CN109207592A - Kit and its application for colorectal cancer detection - Google Patents
Kit and its application for colorectal cancer detection Download PDFInfo
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- CN109207592A CN109207592A CN201811123191.5A CN201811123191A CN109207592A CN 109207592 A CN109207592 A CN 109207592A CN 201811123191 A CN201811123191 A CN 201811123191A CN 109207592 A CN109207592 A CN 109207592A
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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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- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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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
- C12Q2600/154—Methylation markers
Abstract
The invention discloses a kind of kit for colorectal cancer detection and its applications；Wherein kit includes Primer composition, probe compositions；Primer composition includes BS-S9-F, BS-S9-R, BS-N4-F, BS-N4-R, BS-SDC2-F, BS-SDC2-R, BS-ACT-F and BS-ACT-R；Probe compositions include BS-S9-P, BS-N4-P, BS-SDC2-P and BS-ACT-P.Kit of the invention can be applied to colorectal cancer detection, have high sensitivity, the high advantage of specificity.
Description
Technical field
The present invention relates to biotechnology and medical domain more particularly to it is a kind of for colorectal cancer detection kit and its
Using.
Background technique
Cancer has become the maximum killer for threatening human health, and Cancer in China data statistics in 2018 is shown: 2014 complete
State newly send out suffer from cancer number be 380.4 ten thousand, compared with 2013 increase by 120,000, amplification 3.2%.According to another Wanqing Chen at it
Prediction in " the Cancer Statistics in China, 2015 " delivered, 2015, China is newly-increased to suffer from cancer number and is up to
429.2 ten thousand, and newly-increased cancer mortality case is up to 281.4 ten thousand.
Colorectal cancer is a kind of common malignant tumor of digestive tract, has no significant symptom in early days, patient's Non Apparent Abnormality changes
Become, therefore many patients is caused to miss best consultation hours, and has been in cancer middle and advanced stage if making a definite diagnosis.
It is shown according to Cancer in China data statistics in 2018, China's colorectal cancer incidence rate separation all cancer morbidities of men and women
The 4th and third.The colorectal cancer incidence rate in the U.S. is higher than China, but considers the factor of population base, the Colon and rectum in China
The quantity of cancer patient be it is incomparable huge, almost accounted for mondial 1/4.It counts and shows according to WHO: U.S.'s colorectal cancer
Early detection rate reaches 39%, and 5 years survival rates of patient are then up to 65%；But in China, colorectal cancer early detection rate is only
10%, and 5 years survival rates of patient are even more only 32%.
This discovery rate is low, the death rate is high, the reason is that our prevention consciousness is weak, screening technology is developed slowly,
Screening population coverage is low etc..Therefore, promoted the colorectal cancer early screening technology in China, universal common people's self-prevention consciousness,
Increasing town and country screening coverage area seems especially urgent.
DNA methylation refers to that under the catalysis of transmethylase, the cytimidine of two nucleotide of CG of DNA is by selectively
Methyl is added, forms 5-methylcytosine, this is common in the 5'-CG-3' sequence of gene.DNA methylation can cause chromatin knot
The change of structure, DNA conformation, DNA stability and DNA and protein interaction mode, to control gene expression.
A large number of studies show that the methylation of tumor suppressor gene promoter region shows close phase with colorectal cancer
Guan Xing, this develops new colorectal cancer screening technology for us and provides theoretical basis.FDA is in approved products in 2014
Cologuard is used for colorectal cancer screening, but the product is mainly directed towards the crowd of north America region.CFDA also has been approved by blood
The methylation level of SEPT9 can be used for the auxiliary diagnosis of colorectal cancer.Existing blood SEPT9 DNA methylation assay product is upper at present
City, but its sensitivity need to be improved.
Clinically, the goldstandard of diagnosis of colorectal carcinoma is that enteroscopy adds tissue pathological slice to make a definite diagnosis.Carrying out colonoscopy inspection
Before looking into, subject needs fasting and carries out enteron aisle cleaning, at the same this method be it is invasive, have infection and wound risk.
In China, since the screening for cancer consciousness of the common people is not high enough, and to enteroscopy there are biggish resistances psychology, therefore active into
Row enteroscopy is mostly the people for enteron aisle discomfort obviously occur, this causes most of patients with bowel cancer once to be made a definite diagnosis and has entered cancer
Disease middle and advanced stage misses optimal treatment time.
Other kinds of colorectal cancer detection means further includes fecal occult blood detection, protide marker detection etc., but by
Be limited to sensitivity, these methods can not efficiently and accurately to colorectal cancer carry out screening.
Excrement is formed from enteron aisle and is excreted through hepatic portal, wherein there is a large amount of intestinal tissue cast-off cells.To excrement
Humanized's nucleic acid detects in box lunch, according to related gene with the presence or absence of methylation, carries out suffering from cancer risk to subject with this
Assessment, and made a definite diagnosis by goldstandard.The nucleic acid of fecal sample detection is directed to enteron aisle, for blood testing
Performance is sensitiveer, reflection is more intuitive；Cumbersome compared to the complexity of enteroscopy, stool sampling is noninvasive, conveniently, be more easier by by
Inspection person receives, therefore is conducive to a wide range of expansion of colorectal cancer screening.
Currently, China is directed to the product of colorectal cancer related gene DNA methylation assay only based on the SEPT9 of plasma sample
DNA methylation assay, although specificity is very high, sensibility is far from reaching needed for clinic.
Summary of the invention
The technical problem to be solved by the present invention is to overcome the deficiencies in the prior art, provide a kind of for colorectal cancer detection
Kit has the advantages such as high sensitivity, high specific.It additionally provides the kit and is seeing the application detected in colorectal cancer,
Using fecal sample as detection sample, sampling is convenient, can effectively promote the discovery rate of progressive stage adenoma and early stage colorectal cancer.
A kind of kit for colorectal cancer detection, including Primer composition, probe compositions；
The Primer composition includes for detecting the primer pair BS-S9-F and BS-S9-R of SEPT9 gene, for detecting
The primer pair BS-N4-F and BS-N4-R of NDRG4 gene, primer pair BS-SDC2-F and BS-SDC2- for detecting SDC2 gene
R, for detecting the primer pair BS-ACT-F and BS-ACT-R of ACTB gene,
The DNA sequence dna of the BS-S9-F as shown in SEQ ID NO.1,
The DNA sequence dna of the BS-S9-R as shown in SEQ ID NO.2,
The DNA sequence dna of the BS-N4-F as shown in SEQ ID NO.3,
The DNA sequence dna of the BS-N4-R as shown in SEQ ID NO.4,
The DNA sequence dna of the BS-SDC2-F as shown in SEQ ID NO.5,
The DNA sequence dna of the BS-SDC2-R as shown in SEQ ID NO.6,
The DNA sequence dna of the BS-ACT-F as shown in SEQ ID NO.7,
The DNA sequence dna of the BS-ACT-R is as shown in SEQ ID NO.8；
The probe compositions include BS-S9-P, BS-N4-P, BS-SDC2-P and BS-ACT-P,
The DNA sequence dna of the BS-S9-P as shown in SEQ ID NO.9,
The DNA sequence dna of the BS-N4-P as shown in SEQ ID NO.10,
The DNA sequence dna of the BS-SDC2-P as shown in SEQ ID NO.11,
The DNA sequence dna of the BS-ACT-P is as shown in SEQ ID NO.12.
Above-mentioned kit, it is preferred that the kit further includes closing sequence composition, the closing sequence composition
Including BS-S9-C, BS-N4-C and BS-SDC2-C,
The DNA sequence dna of the BS-S9-C is as shown in SEQ ID NO.13；
The DNA sequence dna of the BS-N4-C is as shown in SEQ ID NO.14；
The DNA sequence dna of the BS-SDC2-C is as shown in SEQ ID NO.15.
Above-mentioned kit, it is preferred that respectively closing the concentration of sequence in the closing sequence composition is 0.1 μM.
Above-mentioned kit, it is preferred that the concentration of each primer is 0.2 μM in the Primer composition.
Above-mentioned kit, it is preferred that the concentration of each probe is 0.15 μM in the probe compositions.
Above-mentioned kit, it is preferred that in the probe compositions,
The BS-S9-P is marked using FAM and BHQ1；
And/or the BS-N4-P is marked using ROX and BHQ2；
And/or the BS-SDC2-P is marked using CY5 and BHQ2；
And/or the BS-ACT-P is marked using HEX and BHQ1.
Above-mentioned kit, it is preferred that in the closing sequence composition, the BS-S9-C, the BS-N4-C and institute
It states BS-SDC2-C and carries out phosphorylation modification.
Above-mentioned kit, it is preferred that the kit further includes 2x 360Master Mix, glycerol, tetramethyl chlorination
Ammonium (TMAC) and deionized water.
Above-mentioned kit, it is preferred that the concentration of the tetramethyl ammonium chloride is 10~40mM.
Above-mentioned kit, it is preferred that the volumetric concentration of the glycerol is 8%~20%.
As a total technical concept, the present invention also provides a kind of above-mentioned kits in detection colorectal cancer
Using.
Above-mentioned application, it is preferred that the application method the following steps are included:
(1) fecal sample is dissolved in fecal sample protection liquid, carries out faeces DNA extraction；
(2) faeces DNA of extraction bisulfite is carried out to convert to obtain the faeces DNA of methylation processing；
(3) by it is described methylation processing faeces DNA carry out fluorescence quantitative PCR detection with kit, with SEPT9,
The amplification Ct value of NDRG4, SDC2, ACTB, logically regression formula calculates P value, the logistic regression formula are as follows: P=eScore/
(1+eScore),
Score=w1 × Ct1+w2 × Ct2+w3 × Ct3+w4 × Ct4+B；
Wherein, P is Risk of Colorectal Cancer index,
E is natural constant,
It is -0.12201, w3 is -0.21562, w4 0.20329, B 13.68188 that w1, which is -0.14867, w2,；
Ct1 is that SEPT9 expands Ct value,
Ct2 is that NDRG4 expands Ct value,
Ct3 is that SDC2 expands Ct value,
Ct4 is that ACTB expands Ct value；
When P value is greater than or equal to 0.362, it is judged as positive；When less than 0.362, it is judged as negative.
Above-mentioned application, it is preferred that the response procedures of the fluorescence quantitative PCR detection are as follows: 50 DEG C of 2min；95 DEG C of initial denaturations
10min；Then it is a circulation with 95 DEG C of denaturation 15s, 60 DEG C of annealing 30s, carries out 45 times；Finally in 20 DEG C of holding 2min.
Above-mentioned application, it is preferred that the ingredient of fecal sample protection liquid include: the tris-HCl of 0.25M~0.8M,
EDTA, 0.1m/v%~0.5m/v% lauryl sodium sulfate of the guanidinium isothiocyanate of 1.0M~4.0M, 5mM~20mM
(SDS), the triton x-100 of 0.5v/v%~3v/v%, the polysorbas20 of 0.5v/v%~3v/v%, 3v/v%~10v/v%
Glycerol.
Further, the ingredient of the fecal sample protection liquid includes: the isothiocyanic acid of tris-HCl, 1.5M of 0.5M
Guanidine, the lauryl sodium sulfate (SDS) of EDTA, 0.2m/v% of 10mM, the triton x-100 of 1v/v%, 1v/v% tween
20, the glycerol of 5v/v%.
Testing principle of the invention: unmethylated cytimidine (C) is turned after bisulf iotate-treated in nucleic acid sequence
It turns to uracil (U), and the cytimidine to methylate will not be then converted, and carry out primer for the sequence after methylated genes conversion
The design of probe, the primed probe can only expand methylated genes conversion after nucleic acid, rather than methylated genes conversion after core
It is sour then will not be amplified.In order to further avoid false positive results caused by non-specific amplification, present invention adds with non-methyl
The oligonucleotide sequence of 3 ' the terminal phosphateizations modification of sequence complementary pairing, the sequence can block amplimer after change genetic transformation
And the combination of non-methylated nucleic acid transforming sequence, to play the effect for promoting detection specificity.
Compared with the prior art, the advantages of the present invention are as follows:
(1) the present invention provides it is a kind of for colorectal cancer detection kit, the application using SEPT9, NDRG4,
SDC2 joint-detection monitors humanized's nucleic acid content in sample with this using ACTB house-keeping gene as internal standard；House-keeping gene
Amplified fragments are one section of non-methylated DNA fragments, do internal standard using the amplified fragments, can sufficiently monitor the sample being added when detection
The total amount of humanized's nucleic acid contained by this, can prevent testing result from false positive or false negative occur.SEPT9, NDRG4, SDC2 tri-
The methyl of a gene turns to gene methylation most commonly seen in colorectal cancer patients, but three might not methylate simultaneously,
Therefore positive rate can be effectively promoted by the way of joint-detection, reduced false negative, promoted the sensitivity of detection.This Shen
Kit please, high sensitivity, specificity are high, can effectively promote the discovery rate of progressive stage adenoma and early stage colorectal cancer, from
And the generation or radical cure colorectal cancer of colorectal cancer are prevented, promote 5 years survival rates of patient.
(2) it the present invention provides a kind of kit for colorectal cancer detection, provides and non-methylated DNA fragments is carried out
The closing sequence of shielding, can promote the specificity of detection reagent.
(3) unmethylated in conversion process the present invention provides a kind of kit for colorectal cancer detection
Cytimidine (C) is converted into uracil (U), and the ratio of the guanine (G) and cytimidine (C) that lead to amplification region substantially reduces, and leads
The reduction of amplification efficiency is caused, tetramethyl ammonium chloride (TMAC), glycerine (glycerol) is added in the present invention in the reaction system, to mention
Rise specific amplification efficiency.
(4) the present invention provides a kind of kit for colorectal cancer detection, detection architecture is 4 joint inspection detection reagents,
Using Real-Time Fluorescent Quantitative PCR Technique, four detection genes use different fluorescent markers, convenient for difference, improve detection
Accuracy rate.
(5) application the present invention provides a kind of kit for colorectal cancer detection in detection colorectal cancer, with
Fecal sample is used for colorectal cancer auxiliary diagnosis or screening as detection sample, and sampling is convenient, belongs to Non-invasive detection, Neng Gouti
The acceptance level for rising subject is conducive to the covering crowd for expanding colorectal cancer screening, reduces the incidence of Chinese colorectal cancer,
It is provided safeguard for fitness-for-all.
Detailed description of the invention
In order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below in conjunction with the embodiment of the present invention
In attached drawing, the technical scheme in the embodiment of the invention is clearly and completely described.
Fig. 1 is the schematic illustration of the kit of embodiment 1.
Fig. 2 is that model ROC curve analyzes result figure in embodiment 2.
Fig. 3 is pattern detection internal standard curve synoptic diagram in embodiment 3.
Fig. 4 is positive pattern detection curve synoptic diagram in embodiment 3.
Fig. 5 is not seal up wild-type amplification curve when closing sequence in embodiment 4.
Fig. 6 is wild-type amplification curve when closing sequence and additive being added in embodiment 4.
Fig. 7 is internal standard ACTB detection curve in embodiment 5, and DNA content is 10ng/ reaction.
Fig. 8 is SEPT9 sensitivity technique curve in embodiment 5 (10ng/ reaction, methylate containing 1%SEPT9).
Fig. 9 is NDRG4 sensitivity technique curve in embodiment 5 (10ng/ reaction, methylate containing 1%NDRG4).
Figure 10 is SDC2 sensitivity technique curve in embodiment 5 (10ng/ reaction, methylate containing 1%SDC2).
Specific embodiment
Below in conjunction with Figure of description and specific preferred embodiment, the invention will be further described, but not therefore and
It limits the scope of the invention.
Embodiment
Material employed in following embodiment and instrument are commercially available.
Embodiment 1:
A kind of kit for colorectal cancer detection of the invention, including Primer composition, probe compositions and closing
Sequence composition, 2x 360Master Mix, tetramethyl ammonium chloride (TMAC), glycerol and deionized water.
Wherein the component of Primer composition is listed in Table 1 below.
Table 1: the Primer composition of embodiment 1
| BS-S9-F | 5’-TTTGTATTGTAGGAGCGCGGGC-3’(SEQ ID NO.1) |
| BS-S9-R | 5’-CAACGACGAAAAAACGCCCCCG-3’(SEQ ID NO.2) |
| BS-N4-F | 5’-GTTTCGCGTCGCGGTTTTCGTTC-3’(SEQ ID NO.3) |
| BS-N4-R | 5’-CGTAACTTCCGCCTTCTACGCG-3’(SEQ ID NO.4) |
| BS-SDC2-F | 5’-AGAAATTAATAAGTGAGAGGGCGTCGC-3’(SEQ ID NO.5) |
| BS-SDC2-R | 5’-AACGCTCGCTTCCTCCTCCTACG-3’(SEQ ID NO.6) |
| BS-ACT-F | 5’-GAAAGGGTGTAGTTTTGGGAGGTTAG-3’(SEQ ID NO.7) |
| BS-ACT-R | 5’-CCCAAAACCAACCACAAAAAAAT-3’(SEQ ID NO.8) |
In table 1, primer pair BS-S9-F and BS-S9-R are for expanding SEPT9 gene；Primer pair BS-N4-F and BS-N4-R
For expanding NDRG4 gene；Primer pair BS-SDC2-F and BS-SDC2-R for expanding SDC2 gene, primer pair BS-ACT-F and
BS-ACT-R is for expanding ACTB gene.
The component of probe compositions is listed in Table 2 below.
Table 2: the probe compositions of embodiment 1
In table 2, BS-S9-P probe SEPT9 gene for identification, BS-N4-P NDRG4 gene for identification；BS-SDC2-P
SDC2 gene for identification, BS-ACT-P ACTB gene for identification.
The component of closing sequence composition is listed in Table 3 below.
Table 3: the closing sequence composition of embodiment 1
| BS-S9-C | 5 '-TTTGTATTGTAGGAGTGTGGGT-3 ' phosphorylation modification (SEQ ID NO.13) |
| BS-N4-C | 5 '-GTTTTGTGTTGTGGTTTTTGTTC-3 ' phosphorylation modification (SEQ ID NO.14) |
| BS-SDC2-C | 5 '-AGAAATTAATAAGTGAGAGGGCGTCGC-3 ' phosphorylation modification (SEQ ID NO.15) |
In table 3, BS-S9-C is for closing SEPT9 gene, and BS-N4-C is for closing NDRG4 gene；BS-SDC2-C is used for
Close SDC2 gene.
Fig. 1 is the operation principles of the kit of embodiment 1: when target gene does not methylate, closing sequence is better than primer
In conjunction with its sequence after converting, to block the combination of primer and wild type transforming sequence.It methylates when target gene exists,
Primer and probe is in conjunction with its sequence after converting, during PCR, in archaeal dna polymerase 5 ' to the effect of 3 ' end 5 prime excision enzyme activities
Under, the fluorescent reporter group (FAM, ROX, CY5, HEX) that probe 5 ' is held is separated with 3 ' ends quenching group (BHQ1, BHQ2), is generated
Fluorescence signal；If there is no methylations, primer and probe it is anti-that amplification is not present in conjunction with template for target gene in sample
It answers, therefore fluorescence signal will not be generated.
Embodiment 2:
A kind of application of the kit of embodiment 1 in detection colorectal cancer, application method the following steps are included:
Sampling: for acquisition 5~8g fecal sample as in the fecal sample protection liquid of 15mL, fecal sample protects the ingredient of liquid
It is listed in Table 4 below.
Table 4: fecal sample protects liquid component list
| Component | Concentration |
| tris-HCl | 0.5M |
| Guanidinium isothiocyanate | 1.5M |
| EDTA | 10mM |
| Lauryl sodium sulfate SDS | 0.2% (m/v) |
| Triton x-100 | 1% (v/v) |
| Polysorbas20 | 1% (v/v) |
| Glycerol | 5% (v/v) |
(2) it extracts: reagent is extracted using the Jin Maige excrement genome of people and scientific and technological (Changsha) Co., Ltd of future biological
Box (paramagnetic particle method) extracts, the specific steps are as follows:
2.1, fecal sample (about 20mL) sufficiently oscillation is mixed to homogenate state, 3000g is centrifuged 15min, shifts 10mL
For supernatant into another 50mL centrifuge tube, remaining Sample preservation is spare.
2.2,10mL Lysis Buffer is added in centrifuge tube, is mixed by inversion, 55 DEG C of water-baths are incubated for 20min.
2.3,30 μ L Acryl Carrier, 240 μ L Proteinase Ks are added, 60 μ L magnetic beads mix in vertical blending instrument
30min。
2.4,3000g is centrifuged 3min, and supernatant is poured into waste liquid cylinder, remains about 1.8mL supernatant.
2.5, it after mixing well surplus solution and magnetic bead, is transferred in another 2mL centrifuge tube, is placed on magnetic frame and adsorbs
To clarification, inhales and abandon supernatant.
2.6,800 μ L wash buffer 1 are added, sufficiently oscillation mixes, and stands 1min, is placed on magnetic frame and is adsorbed to
Clarification inhales and abandons supernatant.
2.7,500 μ L wash buffer 2 are added, sufficiently oscillation mixes, and stands 1min, is placed on magnetic frame and is adsorbed to
Clarification inhales and abandons supernatant.
2.8, step 2.7 is repeated, inhales and abandons raffinate.
2.9,800 μ L wash buffer 3 are added, inhale abandoning supernatant after standing 1min.
2.10,60 μ L elution buffer, 55 DEG C of water-bath 15min are added.
2.11, high speed brief centrifugation is placed on magnetic frame and is adsorbed to solution clarification, draws supernatant into new centrifuge tube,
Gained is the DNA extracted.
(3) it converts: using the conversion reagent box EZ-96DNA Methylation-Gold MagPrep (goods of ZYMO company
Number: bisulfite conversion and purifying D5042) are carried out to the faeces DNA of extraction.Conversion processing is to convert unmethylated C
For U, so that methylated DNA fragments and non-methylated DNA fragments are able in post-conversion, there are the differences in sequence, to be examined
It surveys, the two its sequence when not carrying out conversion processing is identical.
SEPT9 wild type converts presequence (SEQ ID NO.16):
TTCATTCAGCTGAGCCAGGGGGCCTAGGGGCTCCTCCGGCGGCTAGCTCTGCACTGCAGGAGCGCGGG
CGCGGCGCCCCAGCCAGCGCGCAGGGCCCGGGCCCCGCCGGGGGCGCTTCCTCGCCGCTGCCCTCCGCGCGACCCG
CTGCCCACCAGCCATCATGTCGGACCCCGCGGTCAACGCGCAGCTGGATGGGATCATTTCGGACTTCGAAGGTGGG
TGCTGGGCTGGCTGCTGCGGCCGCGGACGTGCTGGAGAGGACCCTGCGGGTGGGCCTGGCGCGGGACGGGGGTGCG
CTGAGGGGAGACGGGAGTGCGCTGAGGGGAGACGGGAC。
Sequence (SEQ ID NO.17) after the conversion of SEPT9 methylated genes:
TTTATTTAGTTGAGTTAGGGGGTTTAGGGGTTTTTTCGGCGGTTAGTTTTGTATTGTAGGAGCGCGGG
CGCGGCGTTTTAGTTAGCGCGTAGGGTTCGGGTTTCGTCGGGGGCGTTTTTTCGTCGTTGTTTTTCGCGCGATTCG
TTGTTTATTAGTTATTATGTCGGATTTCGCGGTTAACGCGTAGTTGGATGGGATTATTTCGGATTTCGAAGGTGGG
TGTTGGGTTGGTTGTTGCGGTCGCGGACGTGTTGGAGAGGATTTTGCGGGTGGGTTTGGCGCGGGACGGGGGTGCG
TTGAGGGGAGACGGGAGTGCGTTGAGGGGAGACGGGAT。
NDRG4 methylated genes convert presequence (SEQ ID NO.18):
GGGCCTCGCAGCGCACCCAGCACAGTCCGCGCGGCGGAGCGGGTGAGAAGTCGGCGGGGGCGCGGATC
GACCGGGGTGTCCCCCAGGCTCCGCGTCGCGGTCCCCGCTCGCCCTCCCGCCCGCCCACCGGGCACCCCAGCCGCG
CAGAAGGCGGAAGCCACGCGCGAGGGACCGCGGTCCGTCCGGGACTAGCCCCAGGCCCGGCACCGCCCCGCGGGCC
GAGCG。
Sequence (SEQ ID NO.19) after the conversion of NDRG4 methylated genes:
GGGTTTCGTAGCGTATTTAGTATAGTTCGCGCGGCGGAGCGGGCGAGAAGTCGGCGGGGGCGCGGATC
GATCGGGGTGTTTTTTAGGTTTCGCGTCGCGGTTTTCGTTCGTTTTTTCGTTCGTTTATCGGGTATTTTAGTCGCG
TAGAAGGCGGAAGTTACGCGCGAGGGATCGCGGTTCGTTCGGGATTAGTTTTAGGTTCGGTATCGTTTCGCGGGTC
GAGCG。
SDC2 methylated genes convert presequence (SEQ ID NO.20):
TCCGCGGAGGAGCAAAACCACAGCAGAGCAAGAAGAGCTTCAGAGAGCAGCCTTCCCGGAGCACCAAC
TCCGTGTCGGGAGTGCAGAAACCAACAAGTGAGAGGGCGCCGCGTTCCCGGGGCGCAGCTGCGGGCGGCGGGAGCA
GGCGCAGGAGGAGGAAGCGAGCGCCCCCGAGCCCCGAGCCCGAGTCCCCGAGCCTGAGCCGCAATCGCTGCGGTAC
TCTGCTCCGGATTCGTGTGCGCGGGCTGCGCCGAGCGCTGGGCAGGAGGCTTCGTTTTGCCCTGGTTGCAAGCAGC
GGCTGGGAGCAGCCGGTCCCTGGGGAATATGCGGCGC。
Sequence (SEQ ID NO.21) after the conversion of SDC2 methylated genes:
TTCGCGGAGGAGTAAAATTATAGTAGAGTAAGAAGAGTTTTAGAGAGTAGTTTTTTCGGAGTATTAAT
TTCGTGTCGGGAGTGTAGAAATTAATAAGTGAGAGGGCGTCGCGTTTTCGGGGCGTAGTTGCGGGCGGCGGGAGTA
GGCGTAGGAGGAGGAAGCGAGCGTTTTCGAGTTTCGAGTTCGAGTTTTCGAGTTTGAGTCGTAATCGTTGCGGTAT
TTTGTTTCGGATTCGTGTGCGCGGGTTGCGTCGAGCGTTGGGTAGGAGGTTTCGTTTTGTTTTGGTTGTAAGTAGC
GGTTGGGAGTAGTCGGTTTTTGGGGAATATGCGGCGT。
(4) it detects: nucleic acid of the 5 μ L after step (3) conversion being taken to carry out fluorescence quantitative PCR detection, detection architecture total volume
For 30 μ L, ingredient specific as follows is listed in the table below in 5.
Table 5: the component of detection architecture and scale is used
| Component | Dosage |
| 2x 360Master Mix | 15μL |
| Each primer | 0.2μM |
| Each probe | 0.15μM |
| Tetramethyl ammonium chloride | 20mM |
| Glycerol | 10% (v/v) |
| Each closing sequence | 0.1μM |
| Deionized water | 4.2μL |
| Template | 5μL |
Amplification program are as follows: 50 DEG C of initial denaturation 2min；95 DEG C of denaturation 10min；95 DEG C of denaturation 15s, 60 DEG C of annealing 30s, fluorescence
Acquisition (acquires fluorescence) since first circulation, recycles 45 times；20 DEG C, 2min.
(5) interpretation of result:
Substantially normal for feminine gender with colonoscopy detection, colonoscopy detection polyp is simultaneously verified as adenoma by pathological examination or cancer is
The positive, the goldstandard as detection compare.100 fecal samples are selected, wherein positive sample 50, negative sample 50.With
The amplification Ct value of SEPT9, NDRG4, SDC2, ACTB are that input carries out logistic regression fitting.
Logistic regression formula used is as follows:
P=eScore/(1+eScore),
Score=w1 × Ct1+w2 × Ct2+w3 × Ct3+w4 × Ct4+B.
Wherein, P is Risk of Colorectal Cancer index,
E is natural constant,
W1, w2, w3, w4, B are constant,
Ct1 is that SEPT9 expands Ct value,
Ct2 is that NDRG4 expands Ct value,
Ct3 is that SDC2 expands Ct value,
Ct4 is that ACTB expands Ct value.
W1 therein, w2, w3, the specific value of w4, B are determined by 100 clinical samples, and are analyzed by ROC curve
It selects the whole highest P value of coincidence rate as the threshold value for judging whether there is Risk of Colorectal Cancer, obtains prediction model.The following table 6
It is w1, w2 after 100 clinical samples determine, w3, w4, the specific value of B, P.
The specific value table of table 6:w1, w2, w3, w4, B, P
| w1 | w2 | w3 | w4 | B |
| -0.14867 | -0.12201 | -0.21562 | 0.20329 | 13.68188 |
Fig. 2 is that model ROC curve analyzes result.As can be known from Fig. 2: when P value is selected as 0.362, can obtain highest
Accuracy rate, wherein sensitivity 0.962, specificity is 0.732, so given threshold is 0.362.
When pattern detection, which calculates the P value reached, is greater than or equal to 0.362, it is judged as positive；When less than 0.362, judgement
For feminine gender.
Embodiment 3:
Investigate sensitivity, specificity and the accuracy rate of the method for embodiment 2.Based on the prediction model of embodiment 2,
It is predicted in 90 newly-increased detection samples, and is compared with actual value, obtain sample confusion matrix.The following table 7 is sample moment
Battle array.
Table 7: sample matrix table
According to sensitivity, specificity and the accuracy rate of the predicted value of table 7 and the prediction model of calculated with actual values the application.
Wherein, sensitivity=true positives/(true positives+false negative) × 100%；
Specificity=true negative/(true negative+false positive) × 100%；
Accuracy rate=(true positives+true negative)/detection sum × 100%.
As can be known from the results of Table 7, the prediction model of embodiment 2, sensitivity 0.8857；Specificity is 0.8909；Accurately
Rate is 0.8889；Kappa is 0.7686.
Fig. 3 is pattern detection internal standard curve synoptic diagram；Fig. 4 is positive sample detection curve schematic diagram.
Embodiment 4: specific detection
After wild-type nucleic acid and methylated nucleic acid conversion, still there is high similarity degree, primed probe is still
Non-specific amplification can be may cause in conjunction with the template of wild type.The present embodiment investigation is not added with closing sequence and is added to envelope
Close two kinds of kits of sequence, specific outcome.
Fig. 5 is not seal up wild-type amplification curve when closing sequence；Fig. 6 is that wild type expands when closing sequence and additive is added
Increase curve.As can be seen from Figure 5: when being not added with closing sequence, expand pure wild-type sequence can exist it is faint non-specific
Amplification.As can be seen from Figure 6: after closing sequence and additive is added, non-specific amplification is eliminated, and does not influence this hair
Bright minimum detection limit.
Embodiment 5: sensitivity technique
According to the method for embodiment 2, before conversion, wild type tissue sample DNA (feminine gender) is taken, with CRC cell line dna
(positive) is diluted to 2.5ng/ μ l respectively.By the wild type tissue sample DNA after dilution with CRC cell line dna according to volume ratio
Ratio for 99 ︰ 1 is mixed to get mixed solution.Then 50ng mixed solution is taken to be converted according to the method for embodiment 2,25 μ l
Elution, then 5 μ l (that is, DNA before 10ng conversion) is taken to carry out fluorescence quantitative PCR detection.Testing result is referring to Fig. 7 to figure
10。
Fig. 7 is internal standard ACTB detection curve figure, and Fig. 8 to Figure 10 is respectively SEPT9, and the sensitive amplification of NDRG4, SDC2 are bent
Line chart.It as seen from the figure, is the present invention in the case that 10ng/ reaction target gene methylation ratio is 1% in DNA detection level
Provided reagent can well detect target gene, therefore detection reagent provided by the invention its sensitivity can be low
It methylates up to the target gene in 10ng/ reaction containing 1%, that is, methylates in the sample of nucleic acid of 2ng/ μ L containing 1% target gene.
Embodiment 6: genescreen research
It is selected into KRAS, BMP3, SEPT9, totally 5 genes are pressed as candidate colorectal cancer screening marker by SDC2, NDRG4
It is detected according to the method for embodiment 2.In the research of 100 clinical samples, all genes are carried out using quantitative fluorescent PCR
Detection, obtains the Ct value of each genetic test.It returns progress characteristic value using LASSO to select, as a result referring to table 8.
Table 8: different colorectal cancer screening marker investigation tables
| Gene | Coefficient (coefficient) |
| KRAS | 0 |
| SEPT9 | -0.30444915 |
| BMP3 | 0 |
| NDRG4 | -0.12050771 |
| SDC2 | -0.07247555 |
| ACTB | 0.14267033 |
As can be known from Table 8, KRAS and BMP3 is 0 to model contribution degree, so giving up two markers of KRAS and BMP3.
Embodiment 7: marker combination research
Unlike signal object is combined, the model and ROC curve of various combination is investigated, corresponding AUC is calculated
Value determines optimal marker combination.Specific combination and investigation result are referring to the following table 9.
Table 9: the model AUC value of unlike signal object combination
| Combination | AUC |
| KRAS+BMP3+NDRG4+SEPT9+SDC2 | 0.912 |
| BMP3+NDRG4+SEPT9+SDC2 | 0.862 |
| NDRG4+SETP9+SDC2 | 0.909 |
| SETP9+SDC2 | 0.701 |
| NDRG4+SETP9 | 0.735 |
As can be known from Table 9: with the model of KRAS+BMP3+NDRG4+SEPT9+SDC2 combination and with NDRG4+SETP9+
SDC2 combination model, Detection accuracy highest, for simplified model and guarantee Detection accuracy balance, determine SEPT9,
NDRG4, SDC2 joint-detection.
The above described is only a preferred embodiment of the present invention, being not intended to limit the present invention in any form.Though
So the present invention is disclosed as above with preferred embodiment, and however, it is not intended to limit the invention.It is any to be familiar with those skilled in the art
Member, in the case where not departing from Spirit Essence of the invention and technical solution, all using in the methods and techniques of the disclosure above
Appearance makes many possible changes and modifications or equivalent example modified to equivalent change to technical solution of the present invention.Therefore,
Anything that does not depart from the technical scheme of the invention are made to the above embodiment any simple according to the technical essence of the invention
Modification, equivalent replacement, equivalence changes and modification, all of which are still within the scope of protection of the technical scheme of the invention.
Sequence table
<110>people and scientific and technological (Changsha) Co., Ltd of future biological
<120>kit for colorectal cancer detection and its application
<160> 21
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
tttgtattgt aggagcgcgg gc 22
<210> 2
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
caacgacgaa aaaacgcccc cg 22
<210> 3
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
gtttcgcgtc gcggttttcg ttc 23
<210> 4
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
cgtaacttcc gccttctacg cg 22
<210> 5
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
agaaattaat aagtgagagg gcgtcgc 27
<210> 6
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
aacgctcgct tcctcctcct acg 23
<210> 7
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
gaaagggtgt agttttggga ggttag 26
<210> 8
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
cccaaaacca accacaaaaa aat 23
<210> 9
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
cgttttagtt agcgcgtagg gttcggg 27
<210> 10
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
gttttttcgt tcgtttatcg ggtattttag tcg 33
<210> 11
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
ttttcggggc gtagttgcgg gcgg 24
<210> 12
<211> 33
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<213>artificial sequence (Artificial Sequence)
<400> 12
cctcttctaa taaccacctc cctccttcct aac 33
<210> 13
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
tttgtattgt aggagtgtgg gt 22
<210> 14
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
gttttgtgtt gtggtttttg ttc 23
<210> 15
<211> 27
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<213>artificial sequence (Artificial Sequence)
<400> 15
agaaattaat aagtgagagg gcgtcgc 27
<210> 16
<211> 334
<212> DNA
<213>people (human)
<400> 16
ttcattcagc tgagccaggg ggcctagggg ctcctccggc ggctagctct gcactgcagg 60
agcgcgggcg cggcgcccca gccagcgcgc agggcccggg ccccgccggg ggcgcttcct 120
cgccgctgcc ctccgcgcga cccgctgccc accagccatc atgtcggacc ccgcggtcaa 180
cgcgcagctg gatgggatca tttcggactt cgaaggtggg tgctgggctg gctgctgcgg 240
ccgcggacgt gctggagagg accctgcggg tgggcctggc gcgggacggg ggtgcgctga 300
ggggagacgg gagtgcgctg aggggagacg ggac 334
<210> 17
<211> 334
<212> DNA
<213>people (human)
<400> 17
tttatttagt tgagttaggg ggtttagggg ttttttcggc ggttagtttt gtattgtagg 60
agcgcgggcg cggcgtttta gttagcgcgt agggttcggg tttcgtcggg ggcgtttttt 120
cgtcgttgtt tttcgcgcga ttcgttgttt attagttatt atgtcggatt tcgcggttaa 180
cgcgtagttg gatgggatta tttcggattt cgaaggtggg tgttgggttg gttgttgcgg 240
tcgcggacgt gttggagagg attttgcggg tgggtttggc gcgggacggg ggtgcgttga 300
ggggagacgg gagtgcgttg aggggagacg ggat 334
<210> 18
<211> 225
<212> DNA
<213>people (human)
<400> 18
gggcctcgca gcgcacccag cacagtccgc gcggcggagc gggtgagaag tcggcggggg 60
cgcggatcga ccggggtgtc ccccaggctc cgcgtcgcgg tccccgctcg ccctcccgcc 120
cgcccaccgg gcaccccagc cgcgcagaag gcggaagcca cgcgcgaggg accgcggtcc 180
gtccgggact agccccaggc ccggcaccgc cccgcgggcc gagcg 225
<210> 19
<211> 225
<212> DNA
<213>people (human)
<400> 19
gggtttcgta gcgtatttag tatagttcgc gcggcggagc gggcgagaag tcggcggggg 60
cgcggatcga tcggggtgtt ttttaggttt cgcgtcgcgg ttttcgttcg ttttttcgtt 120
cgtttatcgg gtattttagt cgcgtagaag gcggaagtta cgcgcgaggg atcgcggttc 180
gttcgggatt agttttaggt tcggtatcgt ttcgcgggtc gagcg 225
<210> 20
<211> 333
<212> DNA
<213>people (human)
<400> 20
tccgcggagg agcaaaacca cagcagagca agaagagctt cagagagcag ccttcccgga 60
gcaccaactc cgtgtcggga gtgcagaaac caacaagtga gagggcgccg cgttcccggg 120
gcgcagctgc gggcggcggg agcaggcgca ggaggaggaa gcgagcgccc ccgagccccg 180
agcccgagtc cccgagcctg agccgcaatc gctgcggtac tctgctccgg attcgtgtgc 240
gcgggctgcg ccgagcgctg ggcaggaggc ttcgttttgc cctggttgca agcagcggct 300
gggagcagcc ggtccctggg gaatatgcgg cgc 333
<210> 21
<211> 333
<212> DNA
<213>people (human)
<400> 21
ttcgcggagg agtaaaatta tagtagagta agaagagttt tagagagtag ttttttcgga 60
gtattaattt cgtgtcggga gtgtagaaat taataagtga gagggcgtcg cgttttcggg 120
gcgtagttgc gggcggcggg agtaggcgta ggaggaggaa gcgagcgttt tcgagtttcg 180
agttcgagtt ttcgagtttg agtcgtaatc gttgcggtat tttgtttcgg attcgtgtgc 240
gcgggttgcg tcgagcgttg ggtaggaggt ttcgttttgt tttggttgta agtagcggtt 300
gggagtagtc ggtttttggg gaatatgcgg cgt 333
Claims (10)
1. a kind of kit for colorectal cancer detection, which is characterized in that including Primer composition, probe compositions；
The Primer composition includes for detecting the primer pair BS-S9-F and BS-S9-R of SEPT9 gene, for detecting NDRG4
The primer pair BS-N4-F and BS-N4-R of gene, primer pair BS-SDC2-F and BS-SDC2-R for detecting SDC2 gene, use
In detection ACTB gene primer pair BS-ACT-F and BS-ACT-R,
The DNA sequence dna of the BS-S9-F as shown in SEQ ID NO.1,
The DNA sequence dna of the BS-S9-R as shown in SEQ ID NO.2,
The DNA sequence dna of the BS-N4-F as shown in SEQ ID NO.3,
The DNA sequence dna of the BS-N4-R as shown in SEQ ID NO.4,
The DNA sequence dna of the BS-SDC2-F as shown in SEQ ID NO.5,
The DNA sequence dna of the BS-SDC2-R as shown in SEQ ID NO.6,
The DNA sequence dna of the BS-ACT-F as shown in SEQ ID NO.7,
The DNA sequence dna of the BS-ACT-R is as shown in SEQ ID NO.8；
The probe compositions include BS-S9-P, BS-N4-P, BS-SDC2-P and BS-ACT-P,
The DNA sequence dna of the BS-S9-P as shown in SEQ ID NO.9,
The DNA sequence dna of the BS-N4-P as shown in SEQ ID NO.10,
The DNA sequence dna of the BS-SDC2-P as shown in SEQ ID NO.11,
The DNA sequence dna of the BS-ACT-P is as shown in SEQ ID NO.12.
2. kit according to claim 1, which is characterized in that the kit further includes closing sequence composition, institute
Stating closing sequence composition includes BS-S9-C, BS-N4-C and BS-SDC2-C,
The DNA sequence dna of the BS-S9-C is as shown in SEQ ID NO.13；
The DNA sequence dna of the BS-N4-C is as shown in SEQ ID NO.14；
The DNA sequence dna of the BS-SDC2-C is as shown in SEQ ID NO.15.
3. kit according to claim 2, which is characterized in that in the closing sequence composition, the BS-S9-C,
3 ' the ends of the BS-N4-C and BS-SDC2-C carry out phosphorylation modification.
4. kit according to any one of claim 1 to 3, which is characterized in that in the probe compositions,
The BS-S9-P is marked using FAM and BHQ1；
And/or the BS-N4-P is marked using ROX and BHQ2；
And/or the BS-SDC2-P is marked using CY5 and BHQ2；
And/or the BS-ACT-P is marked using HEX and BHQ1.
5. the kit according to any one of claim 2 to 3, which is characterized in that each primer in the Primer composition
Concentration be 0.2 μM；
And/or the concentration of each probe is 0.15 μM in the probe compositions；
And/or respectively closing the concentration of sequence in the closing sequence composition is 0.1 μM.
6. kit according to any one of claim 1 to 3, which is characterized in that the kit further includes 2x
360Master Mix, tetramethyl ammonium chloride, glycerol and deionized water；The concentration of the tetramethyl ammonium chloride is 10~40mM；Institute
The volumetric concentration for stating glycerol is 8%~20%.
7. a kind of application of kit described in any one of claims 1 to 6 in detection colorectal cancer.
8. application according to claim 7, which is characterized in that the application method the following steps are included:
(1) fecal sample is dissolved in fecal sample protection liquid, carries out faeces DNA extraction；
(2) faeces DNA of extraction is subjected to the faeces DNA after bisulfite is converted；
(3) with kit will carry out fluorescence quantitative PCR detection by resulting faeces DNA after conversion, with SEPT9, NDRG4, SDC2,
The amplification Ct value of ACTB, logically regression formula calculates P value, the logistic regression formula are as follows: P=eScore/(1+eScore),
Score=w1 × Ct1+w2 × Ct2+w3 × Ct3+w4 × Ct4+B；
Wherein, P is Risk of Colorectal Cancer index,
E is natural constant,
It is -0.12201, w3 is -0.21562, w4 0.20329, B 13.68188 that w1, which is -0.14867, w2,；
Ct1 is that SEPT9 expands Ct value,
Ct2 is that NDRG4 expands Ct value,
Ct3 is that SDC2 expands Ct value,
Ct4 is that ACTB expands Ct value；
When P value is greater than or equal to 0.362, it is judged as positive；When less than 0.362, it is judged as negative.
9. application according to claim 8, which is characterized in that the response procedures of the fluorescence quantitative PCR detection are as follows: 50 DEG C
Initial denaturation 2min；95 DEG C of denaturation 10min；It then is that a circulation carries out 45 times with 95 DEG C of denaturation 15s, 60 DEG C of annealing 30s；Finally
In 20 DEG C of holding 2min.
10. application according to claim 8 or claim 9, which is characterized in that the ingredient of fecal sample protection liquid includes:
EDTA, 0.1m/v%~0.5m/ of tris-HCl, 1.0M of 0.25M~0.8M~4.0M guanidinium isothiocyanate, 5mM~20mM
The lauryl sodium sulfate of v%, the triton x-100 of 0.5v/v%~3v/v%, 0.5v/v%~3v/v% polysorbas20,
The glycerol of 3v/v%~10v/v%.
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|---|---|---|---|
| CN201811123191.5A CN109207592B (en) | 2018-09-26 | 2018-09-26 | Kit for colorectal cancer detection and application thereof |
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| CN109207592B CN109207592B (en) | 2020-01-10 |
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