be 0.1～1U/ reaction.
Compared with existing detection method and correlation technique, the present invention has an advantage highlighted below:
(1) application real-time fluorescence PCR technology combined with fluorescent probe, can be with Real Time Observation experimental result.Detect simultaneously and more accelerate
Speed (90min), operates easier, it is easy to automatization, and does not has the misgivings that PCR primer pollutes, and improves the standard of detection
Really property.
(2) just LRRK2 gene 1628 polymorphism can be analyzed in a reaction tube, cost-effective while carry again
High flux.
Accompanying drawing explanation
Fig. 1 be LRRK2 gene 1628 pleomorphism site (rs33949390) c.4883 base be the DNA sample of G/G.
Fig. 2 be LRRK2 gene 1628 pleomorphism site (rs33949390) c.4883 base be the DNA sample of G/C.
Fig. 3 be LRRK2 gene 1628 pleomorphism site (rs33949390) c.4883 base be the DNA sample of C/C.
Fig. 4 is that reaction detection pure water sample is as negative control.
Detailed description of the invention
Following example will the present invention is further illustrated in conjunction with accompanying drawing, and the following example simply illustrates and do not indicates that the present invention
All of probability, the invention is not limited in material mentioned in these embodiments, reaction condition or parameter, any in phase
Field, pass possesses the technical staff of experience, other similar material or reaction condition can be utilized to realize according to the principle of the present invention
LRRK2 gene R1628P polymorphic detection test kit described in the invention.
The LRRK2 1628 pleomorphism site (rs33949390) of embodiment 1 real-time PCR detection 3 type.
One. material
1. instrument: real-time fluorescence PCR instrument, pipettor, centrifuge.
2. primer, probe design: the present invention devises the DNA that can comprise LRRK2 gene 1628 pleomorphism site with specific amplified
The primer of fragment, and the probe of specific recognition LRRK2 gene 1628 pleomorphism site.LRRK2 gene 1628 polymorphism
Site (rs33949390) c.4883 base is that the probe of G is labeled as FAM, LRRK2 gene 1628 pleomorphism site
(rs33949390) c.4883 base is that the probe of C is labeled as HEX.So c.4883 base is that the sample of G/G is as template
Reaction tube in, FAM channel should have amplified signal to produce, and HEX channel should not have amplified signal to produce；With c.4883 base
For the sample of C/C as in the reaction tube of template, HEX channel should have amplified signal to produce, and FAM channel should not expand letter
Number produce；Sample using c.4883 base as G/C is as in the reaction tube of template, FAM and HEX channel should all have amplification letter
Number produce.
Primer and the detecting probe information used are as shown in table 1.
Table 1 primer and detecting probe information list
3. reagent: 1 × PCR buffer；MgCl2；Thermal starting enzyme；UNG enzyme；dATP、dCTP、dGTP、dTTP、dUTP.
Two. method
1. the selection of sample
LRRK2 gene 1628 pleomorphism site (rs33949390) c.4883 base is the sample of G/G base, LRRK2 base
Because 1628 pleomorphism sites (rs33949390) c.4883 base is the sample of G/C, LRRK2 gene 1628 pleomorphism site
(rs33949390) c.4883 base is the sample of C/C.All of sample standard deviation is verified by the method for DNA sequencing.
2. the extraction of genomic DNA
From anticoagulated whole blood, human gene group DNA is extracted with conventional molecular biological method or commercial reagent box.
3. real-time fluorescent PCR amplification and detection
Real-time fluorescence PCR reaction system includes 1 × PCR buffer, 3mmol/L Mg2+, 200 μm ol/L dATP, dCTP,
DGTP, dTTP, the upstream and downstream primer of 400 μm ol/L dUTP, 400nmol/L, each fluorescent probe of 400nmol/L, 1
U thermal starting enzyme, 0.3U UNG enzyme, 25ng human gene group DNA.
Real-time fluorescence PCR reaction is carried out on Roche Light Cycler 480 II instrument, carries out augmentation detection by following condition:
First stage: 50 DEG C of 2min；
Second stage: 95 DEG C of 5min；
Phase III: 95 DEG C of 20sec, 62 DEG C of 20sec (fluorescent collecting), 72 DEG C of 30sec, 40 circulations；
Two fluoroscopic examination channels are respectively FAM and HEX.
4. interpretation of result
LRRK2 gene 1628 polymorphism judges: the sample c.4883 base only having amplified signal to produce in FAM channel as
G/G；The sample c.4883 base only having amplified signal to produce in HEX channel is C/C；In FAM and HEX channel all
The sample c.4883 base having amplified signal to produce is G/C.
It will be seen from figure 1 that only have amplified signal to produce in FAM channel, so this sample c.4883 base is G/G.
Figure it is seen that have amplified signal to produce in FAM and HEX channel, so this sample c.4883 base is
G/C。
From figure 3, it can be seen that only have amplified signal to produce in HEX channel, so this sample c.4883 base is C/C.
From fig. 4, it can be seen that FAM and HEX channel does not all have amplified signal signal to produce, for using pure water right as feminine gender
According to result.
Claims (9)
1.LRRK2 gene 1628 polymorphic detection test kit, it is characterised in that include primer, fluorescent probe and reagent；
Described primer comprises the DNA fragmentation in LRRK2 gene 1628 site, its base sequence such as SEQ for specific amplification
Described in IDNO:1 and SEQ IDNO:2, primer sequence can be replaced by its complementary base sequences thereof；
Described fluorescent probe is used for identifying LRRK2 gene 1628 polymorphism, described fluorescent probe 5 ' end mark fluorescent group, 3 '
End labelling quenching group；The nucleic probe using two kinds of different fluorophor labellings of two labellings indicates in a reaction tube simultaneously
The situation of two kinds of polymorphisms, its base sequence is as described in SEQ IDNO:3 and SEQ IDNO:4, and probe sequence can be by its alkali
Base complementary series is replaced；
Described reagent includes MgCl2, hot start Taq polymerase, UNG enzyme, dATP, dCTP, dGTP, dTTP, dUTP and
PCR reaction buffer.
2. LRRK2 gene 1628 polymorphic detection test kit as claimed in claim 1, it is characterised in that the two difference is glimmering
Light group includes the combination arbitrarily launching two kinds of different fluorophors of wavelength, and conventional fluorophor is including, but not limited to existing
Various fluorescent markers, conventional quenching group is including, but not limited to current various quenchers.
3. LRRK2 gene 1628 polymorphic detection test kit as claimed in claim 2, it is characterised in that described existing various glimmering
Signal thing selected from ALEX-350, FAM, HEX, VIC, TET, JOE, ROX, TEXAS RED, CY3, CY5,
One in CY5.5.
4. LRRK2 gene 1628 polymorphic detection test kit as claimed in claim 2, it is characterised in that described various quenchers
One in DABCYL, BHQ1, BHQ2, BHQ3, TAMRA, ECLIPE.
5. LRRK2 gene 1628 polymorphic detection test kit as claimed in claim 1, it is characterised in that described fluorescent probe bag
Include but be not limited to TaqMan probe, TaqMan-MGB probe, molecular beacon, amelioration regionalization, the displacement of double-strand fluorescence
At least one in probe, LightCycler probe, dicyclo probe.
6. LRRK2 gene 1628 polymorphic detection test kit as claimed in claim 1, it is characterised in that described MgCl2Rub
Your concentration is 1～5mmol/L.
7. as claimed in claim 1 LRRK2 gene 1628 polymorphic detection test kit, it is characterised in that described dATP, dCTP,
The molar concentration of dGTP, dTTP is 50～400 μm ol/L, and the molar concentration of dUTP is 10～1000 μm ol/L.
8. LRRK2 gene 1628 polymorphic detection test kit as claimed in claim 1, it is characterised in that described thermal starting Taq
The unit of enzyme is 1～3U/ reaction.
9. LRRK2 gene 1628 polymorphic detection test kit as claimed in claim 1, it is characterised in that the use of described UNG enzyme
Amount is 0.1～1U/ reaction.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112852954A (en) * | 2021-03-16 | 2021-05-28 | 江苏贝格尔生物医药有限公司 | Primer, probe and kit for detecting polymorphism of LRRK2 gene 1628 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105331721A (en) * | 2015-11-27 | 2016-02-17 | 首都医科大学宣武医院 | Method for detecting PD (PD) pathogenic gene mutation, primer and kit thereof |
-
2016
- 2016-05-19 CN CN201610332890.5A patent/CN105950728A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105331721A (en) * | 2015-11-27 | 2016-02-17 | 首都医科大学宣武医院 | Method for detecting PD (PD) pathogenic gene mutation, primer and kit thereof |
Non-Patent Citations (5)
| Title |
|---|
| AROMA AGAPE GOPALAI 等: "LRRK2 G2385R and R1628P Mutations Are Associated with an Increased Risk of Parkinson’s Disease in the Malaysian Population", 《BIOMED RESEARCH INTERNATIONAL》 * |
| CHIN-SONG LU 等: "The LRRK2 Arg1628Pro variant is a risk factor for Parkinson’s disease in the Chinese population", 《NEUROGENETICS》 * |
| MARINA SIEBERT 等: "Novel Mutations in the Glucocerebrosidase Gene of Brazilian Patients with Gaucher Disease", 《JIMD REPORTS》 * |
| OWEN A. ROSS 等: "Analysis of Lrrk2 R1628P as a Risk Factor for Parkinson’s Disease", 《ANNALS OF NEUROLOGY》 * |
| XI WU 等: "Quantitative assessment of the effect of LRRK2 exonic variants on the risk of Parkinson’s disease: A meta-analysis", 《PARKINSONISM AND RELATED DISORDERS》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112852954A (en) * | 2021-03-16 | 2021-05-28 | 江苏贝格尔生物医药有限公司 | Primer, probe and kit for detecting polymorphism of LRRK2 gene 1628 |
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Application publication date: 20160921 |