CN106399570A - Kit for early stage colorectal cancer auxiliary diagnosis and use method and detection system thereof - Google Patents
Kit for early stage colorectal cancer auxiliary diagnosis and use method and detection system thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING 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
- 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
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
- G01N33/721—Haemoglobin
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- C12Q—MEASURING 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/154—Methylation markers
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/795—Porphyrin- or corrin-ring-containing peptides
- G01N2333/805—Haemoglobins; Myoglobins
Abstract
The invention provides a kit for early stage colorectal cancer auxiliary diagnosis and a use method and a detection system thereof. The kit comprises a nucleic acid isolation and purification reagent, a DNA (deoxyribonucleic acid) sulfite conversion reagent, a KRAS (kirsten rat sarcoma viral oncogene homolog) gene mutation detection reagent, a BMP3 (bone morphogenetic protein 3) and NDRG4 (N-myc downsteam regulated gene 4) gene methylation detection reagent and a fecal occult blood detection reagent, wherein the nucleic acid isolation and purification reagent is used for separating and purifying the human DNA in a faeces sample; the DNA sulfite conversion reagent is used for performing sulfite conversion on the purified partial human DNA, and is used for subsequent BMP3 and NDRG4 gene methylation detection. Through detecting two kinds of indexes of DNA and fecal occult blood in the faeces sample in a combined way, the kit provided by the invention is used for the early stage screening of the colorectal cancer. Compared with an FOBT (fecal occult blood test) detection method, the kit provided by the method can realize higher sensitivity on the detection of colorectal cancer, particularly the developing period tumor.
Description
Technical field
The invention belongs to biotechnology and diagnosis research field, it is related to a kind of examination for early stage colorectal cancer auxiliary diagnosis
Agent box and its using method and detecting system.
Background technology
Colorectal cancer is also referred to as colorectal cancer, occurs in colon (large intestine) or rectum, usual slower development, is Chinatown
Town second cancer occurred frequently, in more than 40 years old Silent cerebral infarction, 6% suffers from early stage intestinal cancer.With changing of food configuration and habits and customs
Become, and the becoming increasingly conspicuous of environmental problem, lead to colorectal cancer incidence rapid growth, according to《China's tumour registration annual report》Report is aobvious
Show, intestinal cancer is the second largest cancer that the current Chinese city incidence of disease is only second to lung cancer, main inclusion colon cancer is big with the carcinoma of the rectum two
Class.China increases colorectal cancer confirmed cases 440,000, annual Died Patients up to 230,000 people every year newly at present.National Cancer is early examined
Zao Zhi project Committee of Experts research report in 2013 is pointed out, Chinese more than 40 years old people at highest risk suffers from progressive stage adenoma and early stage intestines
Cancer ratio is 6%.These canceration focuses are excised not in time, will be converted into pernicious intestinal cancer in the case of 90%.Due to major part
Patient did not find to pay attention to and get timely medical treatment in early stage, and the intestinal cancer death rate remains high.It is screened in commitment
The patient arriving, can be cured more than 90%.
Colorectal cancer has many common features：With polyp (abnormal projection) or adenoma before most of intestinal cancer formation
The form of (galandular epithelium benign tumour) slowly develops for many years in site of pathological change, patient's no any discomfort during this period.Adenoma is one
Plant optimum, non-carcinomatous tumor.As time goes on, part adenoma can be changed into cancer.In Carcinogenesis, tumour cell is invaded
Enter intestinal wall, merge with blood vessel or lymphatic vessel, expanded rapidly by absorbing patient's nutrient.In the later stage, cancer cell is invaded by lymphatic vessel
Neighbouring lymph node or other organs of body, such as liver etc., form the metastasis of cancer.Colorectal cancer be generally developed to late period just have bright
Aobvious manifestation of symptoms (as suffered from abdominal pain, having blood in stool).Because most of Chinese patients do not carry out the consciousness of cancer morning examination, find symptom
Just arrive examination in hospital afterwards, to colorectal cancer make a definite diagnosis when, cancer cell has spread, and Late curative effect is inconspicuous, causes Chinese nearly half
Patients with bowel cancer life cycle is less than 5 years.If intestinal cancer early screening technology is carried out penetration and promotion it is contemplated that Colon and rectum in China
Mortality of carcinoma can reduce by more than 50% from existing level.
Colorectal cancer belongs to the clear and definite malignant tumour of Effect of screening, can be effectively improved by examination early detection colorectal cancer
Survival, reduces the death rate；And the early diagnosis and subsequent excising operation to adenoma before cancer (diameter >=1 centimetre) then may be used
Reduce the incidence of disease of colorectal cancer.Examination object can be divided into general population at risk and people at highest risk.In western developed country, knot is straight
Intestinal cancer examination is the generaI investigation project carried out for general population at risk, encourages the people that all ages were more than 50 years old routinely to accept
Intestines mirror and stool occult blood test (Fecal Occult Blood Test, FOBT) check.And in China although in people at highest risk
In carried out " examination of waiting for an opportunity property ", but not yet have one be directed to general population at risk colorectal cancer examination project.With society
Meeting expanding economy and the raising of people's health consciousness, China's colorectal cancer examination will be from " examination of waiting for an opportunity property " to systematicness sieve
Look into transition.The purpose of colorectal cancer examination will be not only early diagnosis colorectal cancer, and the early detection of adenoma before cancer also will
It is taken seriously；Because the canceration of Colon and rectum adenoma generally takes 10 years about, time enough is had to pass through examination and subsequently
Adenoma excising operation block its Carcinogenesis, reach prevention intestinal cancer purpose.
The screening method of colorectal cancer is mainly colonoscopy and stool occult blood test at present.
Fibro-colonoscope is the most accurate so far Screening Method for Colorectal Cancer, and it is considered as knot that intestines mirror adds pathological biopsy
Carcinoma of the rectum examination and the goldstandard of diagnosis.In some countries of US and European, fibro-colonoscope is widely used in Colon and rectum
The examination of cancer, the people of more than 50 years old is required to do an intestines mirror for every 10 years, but when being because invasive and the INTESTINAL CLEANSING of intestines mirror
Sense of discomfort, the American having more than half is reluctant to accept enteroscopy.In China, of the right age crowd accepts intestines mirror examination means
Less than 10%, cause China to find that intestinal cancer mostly is middle and advanced stage, and after finding 5 annual survival rates less than 50%, less than about 20, U.S.
Percentage point.In addition, there are some researches show recently the intestines mirror flat polyp common to right side colon diagnosis effect bad, intestines mirror sieve
Look into the death rate that can not reduce right side colon.In addition, in China it is contemplated that whether there being enough medical resources to prop up
Hold using this huge project of intestines mirror examination.
CT intestines mirror is a kind of new Screening Method for Colorectal Cancer, there are some researches show that its diagnostic sensitivity can be close to intestines
Mirror；But this method also requires that patient does enteron aisle and reorganizes and outfit, and expensive, be not suitable for popularization and use.In addition, CT intestines mirror is as one kind
Examination means may make whole crowd be subject to x-ray radiation, has the possibility leading to that cancer or disease in the blood system occur.
FOBT detection be uniquely be applied to colorectal cancer big series Mass screening and obtain evidence-based medicine EBM confirm can be effective
The method reducing the death rate, its with hemoglobin in excrement for detecting target, relatively additive method, its can accomplish non-invasive,
Do not need INTESTINAL CLEANSING, conveniently draw materials, and more preferable economy.But there are a lot of drawbacks in also FOBT detection：Its detection be
Tumour simultaneous phenomenon bleeding is in fact it could happen that because suspending bleeding or tumour early stage no failing to pinpoint a disease in diagnosis of causing of bleeding, impact is to swollen
The detection sensitivity of knurl particularly tumour early lesion, the FOBT detection technique of current main flow to the sensitiveness of colorectal cancer is
60-87%, and be 20-43% to advanced tumors sensitiveness.
Content of the invention
The present invention is directed to disadvantages mentioned above, there is provided a kind of kit for early stage colorectal cancer auxiliary diagnosis and its use
Method and detecting system, by taking the fecal sample of patient, combined DNA testing result and fecal occult blood result, for tying in early days
The examination of adenoma before the carcinoma of the rectum and cancer, has higher sensitivity.
For achieving the above object, the present invention takes following technical proposals to realize：
The present invention provides a kind of kit for early stage colorectal cancer auxiliary diagnosis, tries with purifying including separate nucleic acid
Agent, DNA sulphite conversion reagent, KRAS gene mutation detection reagent, BMP3 and NDRG4 gene methylation detection reagent, excrement
Fecal occult blood detection reagent；
Wherein separate nucleic acid is used for separating with purified reagent and is purified into the humanized DNA in fecal sample；
DNA sulphite conversion reagent is used for the part humanized DNA of purifying being carried out sulphite conversion, for follow-up
The detection of BMP3 and NDRG4 gene methylation.
Further, described kit also includes sample collection reagent, and described sample collection reagent is divided into two kinds, is respectively used to
Sample for genetic test and the sample for fecal occult blood detection.
Further, dNTP, 2.5- of MgCl2,0.1-1mM of 1-3mM are wherein comprised in KRAS gene mutation detection reagent
The Taq enzyme of 5U/ reaction, the Tris of KCl, 1-4mM of 20-50mM, the primer of every kind of KRAS type 0.4-0.9uM, every kind of
KRAS type 10uM probe.
Further, comprise dNTP, 2U/ reaction of Mg2+, 0.2mM of 2mM in BMP3 and NDRG4 DNA methylation assay reagent
Taq enzyme, the NH4+ of Tris-HCl, 50Mm of 20mM, the primer of every kind of mutation 0.75mM that methylates, every kind of mutation that methylates
0.25mM probe.
The present invention also provides a kind of using method of the above-mentioned kit for early stage colorectal cancer auxiliary diagnosis, described makes
Comprised the following steps with method：
Sample collection, collects the sample for genetic test and fecal occult blood detection respectively；
The process of sample and detection,
The isolation and purification of nucleic acid, using separate nucleic acid and purified reagent, by paramagnetic particle method seperated nuclear acid, then pass through from
Stem method purification of nucleic acid；
Nucleic acid after purification, a part is used for detecting KRAS gene mutation, using the detection of KRAS gene mutation detection reagent
KRAS gene mutation in sample；
Another part is converted to its DNA through sulphite conversion method using DNA sulphite conversion reagent, then
BMP3 and NDRG4 gene methylation in detection sample；
Fecal occult blood detects, the hemoglobin of sample chapter is detected.
Further, above-mentioned using method also includes detecting the parameter association computational methods obtaining, described computational methods include
Following steps：
With excrement for detecting sample, with KRAS gene mutation, BMP3 and NDRG4 gene methylation and hemoglobin for inspection
Survey target, 4 testing results are endowed different weights, carry out result judgement by logical operation formula；
Described logical operation formula is：P=eK/(1+eK)
K=a*Ct1+b*Ct2+c*Ct3+d*FOBT+X
In above formula, P is colorectal cancer composite index；E is natural constant；A, b, c, d, X are constant；Ct1For KRAS and interior
Ginseng gene C t value difference value；Ct2For BMP3 and reference gene Ct value difference value；Ct3For NDRG4 and B2M gene C t value difference value；FOBT is
Hemoglobinometry value.
Further, described a, b, c, d, X are distributed by clinic test data and determine.
Further, described P value, for detecting composite index, according to the decision threshold of clinical distribution determination, is examined during P value >=threshold value
Surveying result is the positive, is feminine gender during p value ＜ threshold value.In addition, Ct1, the highest with distribution in test crowd respectively in Ct2, Ct3
2.5% contrasts for threshold value, if Ct1, one or more of Ct2, Ct3 value is located in highest 2.5%, then obtain positive knot
Really.
The present invention also provides a kind of detecting system of the examination for early stage colorectal cancer, and described detecting system includes sample
Collection device, target detection unit, difference computational unit, data analysis unit data output unit；Wherein sample collection dress
Put the fecal sample for collecting test crowd；
Wherein target detection unit is for detecting the KRAS gene mutation in test crowd's fecal sample, BMP3 and NDRG4
Gene methylation, fecal hemoglobin；
Difference computational unit be used for calculating respectively the KRAS of test crowd and reference gene Ct value difference value, BMP3 and
Reference gene Ct value difference value；NDRG4 and reference gene Ct value difference value；
Data analysis unit, is analyzed by clinical sample detection statistics, with the Colon and rectum difference course of disease and no relevant disease sample
This above 3 class index after measured, forms the tendency related to pathology and is distributed, thus drawing logic decision calculation by distributed data
Method, sets up the incidence formula of detection data and pathological characters：
Described logical operation formula is：P=eK/(1+eK)
K=a*Ct1+b*Ct2+c*Ct3+d*FOBT+X
In above formula, P is colorectal cancer composite index；E is natural constant；A, b, c, d, X are constant；Ct1For KRAS and interior
Ginseng gene C t value difference value；Ct2For BMP3 and reference gene Ct value difference value；Ct3For NDRG4 and B2M gene C t value difference value；FOBT is
Hemoglobinometry value；Wherein a, b, c, d, X are distributed by clinic test data and determine；
Data outputting unit, P value is detection composite index, and the decision threshold being determined according to clinical distribution, during P value >=threshold value
Testing result is the positive, is feminine gender during p value ＜ threshold value.In addition, Ct1, in Ct2, Ct3 respectively with distribution in test crowd
High 2.5% contrasts for threshold value, if Ct1, one or more of Ct2, Ct3 value is located in highest 2.5%, then obtain the positive
Result.Beneficial effects of the present invention：
The kit of the present invention passes through to detect the gene alteration in fecal sample and fecal occult blood, reaches examination Colon and rectum
Before cancer and cancer, the purpose of adenoma is it is achieved that detection based on excrement, and completely noninvasive, detection process Noninvasive is accurate without enteron aisle
Standby, Silent cerebral infarction is high to this detection acceptance；There are the specificity higher compared to FOBT detection and sensitiveness, meanwhile, not only
It can be found that colorectal cancer it is also possible to find cancer before adenoma, be by extract cancer before adenoma prevent colorectal cancer to provide can
Can property.Compared to intestinal fiberscope, the tumour of left and right sides colon, full enteron aisle detection, no check frequency can be found simultaneously.
Brief description
Fig. 1 is the detecting step figure of kit of the present invention.
Fig. 2 is three target gene fluorescent quantitative PCR curves to be measured in an embodiment.
Fig. 3 is the fluorescent quantitative PCR curve of BMP3 and NDRG4 DNA methylation assay in an embodiment.
Fig. 4 is fecal hemoglobin ELISA examination criteria curve in an embodiment.
Specific embodiment
Due to the growth characteristics of colorectal cancer, it is upheld to enteric cavity first, in its growth course, including adenoma before cancer or
Cancer commitment, constantly has massive tumor cell to shed into excretory system through metabolism and together discharges with excrement, these cells
In there is closely related gene containing tumour, reflect neoplastic process, by detecting these gene markers in excrement,
Just it can be found that presence in intestinal wall for the tumour.Because these genes there is it is thus possible to reflect swollen from the tumour generation initial stage
The process of knurl earliest period.
Used in the present invention, gene target is KRAS (v-Ki-ras2, Kirsten rat sarcoma viral
Oncogene homolog) gene mutation, BMP3 (Bone Morphogenetic Protein 3) and NDRG4 (N-Myc
Downstream-Regulated Gene 4) gene methylation.KRAS gene mutation shows that the colorectal cancer more than 35% is thin
In born of the same parents, 7 kinds of mutation in wherein 2 exons are more up to 98% in colorectal cancer cell.KRAS gene mutation is straight in knot
The formation of intestinal tumor, including the performance all in intestinal cancer and precancerous lesion with higher degree.NDRG4 is oncogene N-myc downstream
One of controlling gene, encodes a kind of plasmosin, and this albumen played an important role in the successive cell cycle, in addition goes back and blood vessel
The mitosis regulation and control of smooth muscle cell are relevant.BMP3 is a member of transforming growth factor β superfamily.Gene promoter area
Methylating of domain dna sequence, causes the change of gene expression, BMP3 and NDRG4 methylates and colorectal cancer and precancerous lesion
There is the association of height.
Applicant is found by numerous studies, KRAS and BMP3, the sign methylating to intestinal cancer and canceration of NDRG4
There is complementarity, use of joining together can aid in raising detection sensitivity.In the present invention, another target is fecal occult blood.Cause
Middle and advanced stage intestinal cancer has discontinuity tumour bleeding, so it is sensitiveer to this stage cancer for hemoglobin is detected in excrement
Label.
In one embodiment of the invention, the main component of kit is included described in following table：
The main component of kit in table 1 one embodiment of the invention
In mentioned reagent, unless hereinafter have illustrated, otherwise all can be real using this area conventional reagent
Existing.
Fig. 1 is the detecting step figure of kit of the present invention.As shown in figure 1, the using method of described kit includes：
Sample collection, collects the sample being respectively used to genetic test and fecal occult blood using sample collection reagent.
Sample process and detection, including：
Separate nucleic acid and purifying, paramagnetic particle method separates with centrifugal column method and is purified into the humanized DNA in fecal sample.
KRAS gene mutation detects, fluorescence quantitative PCR method measures the difference of KRAS gene C t value and reference gene Ct value
Value △ Ct1, target detection target spot is 7 kinds of hot spot mutation types of KRAS the 12nd and 13 codons；Specifically, KRAS gene is preferred
Detection primer and probe, have been recorded in applicant formerly disclosed patent application ZL201510002856.7.
BMP3 and NDRG4 gene methylation detects, through sulphite conversion method, DNA is converted, with quantitative fluorescent PCR
Method carries out the detection of MSP (Mehtylation-Specific PCR) method to gene methylation, measures BMP3 and NDRG4 methyl respectively
Change difference △ Ct2, △ Ct3 of Ct value and reference gene Ct value, target detection target spot is BMP3 and NDRG4 each gene promoter
11-13 neighbouring CpG site；BMP3 and NDRG4 methylates preferred detection primer and probe, has been recorded in applicant formerly
Disclosed patent application ZL 201510486088.7.
Fecal occult blood detects, ELISA method carries out FIT (Fecal Immunochemical Test) detection, quantitative determination excrement
Just content of hemoglobin.
Logical operation and result judgement, according to said determination value, 4 testing results are endowed different weights, by patrolling
Collect operational formula computing, and carry out result judgement according to setting clinical threshold value (Cut-off).
Described logical operation formula is：P=eK/ (1+eK)
In above formula, P is composite index, and K=a* △ Ct1+b* △ Ct2+c* △ Ct3+d*FIT+X, e are natural constant；a,
B, c, d, X are clinical constant.
As P value >=Cut-off, testing result is the positive, is feminine gender during p value ＜ Cut-off.
A in above formula, b, c, d, X pass through sufficient amount of clinical data distribution and determine, this research includes
There is the clinical sample of the different courses of disease distributions of intestines mirror pathological examination, including colorectal cancer, progressive stage adenoma, non-progressive stage gland
Knurl, polyp and Silent cerebral infarction sample.After these samples are carried out with above four detections, by individual event testing result and intestines mirror standard
Result carries out logistic regression analysis, and analysis result provides joint-detection sensitivity and specificity under different threshold values.According to this
The requirement of examination means determines after optimal sensitivity and specificity so that it may determine these constants, and corresponding total score
Threshold value.
In addition, according to clinical study results, result in above-mentioned 4 individual events detection also can with clinical distribution corresponding
High 2.5% is contrasted for threshold value, that is, when the △ Ct1 in detection sample, in △ Ct2, △ Ct3 and FIT any one or more
Result is higher than this threshold value, and result is also judged to the positive, with ensure monomial factor have very high numerical value when to be defined as detection positive.?
The result exponent of detection combines score after regression coefficient multiplied by weight for every factor and whether individual event belongs to highest eventually
2.5% score and calculate final index.
Therefore the present invention provides a kind of kit, realize KRAS gene mutation in qualitative detection fecal sample, BMP3 and
The technology of NDRG4 gene methylation and fecal occult blood totally 4 targets.Meanwhile, this kit provides a kind of patrolling of joint-detection
Collect operation method, the result of 4 individual events detections is carried out synthesis, draws the last total score of sample and testing result.
With reference to specific embodiment, the present invention will be further described, but the present invention is not limited in following embodiments.
Involved experimental technique in described embodiment, if no special instructions, is conventional method in the art.The present embodiment examination used
Agent box is the gained kit of the present invention.
The particular exam flow process of fecal sample：
(1) sample collection, collects the sample being respectively used to genetic test and fecal occult blood using sample collection reagent.
Wherein, the sample consumption for nucleic acid extraction is 1-5g, and the sample for fecal occult blood detection is 10mg, sample collection
Reagent ensures sample collection and the stability of storage.As long as the sample stabilizer in sample collection reagent can maintain sample component phase
It is easy to check to stable, heretofore described sample stabilizer can be from existing it is also possible to select inventor formerly
Stabilizer disclosed in patent application document CN104073564A.
(2) sample process and detection, including：
1) separate nucleic acid and purifying, paramagnetic particle method separates with centrifugal column method and is purified into the humanized DNA in fecal sample.
In order to obtain optimum isolate and purify effect, the present invention use two-stage separate and purification devices, sample cracking after
Nucleic acid is first using paramagnetic particle method concentration and separation, then carries out centrifugal column method and be further purified it is ensured that Nucleic acid quality.Wherein it is desirable to use
The OD260/280 that spectrophotometer method measures DNA should be between 1.8-20., and concentration should be between 10-500ng/ul.
2) KRAS gene mutation detection, fluorescence quantitative PCR method measures KRAS gene C t value and reference gene Ct value
Difference △ Ct1, target detection target spot is 7 kinds of hot spot mutation types of KRAS the 12nd and 13 codons.
In order to obtain sufficiently high detection sensitivity, present invention uses mutation extension retarding system (amplification
Refractory mutation system, ARMS) technology, each factor warp in the PCR reaction system of use and response procedures
Cross optimization, by the functional relation between data fitting and dependent variable, obtain optimal reaction system and response procedures.
In order that this kit using convenient, the present invention is simultaneously used multiple PCR technique, 7 kinds of targeted mutagenesis
Type detection and reference gene detection are placed into same reaction tube, it is possible to achieve primary first-order equation completes to detect.
After optimization, the concrete reaction system of KRAS gene mutation detection is：The MgCl of 1-3mM2, 0.1-1mM dNTP,
The Taq enzyme of 2.5-5U/ reaction, the Tris (Ph8.0) of KCl, 1-4mM of 20-50mM, primer (every kind of mutation of 0.4-0.9uM
Type), 10uM probe (every kind of saltant type).
Further, specific response procedures are：95 DEG C, 5min；95 DEG C, 25sec, 64 DEG C, 20sec, 72 DEG C, 20sec,
15cycles；93 DEG C, 25sec, 60 DEG C, 35sec, 72 DEG C, 20sec, 45cycles；
Fig. 2 is the fluorescent quantitative PCR curve of KRAS detection in the present embodiment.
3) detection of BMP3 and NDRG4 gene methylation, converts to DNA through sulphite conversion method, with fluorescent quantitation
PCR method carries out the detection of MSP (Mehtylation-Specific PCR) method to gene methylation, measures BMP3 and NDRG4 respectively
The Ct value that methylates and difference △ Ct2, △ Ct3 of reference gene Ct value, target detection target spot is that each gene opens BMP3 and NDRG4
11-13 CpG site near mover.
The invention provides a kind of reagent of DNA sulphite conversion, DNA is processed through sulphite, through over cure, deamination
Base, desulfurization chemical reaction, make do not have methylated Cytosines to be uracil, this chemical reaction is by sulfite ion parent
Nuclear reaction is attached on cytimidine, and occurs methylated cytimidine then will not convert, and based on this, specificity is directed to methyl
The probe changing sequence is designed, and detects gene methylation sequence for selectivity.
And specifically, it is preferable to, the sulphite conversion fluid composition in the present invention includes：NaOH、NaHSO3、(NH4)2SO3.H2O、NH4HSO3.
In order to obtain sufficiently high detection sensitivity, the present invention uses Taqman fluorescence probe technology, the PCR reaction of use
Each factor in system and response procedures have passed through optimization, by the functional relation between data fitting and dependent variable, obtains
Good reaction system and response procedures.
In order that this kit using convenient, the present invention is simultaneously used multiple PCR technique, 2 target methyl
Change gene and reference gene is placed into same reaction tube, two genes are distinguished by different fluorescent dyes, it is possible to achieve once anti-
Should complete to detect.
After optimization, the concrete reaction system of BMP3 and NDRG4 DNA methylation assay is：The Mg of 2mM2+, 0.2mM dNTP,
The Taq enzyme of 2U/ reaction, the Tris-HCl (Ph8.0) of 20mM, the NH4 of 50Mm+, 0.75mM primer (every kind of saltant type),
0.25mM probe (every kind of saltant type).
Further, specific response procedures are：95 DEG C, 2min；95 DEG C, 20sec, 60 DEG C, 60sec, 50cycles.
Fig. 3 is the fluorescent quantitative PCR curve of BMP3 and NDRG4 DNA methylation assay in the present embodiment.
4) fecal occult blood detection, ELISA method carries out FIT (Fecal Immunochemical Test) detection, quantitative determination
Fecal hemoglobin content.
Compared to the collaurum qualitative hemoglobin detection method using on market, present invention uses ELISA double antibodies sandwich
Method carries out quantitative hemoglobin detection.
Fecal hemoglobin detection sensitivity in the present invention is 10ng/ml.
Fig. 4 is fecal hemoglobin ELISA examination criteria curve in the present embodiment.
In order to verify beneficial effects of the present invention, the present embodiment has also carried out clinical verification test, by the inspection of the present invention
Survey method and logical operation formula and decision method, are detected as compareing with colloidal gold method FOBT, with detection sensitivity and specificity
Carry out performance comparison for index.
Clinical testing is chosen clinical sample altogether and is amounted to 384 person-portions, collects fecal specimens before entering to choose to carry out enteroscopy
And carry out These parameters detection.
Pathological classification in test and enteroscopy draws, is divided into 5 classifications：
A, colorectal cancer (I～IV phase)；
B, progressive stage adenoma, refer to meet the adenoma of following one or more standard：A) diameter ＞ 10mm；B) contain villous to become
Point；C) there are severe dysplasia or intraepithelial neoplasia.
C, non-progressive stage adenoma (being classified as feminine gender)
D, polyp (is classified as feminine gender)
E, negative (Sigmoidoscope and pathologic diagnosis are all asymptomatic)
Wherein c, d, e class counts negative for pathology, and a, b class counts positive for pathology.
Data analysis employs SAS 9.4and JMP 11.1.1 (64-bit) software (SAS Institute). formula
Set up and four variance factor △ Ct1, △ Ct2, △ Ct3 and FIT testing results and pathological classification have been carried out logistic regression and divided
Analysis, the result obtaining determines clinical coefficient in formula for each factor.In addition, individual event result also with it in test crowd
The highest 2.5% of distribution contrasts for threshold value.If within individual event result belongs to highest 2.5%, enough with individual event result
Facilitate detection positive findings.The result exponent of final detection combine score after regression coefficient multiplied by weight for every factor and
Whether individual event belongs to the score of highest 2.5% and calculates final index.
Described logical operation formula is：P=eK/(1+eK)
In above formula, P is composite index, and K=a* △ Ct1+b* △ Ct2+c* △ Ct3+d*FIT+X, e are natural constant；a,
B, c, d, X are clinical constant.
As P value >=Cut-off, testing result is the positive, is feminine gender during p value ＜ Cut-off.
Concrete clinical test results are following (being shown in Table 2)
Table 2 clinical test results contrast
Note：
Sensitivity (also referred to as True Positive Rate, sensitivity)=true positives number/(true positives number+false negative number) *
100%.
Criticize the degree really judging patient, namely percentage that is actual ill and correctly being diagnosed.
Specificity (also referred to as true negative rate, specificity)=true negative number/(true negative number+false positive number) *
100%.
Criticize the degree really judging non-patient, namely reality is anosis and be correctly diagnosed as anosis percentage.
It will be apparent that the detection kit in the present invention and corresponding method of detection, as a kind of Noninvasive detection side
Formula, it is possible to achieve the detection sensitivity higher compared to FOBT (colloidal gold method), the recall rate especially for adenoma has and significantly carries
Height, has higher recall rate although decreasing on specificity parameter especially for progressive stage adenoma, but for colony
Examination improves sensitiveness and is more of practical significance, and prevents colorectal cancer to provide possibility for by extracing adenoma before cancer.
Although the present invention is open as above with preferred embodiment, it is not for limiting the present invention, any this area
Without departing from the spirit and scope of the present invention, the methods and techniques content that may be by the disclosure above is to this for technical staff
Bright technical scheme makes possible variation and modification, and therefore, every content without departing from technical solution of the present invention, according to the present invention
Technical spirit any simple modification, equivalent variations and modification that above example is made, belong to technical solution of the present invention
Protection domain.
Claims (9)
1. a kind of kit for early stage colorectal cancer auxiliary diagnosis is it is characterised in that described kit includes separate nucleic acid
With purified reagent, DNA sulphite conversion reagent, KRAS gene mutation detection reagent, BMP3 and NDRG4 gene methylation detects
Reagent, fecal occult blood detection reagent；
Wherein separate nucleic acid is used for separating with purified reagent and is purified into the humanized DNA in fecal sample；
DNA sulphite conversion reagent is used for the part humanized DNA of purifying being carried out sulphite conversion, for follow-up BMP3
Detection with NDRG4 gene methylation.
2. the kit for early stage colorectal cancer auxiliary diagnosis according to claim 1 is it is characterised in that described reagent
Box also includes sample collection reagent, and described sample collection reagent is divided into two kinds, is respectively used to for the sample of genetic test and is used for
The sample of fecal occult blood detection.
3. the kit for early stage colorectal cancer auxiliary diagnosis according to claim 1 is it is characterised in that wherein KRAS
The MgCl of 1-3mM is comprised in detection in Gene Mutation reagent2, the Taq enzyme of dNTP, 2.5-5U/ reaction of 0.1-1mM, 20-50mM
The Tris of KCl, 1-4mM, the primer of every kind of KRAS type 0.4-0.9uM, every kind of KRAS type 10uM probe.
4. the kit for early stage colorectal cancer auxiliary diagnosis according to claim 1 it is characterised in that BMP3 and
Comprise in NDRG4 DNA methylation assay reagent 2mM Mg2+, 0.2mM dNTP, 2U/ reaction Taq enzyme, the Tris-HCl of 20mM,
The NH4+ of 50Mm, every kind of methylate mutation 0.75mM primer, every kind of methylate mutation 0.25mM probe.
5. the user of the kit for early stage colorectal cancer auxiliary diagnosis as described in a kind of any one as claim 1-4
Method is it is characterised in that described using method comprises the following steps：
Sample collection, collects the sample for genetic test and fecal occult blood detection respectively；
The process of sample and detection,
The isolation and purification of nucleic acid, using separate nucleic acid and purified reagent, by paramagnetic particle method seperated nuclear acid, then passes through centrifugal column
Method purification of nucleic acid；
Nucleic acid after purification, a part is used for detecting KRAS gene mutation, detects sample using KRAS gene mutation detection reagent
Middle KRAS gene mutation；
Another part is converted to its DNA through sulphite conversion method using DNA sulphite conversion reagent, then detects
BMP3 and NDRG4 gene methylation in sample；
Fecal occult blood detects, the hemoglobin of sample chapter is detected.
6. the using method of the kit for early stage colorectal cancer auxiliary diagnosis according to claim 5, its feature exists
In the computational methods of the data that pattern detection obtains comprise the following steps：
With excrement for detecting sample, with KRAS gene mutation, BMP3 and NDRG4 gene methylation and hemoglobin for detecting target
Mark, 4 testing results are endowed different weights, carry out result judgement by logical operation formula；
Described logical operation formula is：P=eK/(1+eK)
K=a*Ct1+b*Ct2+c*Ct3+d*FOBT+X
In above formula, P is colorectal cancer composite index；E is natural constant；A, b, c, d, X are constant；Ct1For KRAS and reference gene
Ct value difference value；Ct2For BMP3 and reference gene Ct value difference value；Ct3For NDRG4 and reference gene Ct value difference value；FOBT is blood red egg
White measured value.
7. the using method of the kit for early stage colorectal cancer auxiliary diagnosis according to claim 6, its feature exists
In, described a, b, c, d, X are distributed by clinic test data and determine.
8. the using method of the kit for early stage colorectal cancer auxiliary diagnosis according to claim 6, its feature exists
In P is colorectal cancer composite index, the decision threshold being determined according to clinical distribution, and during P value >=threshold value, testing result is the positive, p
It is feminine gender during value ＜ threshold value；In addition, Ct1, Ct2, Ct3Middle respectively with test crowd distribution highest 2.5% do for threshold value right
Ratio is if Ct1, Ct2, Ct3One or more of value be located at highest 2.5% in, then obtain positive findings.
9. a kind of detecting system of the examination for early stage colorectal cancer is it is characterised in that described detecting system includes sample receipts
Acquisition means, target detection unit, difference computational unit, data analysis unit data output unit；Wherein sample collection device
For collecting the fecal sample of test crowd；
Target detection unit is for detecting the KRAS gene mutation in test crowd's fecal sample, BMP3 and NDRG4 gene methyl
Change, fecal hemoglobin；
Difference computational unit is used for calculating KRAS and reference gene Ct value difference value, the BMP3 and reference gene Ct of test crowd respectively
Value difference value；NDRG4 and reference gene Ct value difference value；
Data analysis unit, is analyzed by clinical sample detection statistics, with the Colon and rectum difference course of disease and no relevant disease sample warp
Measure KRAS gene mutation, BMP3 and NDRG4 gene methylation, the index of fecal hemoglobin, form become related to pathology
Gesture is distributed, thus drawing logic decision algorithm by distributed data, sets up the incidence formula of detection data and pathological characters：
Described logical operation formula is：P=eK/(1+eK)
K=a*Ct1+b*Ct2+c*Ct3+d*FOBT+X
In above formula, P is colorectal cancer composite index；E is natural constant；A, b, c, d, X are constant；Ct1 is KRAS and internal reference base
Because of Ct value difference value；Ct2 is BMP3 and reference gene Ct value difference value；Ct3 is NDRG4 and B2M gene C t value difference value；FOBT is blood red
Protein determination value；Wherein a, b, c, d, X are distributed by clinic test data and determine；
Data outputting unit, P value is detection composite index, according to the composite index positive decision threshold setting, during P value >=threshold value
Testing result is the positive, is feminine gender during p value ＜ threshold value；In addition, Ct1, in Ct2, Ct3 respectively with distribution in test crowd
High 2.5% contrasts for threshold value, if Ct1, one or more of Ct2, Ct3 value is located in highest 2.5%, then obtain the positive
Result.
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