CN108004315A - Appraisal procedure for Alzheimer's disease risk - Google Patents

Appraisal procedure for Alzheimer's disease risk Download PDF

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CN108004315A
CN108004315A CN201711399846.7A CN201711399846A CN108004315A CN 108004315 A CN108004315 A CN 108004315A CN 201711399846 A CN201711399846 A CN 201711399846A CN 108004315 A CN108004315 A CN 108004315A
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alzheimer
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裘锋
章明晔
裘武军
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Bai Shi Kai (hangzhou) Biological Technology Co Ltd
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    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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    • C12Q1/6883Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/156Polymorphic or mutational markers

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Abstract

The present invention relates to biological gene detection field, disclose a kind of appraisal procedure for Alzheimer's disease risk, DNA is extracted from human body cell, PCR reactions are carried out to APOE genetic fragments as template using the dissociative DNA of extraction and the base type of two SNP sites of rs429358 and rs7412 of APOE genes is identified by fluoroscopic examination, and the Alzheimer's disease risk of receipts detection crowd is assessed by the result of fluoroscopic examination.The advantage of the invention is that, solve the problems, such as that existing detection means is costly, the risk of substantial amounts of examined crowd can be predicted with relatively low cost and expense, for next step medical treatment detection or social developmental plan provides low cost, effectively, there is higher application value.

Description

Appraisal procedure for Alzheimer's disease risk
Technical field
The present invention relates to biological gene detection field, more particularly to a kind of commenting for Alzheimer's disease risk Estimate method, human body gene detection and risk assessment are combined.
Background technology
Alzheimer's disease (Alzheimer ' s Disease, abbreviation AD), Chinese former name " senile dementia ", is A kind of nerve cell death due to brain and caused by nerve degenerative diseases, occurred in over-65s crowd.According to U.S. The statistics of state, the ratio with AD is about 4% in 65~69 years old crowd, and in the elderly population more than 85 years old, the ratio Example is then up to 36%.Meanwhile over-65s old man it is lethal the reason in, AD rankings the 9th.The whole America there are about 4,000,000 AD patients.And China, patient populations are up to 9,000,000.The AD patient populations in the whole world are up to 2100~35,000,000.Expect the year two thousand thirty, global AD patient Quantity may increase to 70,000,000.With age, the risk for suffering from AD is consequently increased.
AD is a kind of gradual process.The symptom of early stage may be mistakenly considered caused by old It is forgetful so as to ignored.With the progressively development of the state of an illness, ability of learning and memory, cognition judgement and many days of patient Often the basic capacity of activity is all gradually degenerated, while may occur in which the symptoms such as disposition change, behavior difficulty.In diseased late period, AD can Patient is caused to lose intelligence, final death.Average each AD patient makes a definite diagnosis that follow-up to renew the time living be about 8 years.In the U.S., except medicine Outside expense, the expense only nursed to AD patient is just up to 100,000,000,000 dollars.Averagely for the various of each AD patient Expense is about 174,000 dollars.So high expense is all extremely white elephant to personal and society.
Gradual due to AD, sufferers themselves and its family members often do not recognize to have shown to suffer from its morbidity early stage The symptom of disease, and usually it is mistakenly considered the normal phenomenon that is producing because of old.And when patient has shown obvious abnormality disease When shape starts to see a doctor, the detection in terms of behaviouristics is typically carried out, and with the brain of definite patient the methods of combination clinical detection Whether nerve has exception.And at this moment AD has had evolved to certain phase, best occasion for the treatment has often been had already passed by.It is so early The examination of phase is extremely important.And traditional clinical testing procedure includes expensive position emissron tomography (abbreviation PET), or carry out The lumbar puncture of pain carries out cerebrospinal fluid sampling etc..It is such detection no matter in economic capability or body it is physiologically tested Person is often difficult to bear.And the clinical detection precision of early stage is not high, possible mistaken diagnosis.So carry out a kind of convenient and efficient And cheap initial in vitro molecular level detection method is just very necessary.
The method of traditional live body DNA detections APOE is genomic DNA in extraction cerebrospinal fluid or haemocyte etc..It is such as existing The method with blood DNA identification APOE that technology proposes, is to extract the genomic DNA in haemocyte as detection method, at the same time This method can not know the somatic variation information in brain tissue source.
Wide concerned cell free DNA (cell free DNA, abbreviation cfDNA) in recent years, in lesion tissue The somatic variation accumulated in apoptosis or non-viable non-apoptotic cell, with elderly patients' brain tissue is closely related.Current liquid Biopsy Operation can not depended on, non-invasively obtaining human body fluid (including blood plasma and other various body fluid, such as urine, saliva, sweat, chest Hydrops etc.) in derive from brain tissue micro metabolic DNA fragment, so as to carry out disease correlation body cell base in brain tissue for us Because variation detection provides more more options space and great convenience.Detected different from traditional genomic DNA, this technology can Reflection patient's body lesion situation in real time, so that the assessment of progression of the disease situation is dynamically carried out according to the state of an illness in real time, and and When formulate corresponding therapeutic scheme.So being detected compared to traditional genomic DNA, Alzheimers are carried out using cfDNA The diagnosis of disease has big advantage in clinical practice.
The content of the invention
The present invention in the prior art for alzheimer's disease testing cost it is excessive the shortcomings that, there is provided Yi Zhongyong In the appraisal procedure of Alzheimer's disease risk, there is provided a kind of appraisal procedure that can be applied in different age group, This method can be assessed with regard to the examined individual risk in specific crowd, so as to instruct the examined individual Next step medical treatment detection.
To achieve the above object, the present invention can take following technical proposals:
A kind of appraisal procedure for Alzheimer's disease risk, including step in detail below:From human body cell Middle extraction DNA, the DNA come from human body fluid and are located at extracellular dissociative DNA;Using the dissociative DNA of extraction as template PCR reactions are carried out to APOE genetic fragments and two SNP of rs429358 and rs7412 of APOE genes are identified by fluoroscopic examination The base type in site, PCR reactions use the probe with FAM and TET fluorescence radiation groups;After PCR reactions start, detection Fluorescence results;Risk is excessive risk when fluoroscopic examination result is following result:1) rs429358 sites FAM and TET is glimmering The detection of light luminophore has reaction, and rs7412 sites FAM and the detection of TET fluorescence radiations group have reaction;2) Rs429358 sites FAM and the detection of TET fluorescence radiations group have reaction, and rs7412 sites only TET fluorescence radiations group is examined Survey has reaction;3) rs429358 sites only TET fluorescence radiations group detection has reaction, rs7412 sites only TET fluorescence radiations base Group's detection has reaction.
Further, as a kind of optional scheme, in embodiments herein, when rs429358 sites, only TET is glimmering The detection of light luminophore has reaction, and the only TET fluorescence radiations group detection of rs7412 sites has reaction, then it is pole to evaluate risk It is high.
Further, as a kind of optional scheme, in embodiments herein, it is used in PCR reactions Rs429358 sites amplification primer sequence and probe sequence be:
rs429358F:5’-ACGGCTGTCCAAGGAGCTGC-3’
rs429358R:5’-AGGCGCACCCGCAGCTCCTC-3’
prob-1T:5’-FAM-AGGACGTGTGCGGCCGCCTG-MGB Eclipse-3’
prob-1C:5’-TET-AGGACGTGCGCGGCCGCCTG-MGB Eclipse-3’
Further, as a kind of optional scheme, in embodiments herein, it is used for rs7412 in PCR reactions Site amplification primer sequence and probe sequence be:
rs7412F:5’-TGCGGGTGCGCCTCGCCTCCC-3’
rs7412R:5’-AGGCGCTCGCGGATGGCGCTG-3’
prob-2T:5’-FAM-CAGAAGTGCCTGGCAGTGTA-MGB Eclipse-3’
prob-2C:5’-TET-CAGAAGCGCCTGGCAGTGTA-MGB Eclipse-3’
Further, as a kind of optional scheme, in embodiments herein, the human body fluid includes peripheral blood Liquid, saliva, cerebrospinal fluid, chest hydrops and urine.
The present invention has following notable technique effect:
Relatively low cost can be used to complete to comment for the alzheimer's disease risk of big quantity particular range crowd Estimate prediction.
Brief description of the drawings
Fig. 1 is PCR fluoroscopic examination result schematic diagrams.
Embodiment
With reference to embodiment, the present invention is described in further detail.
Embodiment 1
A kind of appraisal procedure for Alzheimer's disease risk, be mainly useful for the larger crowd of quantity into Capable Alzheimer's disease risk is deleted and classified, including step in detail below:Extracted from human body cell DNA, the DNA come from human body fluid and are located at extracellular dissociative DNA;It is template to APOE using the dissociative DNA of extraction Genetic fragment carries out PCR reactions and two SNP sites of rs429358 and rs7412 of APOE genes is identified by fluoroscopic examination Base type, PCR reactions use the probe with FAM and TET fluorescence radiation groups;After PCR reactions start, fluorescence knot is detected Fruit;Risk is excessive risk when fluoroscopic examination result is following result:1) rs429358 sites FAM and TET fluorescence radiation Group detection has reaction, and rs7412 sites FAM and the detection of TET fluorescence radiations group have reaction;2) rs429358 sites The detection of FAM and TET fluorescence radiations group has reaction, and the only TET fluorescence radiations group detection of rs7412 sites has reaction;3) The only TET fluorescence radiations group detection of rs429358 sites has reaction, and the only TET fluorescence radiations group detection of rs7412 sites has anti- Should.The above method is only used for risk assessment, in particular for the crowd of classification to(for) big quantity, when above-mentioned detection knot Fruit not definitely indicates whether detected person is diseased, it is necessary to carry out further detection in order to determine if illness.This Outside, the methods of risk assessment in the application also contemplates the diseased probability of overall patient groups, the economy of diagnosis and is examined The age of survey crowd.In addition, the age of onset in the application for risk is preset as average 76 one full year of life, therefore for difference It is only to predict for the examined people of age bracket, does not can confirm that whether it can diseased and illness age.
Further, when rs429358 sites, only TET fluorescence radiations group detection has reaction, and rs7412 sites only TET is glimmering The detection of light luminophore has reaction, then it is high to evaluate risk.In the case, the diseased age of prediction falls to average 68 one full year of life, it is contemplated that carrying crowd's quantity of gene, therefore, needs the risk to Alzheimer's disease in such cases Carry out additional attention or carry out the medicine detection of early stage.
Further, the primer sequence of rs429358 sites amplification is used in PCR reactions and probe sequence is:
rs429358F:5’-ACGGCTGTCCAAGGAGCTGC-3’
rs429358R:5’-AGGCGCACCCGCAGCTCCTC-3’
It is primer sequence above.
prob-1T:5’-FAM-AGGACGTGTGCGGCCGCCTG-MGB Eclipse-3’
prob-1C:5’-TET-AGGACGTGCGCGGCCGCCTG-MGB Eclipse-3’
It is probe sequence above, probe carries FAM or TET fluorescence radiations group and MGB Eclipse fluorescent quenchings Group, it is ensured that when PCR reactions do not carry out, PCR system does not send fluorescence;After PCR reactions proceed by, PCR product Combined with fluorescence probe and begin to send out fluorescence, and can be detected and record with sufficiently strong luminous intensity.
Further, the primer sequence of rs7412 sites amplification is used in PCR reactions and probe sequence is:
rs7412F:5’-TGCGGGTGCGCCTCGCCTCCC-3’
rs7412R:5’-AGGCGCTCGCGGATGGCGCTG-3’
It is primer sequence above.
prob-2T:5’-FAM-CAGAAGTGCCTGGCAGTGTA-MGB Eclipse-3’
prob-2C:5’-TET-CAGAAGCGCCTGGCAGTGTA-MGB Eclipse-3’
It is probe sequence above.
Above-mentioned primer and probe are directed to specific site and use, and above-mentioned primer and probe can be by commercially available Obtain.
Further, the human body fluid includes peripheral blood, saliva, cerebrospinal fluid, chest hydrops and urine.
In addition, what the extracellular DNA that embodiments herein further comprises in the above-mentioned body fluid by human peripheral was extracted Step:
200 μ l blood plasma are taken into the centrifuge tube of 2ml, add 20 μ l Proteinase K solution, is vortexed and mixes;Add 200 The buffer solution GB of μ l, gently overturns and mixes, and 56 DEG C are incubated 10 minutes, and shake sample frequently.Brief centrifugation is to remove in tube cover The drop of wall;Add the ethanol (100%) of 200 μ l precoolings.Gently overturn mix sample, room temperature place 5 minutes, brief centrifugation with Remove the drop of cap wall;Previous step resulting solution is added in an adsorption column CR2 (adsorption column is put into collecting pipe), 12,000rpm (~13,400 × g) are centrifuged 30 seconds, abandon waste liquid, adsorption column CR2 is put back in collecting pipe;Add into adsorption column CR2 Enter 500 μ l buffer solutions GD, 12,000rpm (~13,400 × g) centrifugation 30 seconds, abandon waste liquid, adsorption column CR2 is put back into collecting pipe In;600 μ l rinsing liquids PW, 12,000rpm (~13,400 × g) centrifugation 30 seconds is added into adsorption column CR2, waste liquid is abandoned, will inhale Attached column CR2 is put back in collecting pipe, repeats this step;12,000rpm (~13,400 × g) are centrifuged 2 minutes, outwell waste liquid.It will inhale Attached column CR2 is placed in room temperature and places 2-5 minutes, thoroughly to dry rinsing liquid remaining in sorbing material;Adsorption column CR2 is transferred to one In a clean centrifuge tube, 20-50 μ l elution buffer TB are vacantly added dropwise to adsorbed film centre position, room temperature is placed 2-5 minutes, 12,000rpm (~13,400 × g) are centrifuged 2 minutes, solution are collected into centrifuge tube, up to cfDNA.
Further, PCR reactions are carried out to cfDNA obtained above, comprised the following steps that:In above-mentioned cfDNA, take 5ng is template, and primer combines every kind of each 0.9uM (final concentration), the every kind of each 0.2uM (final concentration) of probe combinations, 2 × MasterMix 5ul, with ddH2O polishings cumulative volume to 10ul, PCR reaction conditions are 95 DEG C, 10 minutes, and with 95 DEG C 15 seconds, 60 DEG C of frequencies of 30 seconds carry out 30-40 circulation.The fluorescence results recorded in PCR reaction process are as shown in Figure 1.
In short, the foregoing is merely presently preferred embodiments of the present invention, all equalizations made according to scope of the present invention patent Change and modification, should all belong to the covering scope of patent of the present invention.

Claims (5)

1. a kind of appraisal procedure for Alzheimer's disease risk, it is characterised in that including step in detail below:From DNA is extracted in human body cell, the DNA comes from human body fluid and is located at extracellular dissociative DNA;With the free of extraction DNA be template to APOE genetic fragments carry out PCR reactions and by fluoroscopic examination identify APOE genes rs429358 and The base type of two SNP sites of rs7412, PCR reactions use the probe with FAM and TET fluorescence radiation groups;PCR is anti- After should starting, fluorescence results are detected;Risk is excessive risk when fluoroscopic examination result is following result:1) rs429358 sites The detection of FAM and TET fluorescence radiations group has reaction, and rs7412 sites FAM and the detection of TET fluorescence radiations group have anti- Should;2) rs429358 sites FAM and TET fluorescence radiation group detection has reaction, rs7412 sites only TET fluorescence radiations base Group's detection has reaction;3) rs429358 sites only TET fluorescence radiations group detection has reaction, and rs7412 sites only TET fluorescence is sent out The detection of light group has reaction.
2. the appraisal procedure according to claim 1 for Alzheimer's disease risk, it is characterised in that when The only TET fluorescence radiations group detection of rs429358 sites has reaction, and the only TET fluorescence radiations group detection of rs7412 sites has anti- Should, then it is high to evaluate risk.
3. the appraisal procedure according to claim 1 for Alzheimer's disease risk, it is characterised in that Primer sequence and probe sequence in PCR reactions for the amplification of rs429358 sites are:
rs429358F:5’-ACGGCTGTCCAAGGAGCTGC-3’
rs429358R:5’-AGGCGCACCCGCAGCTCCTC-3’
prob-1T:5’-FAM-AGGACGTGTGCGGCCGCCTG-MGB Eclipse-3’
prob-1C:5’-TET-AGGACGTGCGCGGCCGCCTG-MGB Eclipse-3’.
4. the appraisal procedure according to claim 1 for Alzheimer's disease risk, it is characterised in that Primer sequence and probe sequence in PCR reactions for the amplification of rs7412 sites are:
rs7412F:5’-TGCGGGTGCGCCTCGCCTCCC-3’
rs7412R:5’-AGGCGCTCGCGGATGGCGCTG-3’
prob-2T:5’-FAM-CAGAAGTGCCTGGCAGTGTA-MGB Eclipse-3’
prob-2C:5’-TET-CAGAAGCGCCTGGCAGTGTA-MGB Eclipse-3’.
5. the appraisal procedure according to claim 1 for Alzheimer's disease risk, it is characterised in that described Human body fluid includes peripheral blood, saliva, cerebrospinal fluid, chest hydrops and urine.
CN201711399846.7A 2017-12-22 2017-12-22 Appraisal procedure for Alzheimer's disease risk Pending CN108004315A (en)

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CN114736963A (en) * 2022-05-25 2022-07-12 首都医科大学宣武医院 Substance combination for predicting Alzheimer disease onset risk, kit and application

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CN114736963A (en) * 2022-05-25 2022-07-12 首都医科大学宣武医院 Substance combination for predicting Alzheimer disease onset risk, kit and application

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