CN109355390A

CN109355390A - For detecting kit and its application of colorectal cancer

Info

Publication number: CN109355390A
Application number: CN201811478521.2A
Authority: CN
Inventors: 林有升; 张淑娟; 丁崴
Original assignee: Suzhou Ada Health Care Technology Co Ltd
Current assignee: Suzhou Ada Health Care Technology Co Ltd
Priority date: 2018-12-05
Filing date: 2018-12-05
Publication date: 2019-02-19

Abstract

The invention discloses the kits for detecting colorectal cancer, including oligonucleotides premixed liquid I group nucleotide sequence, the nucleotide sequence includes SEQ ID NO.01, SEQ ID NO.02, SEQ ID NO.03, SEQ ID NO.04, SEQ ID NO.05, SEQ ID NO.06, SEQ ID NO.07, SEQ ID NO.08, SEQ ID NO.09, SEQ ID NO.10.The diagnosis of colorectal cancer can be achieved in the present invention, has high sensitivity, the advantage that specificity is good, detection time is short, popularization degree is high, testing result is stable.

Description

For detecting kit and its application of colorectal cancer

Technical field

The invention belongs to oncology field of biological detection, more particularly, to detect the kit of colorectal cancer and its answer With.

Background technique

Colorectal cancer (CRC) is one of current most common disease, and the annual whole world has about 1,200,000 patients to be diagnosed as tying The carcinoma of the rectum, and have more than 600,000 patients and directly or indirectly die of colorectal cancer.The disease incidence of colorectal cancer can be with the age Increase and increase, for example the Colorectal Cancer the median age of developed country is 70 years old.China's colorectal cancer incidence rate and death Rate increases trend in overall, and disease incidence of the colorectal cancer between 10 years shows as significantly raised trend, and in terms of the death rate, male Property patient equally show as rising year by year trend, and female patient is then opposite tends to be steady.There are about 281.4 ten thousand people for China in 2015 Cancer is died of, colorectal cancer death rate in male and female is the 5th.

5 years survival rates of I phase colorectal cancer are more than 90%, the second stage of 80% or so, and three phases also had 60% or so, but if The Section IV phase is arrived, survival rate only has 10% within 5 years.And the recurrence probability of colorectal cancer has a correlation with when making a definite diagnosis by stages, generally, more It is the colorectal cancer of early detection, recurrence rate will be lower after treatment.In terms of life cycle, 5 years survival rates of I phase patient are reachable 90% or more, and IV phase patient is only slightly larger than 10% 5 years survival rates.

Tumor recurrence and transfer are the major causes of death of colorectal cancer patients, and clinic is temporarily without the treatment of significant effective at present Measure, recurrence or transfer after preventing treatment of colorectal cancer, the adjuvant chemotherapy standardized after treatment and radiotherapy can reduce colorectal cancer Relapse and metastasis or extend time of relapse and metastasis, but the recurrence of tumour can not be avoided even to shift completely, therefore generally faced Bed doctor can suggest that patient inspects periodically.Colorectal cancer CRC has higher recurrence rate simultaneously, implements root value criterion, there is 50% Recurrence may, recurrence rate highest in two years after treatment of colorectal cancer, therefore clinical doctor suggests check one in the every 3-6 of patient months Secondary, being changed to half a year after 2 years is once continued until the 3rd year.

The method and its relevant feature that colorectal cancer occurs at present or recurrence detects include as described below: (1) stool occult blood is tried It tests (Fecal Occult Blood Test, FOBT): having many advantages, such as that convenient, noninvasive, patient acceptance is high, but it examines knot The false positive rate and false negative rate of fruit are higher, easily cause missing inspection or false retrieval；(2) Overall Colonoscope/sigmoidoscopy: being mesh Preceding diagnosis/recurrence detection goldstandard inspection method, the sensibility and specificity of inspection result is higher, but some patientss are examined It looks into process and needs anesthesia or calm, and be likely to occur the complication such as bleeding, perforation (0.1%) in inspection, these checking processes Contraindication that is uncomfortable and checking makes many patients have doubt to the colonoscopy heart is received, and as extensive Colon and rectum For cancer methods for screening, the social medical resource of consuming will be excessively huge；(3) serum such as CEA, CA99 routine tumor-marker Object: it is widely used is monitored with tumour auxiliary diagnosis, recurrence now, detection sensitivity is lower；(4) PET-CT is checked: for disease The patient that feelings are complicated, routine inspection can not clarify a diagnosis can be used as effective auxiliary examination.It is more than the phase to prompt for III for preoperative planning Tumour, to understand, whether there is or not DISTANT METASTASES IN uses；(5) per rectum Intracavitary ultrasonography, Type B ultrasound, CT, MRI: for conventional detection, With conveniently superiority；Specify the depth that lesion invades intestinal wall, the position of the range and DISTANT METASTASES IN that spread to outside wall.

Now studies have found that cancer cell can release Circulating tumor DNA (circulating in rupture and death Tumor DNA, ctDNA).Free Circulating tumor DNA is a kind of extracellular dna, is primarily present in blood (serum or blood plasma), synovial membrane In the body fluid such as liquid and cerebrospinal fluid.CtDNA is released into the mechanism of blood at present still without final conclusion, generally believes the apoptosis of cell and bad now The dead main source for ctDNA in blood.In recent years, research of a large amount of ctDNA detections in diagnosing tumor, treatment is reported.

The Septin9 gene methylation detection kit EpiproColon 2.0 of Epigenomics AG company is that knot is straight Intestinal cancer auxiliary diagnosis provides a good detection means, but too big in the presence of detection blood volume, and detection sensitivity is lower to ask Topic.

In conclusion it is still necessary to providing that a kind of sample to be tested blood volume is few, gene methylation detection examination of high sensitivity Agent box.

Summary of the invention

In view of the problems of the above-mentioned prior art, the purpose of the present invention is to propose to a kind of for detecting the examination of colorectal cancer Agent box.The present invention is to detect the method that blood plasma ctDNA discovery colorectal cancer occurs, by Circulating tumor DNA (ctDNA) methyl Change abnormal highly sensitive detection, the aberrant methylation of multiple colorectal cancer markers can be analyzed simultaneously, to ctDNA methyl Change the abnormal detection sensitivity having down to 0.1%, it can be achieved that early detection, the recurrence of tumour real time monitoring, there is sensitivity The advantage high, specificity is good, detection time is short, popularization degree is high, testing result is stable.

The purpose of the invention will be achieved through the following technical solutions:

For detecting the kit of colorectal cancer, including oligonucleotides premixed liquid I group nucleotide sequence, the nucleotide Sequence SEQ ID NO.01, SEQ ID NO.02, SEQ ID NO.03, SEQ ID NO.04, SEQ ID NO.05, SEQ ID NO.06、SEQ ID NO.07、SEQ ID NO.08、SEQ ID NO.09、SEQ ID NO.10。

In some embodiments, further including includes oligonucleotides premixed liquid II group nucleotide sequence, the nucleotides sequence Column include including SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16、SEQ ID NO.17、SEQ ID NO.08、SEQ ID NO.09、SEQ ID NO.10。

It in some embodiments, further include dissociative DNA methylation solvent.

It in some embodiments, further include PCR detection premixed liquid, PCR detection nucleotide premixed liquid, quality-control product reagent；Institute Stating PCR detection nucleotide premixed liquid includes PCR detection nucleotide premixed liquid I and PCR detection nucleotide premixed liquid II；The PCR Detecting nucleotide premixed liquid I includes first group of nucleotide sequence, and PCR detection nucleotide premixed liquid II includes described the Two groups of nucleotide sequences.

In some embodiments, the 3 ' ends of the nucleotide sequence SEQ ID NO.04 are modified with partition 3；The nucleosides The the 16th, 23,25,32 adenylic acid of acid sequence SEQ ID NO.04 is modified with lock nucleic acid.Lock nucleic acid modification can be with Increase checks segment (blocker) for the binding ability of template, so that negative template is easier to be thwarted.

In some embodiments, the 3 ' ends of the nucleotide sequence SEQ ID NO.15 are modified with partition 3；The nucleosides The the 4th, 8,11,21 adenylic acid of acid sequence SEQ ID NO.15 is modified with lock nucleic acid.

In some embodiments, dissociative DNA methylation solvent include bisulphite modified reagent, DNA protection liquid, De- sulphur liquid, rinsing liquid, eluent, and/or silica bead suspension.

In some embodiments, PCR detection premixed liquid include polymerase buffer, Taq polymerase, divalent sun from Son, and/or dNTPs.

In some embodiments, the quality-control product includes positive quality control product, negative quality-control product, and/or blank quality-control product.

In some embodiments, the positive quality control product includes manual simulation's blood plasma, and/or HCT116 cell line dna；Institute Stating negative quality-control product includes artificial blood plasma, and/or HCT116 cell line dna；The blank quality-control product includes manual simulation's blood plasma.

A kind of kit according to above-mentioned any one is in diagnosis of colorectal carcinoma, colorectal cancer real-time monitoring or postoperative Application in relapse diagnosis.

In some embodiments, it is detected using Q-PCR detection method, the Q-PCR detection method is triple PCR detection Combination.

In some embodiments, the combination of triple PCR detection include: detection pipe I be SEPTIN9, BCAT1, And ACTB；And/or detection pipe II is IKZF1, SDC2, ACTB.

In some embodiments, the fluorescence signal for using PCR to detect is three channel, the fluorescence of three channels acquisition Signal is respectively 513nm-523nm, 543nm-553nm and 662nm-672nm.

Compared with prior art, the kit provided by the present invention for detection colorectal cancer and its application, the skill reached Art effect is: (1) kit of the invention can be used for the early detection or postoperative recurrence detection of colorectal cancer；(2) false positive Rate is low: protein labeling blood content is also possible to increase under the pathology or physiological condition of non-tumour, and kit of the present invention Target only just will appear in the presence of the cell of canceration and precancerous lesion, false positive probability is low；(3) can reflect in real time swollen The dynamic of tumor: most protein biological identification can have several weeks in blood, and less than two hours of the half-life period of ctDNA, Therefore tumour progresses in real time situation can be presented in detection kit of the present invention；(4) sensibility is higher: dependent on highly sensitive detection skill Art and polygenes methylation markers, detection kit of the present invention can carry out quantitative, qualitative analysis to single copy ctDNA, by This realizes higher detection sensitivity；(5) smaller blood usage amount: patient's interdependence is poor when acquisition blood volume is larger, inspection When surveying 4 target genes using 3 weight PCR amount of blood collected can be reduced to 10ml by 20ml, effectively raise patient according to Sustainability；(6) a more double, re-detection, triple PCR detection can be used same blood volume and detect 3 gene (1 internal reference bases Cause and two target genes), recall rate is improved by multiplex detection while not improving blood sampling volume.

Detailed description of the invention

Fig. 1 is according to some embodiments of the application, BCAT1, and SEPT9 genetic test result is positive pattern detection result Figure；

Fig. 2 is according to some embodiments of the application, SDC2, and IKZF1 genetic test result is positive pattern detection result Figure；

Fig. 3 is according to some embodiments of the application, BCAT1, and SEPT9 genetic test result is negative pattern detection result Figure；

Fig. 4 is according to some embodiments of the application, SDC2, and IKZF1 genetic test result is negative pattern detection result Figure.

Below just in conjunction with the embodiments, the embodiment of the present invention is described in further detail, so that technical solution is more It should be readily appreciated that, grasp.

Specific embodiment

The present invention will be described below by way of specific embodiments, but the present invention is not limited thereto.In following embodiments The experimental method is unless otherwise specified conventional method；The reagent and material unless otherwise specified can be from business Approach obtains, the scope of the patents that following example is not intended to limit the invention, all equivalence enforcements without departing from carried out by the present invention Or change, it is intended to be limited solely by the scope of this patent.

Embodiment 1 is used to detect the kit forms of colorectal cancer

It may include as shown in table 1 that the kit forms of colorectal cancer are detected in the application:

The composition of 1 kit of table

Embodiment 2 is used to detect the kit forms of colorectal cancer

It may include as shown in table 2 that the kit forms of colorectal cancer are detected in the application:

The composition of 2 kit of table

It will be specifically described the composition of each section used in embodiment 1, embodiment 2 below:

It includes: that (1) bisulphite modified reagent contains 50g sodium hydrogensulfite solid that plasma DNA, which modifies reagent, is made Used time need to be dissolved in 1ml deionized water；(2) DNA protects liquid to may include the water-soluble VE being dissolved in dimethyl ether, concentration It can be 2-4M.Wherein, in some embodiments, optimal concentration 3.2M；(3) combining liquid may include different hydrogen sulfate guanidine, Its concentration is 5-8M.Wherein, in some embodiments, optimal concentration can be 6.6M；(4) taking off sulphur liquid may include hydroxide Sodium, concentration 1.0-2.0M.Wherein, in some embodiments, optimal concentration can be 1.6M；(5) rinsing liquid can wrap 75% dehydrated alcohol, Tri-Hcl are included, the concentration of Tri-Hcl can be 8-12mM.Wherein, in some embodiments, optimal dense Degree can be 10.5mM；(6) eluent may include Tri-Hcl, and concentration can be 10mmol/L；(7) bead suspension can Think silica bead suspension, in some embodiments, silica bead suspension can contain 60-100mg/ml silica Magnetic bead.In some embodiments, optimal concentration can be 80mg/ml.

It may include: polymerase buffer that PCR, which detects premixed liquid, it can be to contain 2 × polymerization in some embodiments Enzyme buffer liquid；Taq polymerase, in some embodiments, content can be 0.27U/ μ l；Bivalent cation, in some implementations In example, bivalent cation can be MgCl₂, concentration 24mM；DNTPs concentration can be 4.0mM.

It may include nucleotide sequence and its content as shown in table 3 that PCR, which detects nucleotide premixed liquid I,.

Table 3PCR detects nucleotide premixed liquid I

It may include nucleotide sequence and its content as shown in table 4 that PCR, which detects nucleotide premixed liquid II,.

Table 4PCR detects nucleotide premixed liquid II

Wherein, the end of SEQ ID NO.04 nucleotide sequence 3 ' is modified with partition 3；16th, 23,25,32 adenine core Thuja acid is modified with lock nucleic acid.The end of SEQ ID NO.15 nucleotide sequence 3 ' is modified with partition 3, and the 4th, 8,11,21 gland is fast Purine nucleotide is modified with lock nucleic acid.

Quality-control product reagent includes: (1) positive quality control product reagent: may include manual simulation's blood plasma, HCT116 cell line DNA, DNA concentration 10ng/ml；(2) negative quality-control product reagent: may include manual simulation's blood plasma, Juarket cell line dna, DNA concentration is 10ng/ml；(3) blank quality-control product reagent: can be artificial simulating blood plasma (outsourcing).

The detection method of the kit of the present invention of embodiment 3

1) nucleic acid enriching is carried out to sample to be tested, quality-control product, carry out after elution it is bisulphite modified, after purification Obtain sample to be tested template, quality-control product template；

2) PCR detection is carried out to sample to be tested template, quality-control product template, sample to be tested template, quality-control product template is distinguished It is added in two groups of PCR detection pipes；

3) sample to be tested template, quality-control product template are divided into detection pipe I, detection pipe II and are carried out；

Detection pipe I composition such as table 5:

5 detection pipe I of table composition

PCR premixed liquid	15μl
		PCR detects nucleotide premixed liquid I	1μl
ddH₂O	6μl
		DNA/ quality-control product	8μl

Detection pipe II composition such as table 6:

6 detection pipe II of table composition

4) PCR detection pipe is put into QPCR instrument, it is as follows is related to program:

It 1:35 DEG C -39 DEG C of step, reacts 30 minutes；2:92 DEG C -96 DEG C of step, preferably 94 DEG C are reacted 2 minutes；Step 3: It 54 DEG C -58 DEG C, reacts 40 seconds, then reacts 30 seconds for 94 DEG C, recycle 15 times: 4:65 DEG C of step and react 2 seconds, 56 DEG C are reacted 40 seconds (collect fluorescence), 94 DEG C react 20 seconds, recycle 30 times；5:40 DEG C of step reaction: 30 seconds.

In some specific embodiments, program can be such that 1:37 DEG C of step is reacted 30 minutes；2:94 DEG C of step reaction 2 minutes；3:56 DEG C of step is reacted 30 seconds for reaction 40 seconds, 94 DEG C, is recycled 15 times: 4:65 DEG C of step and is reacted 2 seconds, 56 DEG C and reacts 40 seconds (collect fluorescence), 94 DEG C react 20 seconds, recycle 30 times；5:40 DEG C of step reaction: 30 seconds.

Wherein, the fluorescence signal of detection pipe I detection is three channels, and the fluorescence signal of acquisition is 518nm, 548nm, 667nm；The fluorescence signal of detection pipe II detection is three channels, and the fluorescence signal of acquisition is 518nm, 548nm, 667nm；

Sample to be tested may include that tumor tissues genomic DNA, peripheral blood dissociative DNA, urine dissociative DNA or excreta are thin One of born of the same parents DNA or any combination.

5) detection data is analyzed:

Detect invalid interpretation: I group: the amplification Ct value > 23 or NTC of ACTB has in amplification deta Rn > 0.1 or NC SEPT9, BCAT1 amplification, fluorescence increase deta Rn > 0.1；

II group: the amplification Ct value > 23 or NTC of ACTB has amplification, and fluorescence increases deta Rn deta Rn > 0.1；In NC Any gene of SDC2, IKZF1 has amplification, and fluorescence increases deta Rn > 0.1.

It detects positive interpretation: under the premise of without invalid interpretation is detected, bringing the detection Ct value of 4 target genes into recurrence side Journey: the detection Ct value of 4 genes is brought into regression equation: Y=-42.40+42.76*SEPT9+21.82*IKZF1+21.82* BCAT1+20.59*SDC2, obtained numerical value > 0.

It detects negative interpretation: under the premise of without invalid interpretation is detected, bringing the detection Ct value of 4 target genes into recurrence side Journey: Y=-42.40+42.76*SEPT9+21.82*IKZF1+21.82*BCAT1+20.59*SDC2, obtained numerical value≤0.

Threshold value is more than or equal to for individual gene and is calculated as " 1 ", is calculated as " 0 " less than threshold value；4 target gene threshold values are as follows: SEPT9 For 23.2, BCAT1 24.1, SDC2 23.4, IKZF1 24.5.Reference gene threshold value is 21.0.

The 4 bisulf iotate-treated DNA rate of recovery of embodiment, conversion ratio

Sample to be tested: reference material DNA: tumor tissues are derived from, is smashed between 50-300bp segment, is made using ultrasonic wave It is quantitative with ddPCR

Instrument: Agilent 2200TapeStation, supercentrifuge, PCR instrument, whirlpool concussion instrument, brief centrifugation machine, Refrigerator

Step: (1) taking 200ul EP pipe, sequentially add the reagent in table 7, after cover tightly pipe lid and be vortexed and mix, instantaneously Centrifugation.The amount of DNA of addition is followed successively by 10ng, 20ng, 40ng；

7 reaction system of table

(2) EP pipe is put into PCR instrument, carries out bisulf iotate-treated reaction by following procedure.

8 response procedures of table

(3) DNA is purified

A, in 1.5ml EP pipe, reaction mixture is walked in addition, adds 1.8ml combination liquid, is vortexed and mixes, instantaneously from The heart；B, be added 50ul magnetic bead suspension, rotate 30 minutes, after be placed on magnetic frame, pour out liquid after 30 seconds；C, 100ul drift is added Washing lotion shakes 30s on vortex oscillation instrument, after be placed on magnetic frame, pour out liquid after 30 seconds；D, 200 μ l are added and take off sulphur liquid, room temperature (20-25) place 20min, after be placed on magnetic frame, pour out liquid after 30 seconds；E, 200ul rinsing liquid is added, on vortex oscillation instrument Shake 30s, after be placed on magnetic frame, pour out liquid after 30 seconds；F, last time is repeated；G, be added 40ul eluent, after be placed on magnetic frame, Sucking liquid after 30 seconds；DNA solution as after purification.

(4) rate of recovery calculates

It is quantified using the DNA that ddPCR returns to purification and recovery, the obtained rate of recovery:

The table 9DNA rate of recovery

Input amount (ng)	Yield (ng)	The rate of recovery
			10	6.89	68.9%
20	14.65	73.25%
			40	29.75	74.38%

(5) conversion ratio calculates

The DNA got using purifying learns that C turns the conversion ratio of U after carrying out sanger sequencing, with former alignment.System altogether 474 C are counted, only one C is not changed into U, conversion ratio 99.79%.

Embodiment 5PCR detection architecture performance evaluation

Instrument: ABI 7500, supercentrifuge, water-bath, whirlpool concussion instrument, refrigerator.

Configure various concentration positive reference product: 297copies/10ng, 99copies/10ng, 33copies/10ng, 11copies/10ng。

PCR detection method:

A. detection pipe I: taking a 1.5ml EP pipe, sequentially adds 15 μ l PCR detection premixed liquid, 1 μ l nucleotide premixed liquid I、6μl dd H₂O is vortexed and mixes, brief centrifugation.

Detection pipe II: a 1.5ml EP pipe is taken, 15 μ l PCR premixed liquids, 1 μ l nucleotide premixed liquid II, 6 μ are sequentially added l dd H₂O is vortexed and mixes, brief centrifugation.

B. it will test pipe I, detection pipe II premixed liquid is dispensed into 96 orifice plates, rear addition template DNA and quality-control product.

C. it by 96 orifice plate brief centrifugations, is put into ABI7500 instrument, setting fluorescence quantitative PCR instrument program carries out instead as follows It answers:

1:37 DEG C of step is reacted 30 minutes；2:94 DEG C of step is reacted 2 minutes；3:56 DEG C of step is reacted 40 seconds, 94 DEG C of reactions It 30 seconds, recycles 16 times: 4:65 DEG C of step and reacts to react within 2 seconds, 56 DEG C 40 seconds (collecting fluorescence), 94 DEG C and react 20 seconds, recycle 30 times； 5:40 DEG C of step reaction: 30 seconds.

Wherein, the fluorescence signal of detection pipe I detection is three channels, the fluorescence signal of acquisition be 518nm, 548nm, 667nm；The fluorescence signal of detection pipe II detection is three channels, and the fluorescence signal of acquisition is 518nm, 548nm, 667nm；

(2) testing result

3 weight PCR amplifications are noiseless in detection pipe I, detection pipe II, and gene magnification efficiency is respectively SEPT9:105%； BCAT1:98%；SDC2:108%；IKZF1:105%.

Application of the kit of 6 the application of embodiment in diagnosis of colorectal carcinoma, post operative diagnosis

Sample to be tested: plasma sample (4ml) 40 to be checked, positive quality control product (4ml), negative quality-control product (4ml), blank matter Control product (4ml), detailed sample to be tested are as shown in table 10:

The essential information of 10 sample to be tested of table

Instrument: ABI 7500, supercentrifuge, PCR instrument, water-bath, whirlpool concussion instrument, refrigerator.

Operating method: 1. nucleic acid enrichings: QIAamp Circulating Nucleic Acid (article No. 55114) is used, is pressed Book extracts plasma DNA as directed.Using Agilent 2200TapeStation electrophoresis platform, QC matter is carried out to cfDNA Control.

2 pairs of dissociative DNAs carry out sodium hydrogensulfite processing

(1) take 200ul EP pipe, following reagent be once added, after cover tightly pipe lid and be vortexed and mix, brief centrifugation is anti- Answer system such as table 7；

(2) EP pipe is put into PCR instrument, carries out bisulf iotate-treated reaction by following procedure.Its program is as shown in table 8；

(3) DNA is purified

3.PCR detection

(1) detection pipe I: a 1.5ml EP pipe is taken, 15 μ l PCR premixed liquids, 1 μ l nucleotide premixed liquid I, 6 are sequentially added μl dd H₂O, it is vortexed and mixes, brief centrifugation；

Detection pipe II: a 1.5ml EP pipe is taken, 15 μ l PCR premixed liquids, 1 μ l nucleotide premixed liquid II, 6 μ are sequentially added l dd H₂O, it is vortexed and mixes, brief centrifugation；

(2) will test pipe I, detection pipe II premixed liquid is dispensed into 96 orifice plates, plate-laying is as follows, rear that template DNA and matter is added Control product.

I-S1

I-S9

I-S17

I-N5

I-N13

I-NC

II-S1

II-S9

II-S17

II-N5

II-N13

II-NC

I-S2

I-S10

I-S18

I-N6

I-N14

I-PC

II-S2

II-S10

II-S18

II-N6

II-N14

II-PC

I-S3

I-S11

I-S19

I-N7

I-N15

I-NTC

II-S3

II-S11

II-S19

II-N7

II-N15

II-NTC

I-S4

I-S12

I-S20

I-N8

I-N16

II-S4

II-S12

II-S20

II-N8

II-N16

I-S5

I-S13

I-N1

I-N9

I-N17

II-S5

II-S13

II-N1

II-N9

II-N17

I-S6

I-S14

I-N2

I-N10

I-N18

II-S6

II-S14

II-N2

II-N10

II-N18

I-S7

I-S15

I-N3

I-N11

I-N19

II-S7

II-S15

II-N3

II-N11

II-N19

I-S8

I-S16

I-N4

I-N12

I-N20

II-S8

II-S16

II-N4

II-N12

II-N20

(3) it by 96 orifice plate brief centrifugations, is put into ABI7500 instrument, setting fluorescence quantitative PCR instrument program is reacted；

4. testing result is as shown in table 11 and shown in Fig. 1-Fig. 4:

Testing result of 11 kit of table in postoperative recurrence of colorectal cancer patient

5. testing result discussion

According to interpretation standard, 40 detection ACTB Ct values are both less than 32, and each gene NTC is without amplification, in addition to reference gene is each For gene NC without amplification, each gene PC amplification is normal, and showing detection all is effectively detection, specific testing result such as table 11.Show at this 8 colorectal cancer clinical definite patients, 7 test positive are detected in example property experiment；17 clinical examinations are no colorectal cancer Crowd, 16 are detected as feminine gender；Detect 12 postoperative recurrence of colorectal cancer patients, 10 test positive；3 colorectal cancer arts Non- patients with recurrent afterwards, 3 are all detected as feminine gender.Detecting total sensitivity is 85%, and specificity is 95%.Thus illustrate this hair Bright kit has good detection performance.

Kit of the invention can be also used for the dynamic of real time monitoring colorectal cancer, such as monthly monitor colorectal cancer Progress dynamic.

Sequence table

<110>Suzhou Ida health medical science and technology Co., Ltd

<120>for detecting kit and its application of colorectal cancer

<141> 2018-12-05

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Claims

1. for detecting the kit of colorectal cancer, which is characterized in that including oligonucleotides premixed liquid I group nucleotide sequence, The nucleotide sequence includes SEQ ID NO.01, SEQ ID NO.02, SEQ ID NO.03, SEQ ID NO.04, SEQ ID NO.05、SEQ ID NO.06、SEQ ID NO.07、SEQ ID NO.08、SEQ ID NO.09、SEQ ID NO.10。

2. kit according to claim 1, which is characterized in that including oligonucleotides premixed liquid II group nucleotide sequence Column, the nucleotide sequence includes SEQ ID NO.08, SEQ ID NO.09, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12、SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.16、SEQ ID NO.17。

3. kit according to claim 1, which is characterized in that further include dissociative DNA methylation solvent.

4. kit according to claim 4, which is characterized in that further include PCR detection premixed liquid, PCR detection nucleotide Premixed liquid, quality-control product reagent；The PCR detection nucleotide premixed liquid includes PCR detection nucleotide premixed liquid I and PCR detection core Thuja acid premixed liquid II；The PCR detection nucleotide premixed liquid I includes first group of nucleotide sequence, and the PCR detects nucleosides Sour premixed liquid II includes second group of nucleotide sequence.

5. kit according to claim 1, which is characterized in that 3 ' the end bands of the nucleotide sequence SEQ ID NO.04 There are the modification of partition 3 or phosphorylation modification；The the 16th, 23,25, the 32 adenine core of the nucleotide sequence SEQ ID NO.04 Thuja acid is modified with lock nucleic acid.

6. kit according to claim 4, which is characterized in that 3 ' the end bands of the nucleotide sequence SEQ ID NO.17 There are the modification of partition 3 or phosphorylation modification；The the 4th, 8,11,21 adenosine of the nucleotide sequence SEQ ID NO.15 Acid band has lock nucleic acid modification.

7. kit according to claim 4, which is characterized in that the dissociative DNA methylation solvent includes bisulfite Salt modifies reagent, DNA protection liquid, de- sulphur liquid, rinsing liquid, eluent, and/or silica bead suspension.

8. kit according to claim 4, which is characterized in that PCR detection premixed liquid include polymerase buffer, Taq polymerase, bivalent cation, and/or dNTPs.

9. kit according to claim 4, which is characterized in that the quality-control product includes positive quality control product, negative Quality Control Product, and/or blank quality-control product.

10. kit according to claim 10, which is characterized in that the positive quality control product include manual simulation's blood plasma, And/or HCT116 cell line dna；The feminine gender quality-control product includes artificial blood plasma, and/or HCT116 cell line dna；The blank Quality-control product includes manual simulation's blood plasma.

11. a kind of -10 described in any item kits according to claim 1 diagnosis of colorectal carcinoma, Colon and rectum real-time monitoring or Application in postoperative recurrence diagnosis.

12. application according to claim 11, which is characterized in that detected using Q-PCR detection method, the Q-PCR Detection mode is the combination of triple PCR detection.

13. application according to claim 12, which is characterized in that the combination of the triple PCR detection includes: detection Pipe I is SEPTIN9, BCAT1 and ACTB；And/or detection pipe II is IKZF1, SDC2, ACTB.

14. application according to claim 13, which is characterized in that the fluorescence signal for using PCR to detect is three channels, institute The fluorescence signal for stating the acquisition of three channels is respectively 513nm-523nm, 543nm-553nm and 662nm-672nm.