CN108179195A - A kind of kit of colorectal cancer extreme early lesion detection, method and its application - Google Patents
A kind of kit of colorectal cancer extreme early lesion detection, method and its application Download PDFInfo
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Abstract
The present invention relates to colorectal cancer lesion detection technologies, more particularly to a kind of kit of colorectal cancer extreme early lesion detection, method and its application, the kit includes three pairs for the specific primer of Septin9 gene promoter region CpG islands joint-detection, specificity fluorescent probe and specificity closing probe；RT PCR react buffer；RT PCR react Taq enzyme；Negative quality-control product；Positive quality control product and separation and the packing box for packing reagent bottle or pipe.The present invention have many advantages, such as to the detection method of colorectal cancer lesion high specificity, high sensitivity, it is contaminated it is small, security performance is high, testing result have preferable accuracy and repeatability.Can lesion whether accurately occur to colorectal cancer to judge, clinical assistant diagnosis is provided, the treatment and prognosis to colorectal cancer patients have important value.
Description
Technical field
The present invention relates to colorectal cancer extreme early lesion detection technologies, and in particular to a kind of colorectal cancer extreme early lesion inspection
The kit of survey, method and its application.
Background technology
Colorectal cancer (colorectal cancer, CRC) is a kind of common malignant tumour, typically occurs in Colon and rectum
Mucosal epithelium position.2016, Cancer Hospital of Chinese Academy of Medical Sciences, National Cancer Center He Jie academician, national tumour registration
2015 Cancer in China statistical data, report display have been delivered in center professor Chen Wanqing report, and China's colorectal cancer in 2015 is newly sent out
Case about 37.63 ten thousand, death about 19.11 ten thousand, occupies first five in malignant tumour.Therefore, China's colorectal cancer is reduced
Morbidity and mortality are very urgent important clinical problem in science.But the early diagnostic rate of current China's colorectal cancer compared with
It is low, hence it is evident that less than American-European countries.Therefore, it popularizes colorectal cancer early screening and realizes that early diagnosis is early controlled, be to improve China's colorectal cancer
Early diagnostic rate, the effective way for reducing colorectal cancer related mortality.
The definition of early stage colorectal cancer refers to be confined to the cancer of Colorectal mucosa layer and submucosa, also referred to as in situ
Cancer in cancer, mucous membrane.It is cancer in mucous membrane to be wherein confined to mucous layer, infiltrates to submucosa but does not invade muscularis propria person is glutinous
Cancer under film.The goldstandard of colorectal cancer early diagnosis at present is fiber Colon and rectum mirror, which has invasive, and requires
Fully and completely INTESTINAL CLEANSING, the inconveniences such as the exposure and calmness of privacy, detection compliance are low.Fecal occult blood testing (FOBT)
It is the standard non-intruding means of the current clinical colorectal cancer early screening recommended, however its sensitivity is relatively low, is usually no more than
50%, specificity is relatively low, it is impossible to realize early screening and the diagnosis of colorectal cancer.
In recent years, numerous studies show the unconventionality expression of epigenetics modification modulate tumor related gene, are colorectal cancers
One of main mechanism in occurrence and development.DNA methylation is one of most common epigenetics modification mode, tumour cell
Middle methylation state of DNA is abnormal, and showing as genome entirety methylation level reduces and specific gene promoter region CpG islands
Methylation level increases extremely.DNA abnormal methylations can promote mutation and the unconventionality expression of tumor-related gene, some suppression cancers
Gene makes generation of the gene expression missing so as to cause tumour due to methylating for promoter region.Existing research is reported in knot
The DNA variation identical with primary tumo(u)r is found in rectal cancer patient peripheral blood, shows the generation to methylate in tumour, the hair of DNA
Process is opened up and lapses to play an important role.
Numerous about the test in laboratory method to methylate, bisulphite modified rear PCR sequencing PCR is considered as the inspection that methylates
The goldstandard looked into.It is a kind of reliability and accurate because this method can disclose the methylation state in each CpG sites of monospecific polyclonal
The very high method of degree, but also have the shortcomings that detecting step complexity, somewhat expensive simultaneously.At present in the methylation state of research gene
In be most widely used mainly methylation status of PTEN promoter, basic principle is with bisulf iotate-treated ctDNA, non-methyl
The cytimidine of change becomes uracil, and the cytimidine to methylate is constant, then for CpG islands regional sequence design height specificity
Primer pair and fluorescent marker probe, institute's cls gene is expanded.By the release of fluorescence signal in amplification procedure and adopt
Collection, discriminatory analysis result.PCR method has very high susceptibility, can detect the minimum methylate DNA chain of accounting in sample, should
Method has the advantages of design is simple, easy to operate, low-cost.
MLH1 genes are that the abnormal blood gene DNA reported earliest in colorectal cancer methylates index, hereafter, in Colon and rectum
In the blood sample of cancer patient, the abnormal methylation of different genes promoter, such as CDKN2A, ALX4, RUNX3 are detected,
The frequency to methylate is obviously higher than normal healthy controls person.The detection of Septin9 gene methylations is earliest commercialization and enters clinical
The methylation detection kit of experiment carries out DNA methylation assay by the plasma dna to colorectal cancer and healthy patients, judges
Whether Septin9 genes methylate, so as to predict whether that colorectal cancer lesion occurs.But Septin9 gene promoters
Many places CpG islands sequence is contained in region, and existing Septin9 gene methylations detection is only with Septin9 gene promoter v2 regions
Sequence is detected at one, can not effectively assess Septin9 gene promoter entirety methylations, and omission factor is higher, so as to
It causes false negative rate high, causes sensitivity relatively low, generally 68% or so, it can not realize the extreme early screening of colorectal cancer.
The present invention integrates Septin9 gene promoter region whole CpG islands sequence using bioinformatics technique
Analysis and scoring, three pairs of specific primers of design, specificity fluorescent probe and specificity closing probe, to Septin9 genes
The methylated DNA fragments of promoter region carry out joint acquisition and detection, can realize extreme early it is special, it is sensitive, accurately screening and examine
Disconnected colorectal cancer, while follow up for the patient and prognosis provide the foundation of science.
At present, there are no the open and relevant Septin9 gene promoters of colorectal cancer extreme early sieving and diagnosis both at home and abroad
The research report of region methylation combined detection kit.
Invention content
In view of the deficiencies of the prior art, the object of the present invention is to provide a kind of reagents of colorectal cancer extreme early lesion detection
Box, method and its application, the kit precisely can sensitively detect Septin9 gene methylation degree, be colorectal cancer
Extreme early screening is laid a good foundation.
For this purpose, the present invention uses following technical scheme：
In a first aspect, the present invention provides a kind of kit of colorectal cancer extreme early lesion detection, the kit packet
Include three couples of specific primer, specificity fluorescent probe and spies for being directed to Septin9 gene promoter region CpG islands joint-detection
Opposite sex closing probe；RT-PCR reacts buffer；RT-PCR reacts Taq enzyme；Negative quality-control product；Positive quality control product and separation are simultaneously
Pack the packing box of reagent bottle or pipe.
It is first public to be methylated journey by joint-detection Septin9 gene promoter regions many places CpG islands in the present invention
Degree, for the kit of extreme early screening colorectal cancer lesion.Pass through the specific primer sequence of unique design, specificity fluorescent
Probe and specificity closing probe, using two step method PCR, are realized in same reaction tube to Septin9 gene promoter areas
The different CpG islands in domain carry out joint-detection, it will be apparent that detection sensitivity and specificity are improved, when greatly shortening detection
Between.
Preferably, the nucleotide sequence packet of the specific primer, specificity fluorescent probe and specificity closing probe
Containing the segment as shown in SEQ ID NO.1-15.
Preferably, the SEQ ID NO：1-4, SEQ ID NO：5-8, SEQ ID NO：Sequence shown in 9-12 is used respectively
The different CpG islands methylation at detection Septin9 gene promoter regions three；
Preferably, the SEQ ID NO：Sequence shown in 13-15 is examined for detecting internal reference ACTB genes for assessing
Whether sample DNA amount meets detection demand during survey.
The nucleotide sequence of the specific primer and specificity fluorescent probe is as follows：
| Primer | Sequence |
| SEQ ID NO：1 | 5’-GTTGTTTGTGTTTGTTAAGT-3’ |
| SEQ ID NO：2 | 5’-FAM-CGCGAACCCTCGAAAAAATCGC-TAMRA-3’ |
| SEQ ID NO：3 | 5’-TTTCCCAAAATAAAACTATA-3’ |
| SEQ ID NO：4 | 5’-GTGATAGTGATTTTTTTGAGGGTTTGTGAG-3’ |
| SEQ ID NO：5 | 5’-GTGGAGTTTTCGGGTTTTAAG-3’ |
| SEQ ID NO：6 | 5’-FAM-CGCCGAAACACCGACTCGACCGC-TAMRA-3’ |
| SEQ ID NO：7 | 5’-ATATCAATAAACAGCTCAAT-3’ |
| SEQ ID NO：8 | 5’-TAAGGTGGTTGAGTTGGTGTTTTGGTGT-3’ |
| SEQ ID NO：9 | 5’-GGTTTTGAGTTATGTGATT-3’ |
| SEQ ID NO：10 | 5’-FAM-CGCGCCGAAAACCGCGACCCGCCCACCG-TAMRA-3’ |
| SEQ ID NO：11 | 5’-AACAAAACTCACCCCAACAA-3’ |
| SEQ ID NO：12 | 5’-TGGTGGGTGGGTTGTGGTTTTTGGTGTGTTTAGTGT-3’ |
| SEQ ID NO：13 | 5’-GTGATGGAGGAGGTTTAGTAAGTT-3’ |
| SEQ ID NO：14 | 5’-CCAATAAAACCTACTCCTCCCTTAA-3’ |
| SEQ ID NO：15 | 5’-HEX-ACCACCACCCAACACACAATAACAAACACA-BHQ1-3’ |
The specificity fluorescent probe and specificity closing probe have modification.
Preferably, the end of specificity fluorescent probe 5 ' is at least modified with FAM, HEX, JOE, Texas Red, Cy5 group
In one kind；
Preferably, the end of specificity fluorescent probe 3 ' is at least modified with BHQ1, BHQ2, BHQ3, DABCYL, TAMRA base
One kind in group；
Preferably, the specificity closing terminal modified arm between having C3 of probe 3 '.
In the present invention, three specificity fluorescent probe modifications of unique design have identical fluorescence generation group and fluorescence to quench
Go out group, and three specificity closing probe modifications have C3 to ask arm, three species specificity fluorescence probes and specificity closing probe mixing
In a detection pipe, for joint-detection.In PCR reaction process, if first does not occur for Septin9 gene promoter regions
Base, then closing probe can specifically bind at non-methylated DNA fragments, 3 ' C3 ' arms in end of modification prevent 3 ' end excision enzymes and
3 ' end polymerase effects, so as to the progress that PCR is blocked to react, without the release of fluorescence signal.Septin9 gene promoters
If region methylates, then specific combination is at place to be detected for specificity fluorescent probe, with the progress that PCR reacts,
Fluorescence signal can be released.The change of fluorescence signal is collected by fluorescence quantitative PCR instrument, is converted into Ct values, you can judge
Septin9 gene methylation degree.Detection method on the market is only with CpG islands at Septin9 gene promoter regions one at present
For sequence to be checked, false negative is high, and sensitivity is low, temporarily without the examination to Septin9 gene promoter regions many places CpG islands joint-detection
Agent box.
Preferably, the kit further includes：RT-PCR reaction buffer, RT-PCR reaction enzymes, ddH2O, negative Quality Control
Product, wild type quality-control product, saltant type quality-control product.
Preferably, RT-PCR reaction buffer pH value is 7.5~8.5, by the Tris-HCl of 20~100mmol/L,
The MgCl of 5~20mmol/L2And the KCl compositions of 0.1~0.5mmol/L.
The RT-PCR reaction enzymes are made of hot start Taq polymerase and dNTPs；
Preferably, the dosage of hot start Taq polymerase is 2~10U；DNTPs is 2~5mmol/L.
The feminine gender quality-control product is the artificial synthesized non-Septin9 gene DNAs that methylate, and uses final concentration of 0.2mM.
The positive quality control product for the HeLa genomic DNAs to methylate, uses final concentration of 0.2mM.
Second aspect, the present invention provide a kind of kit utilized as described in relation to the first aspect for colorectal cancer extreme early disease
Become the method for detection, include the following steps：
1) using plasma dna as detection sample, sulphite conversion is carried out；
2) each component is taken to mix, adds in the DNA sample after 15 μ L conversions, carry out pcr amplification reaction；
3) PCR amplification：Condition for 95 DEG C 5 minutes；95 DEG C 15 seconds, 60 DEG C 30 seconds (acquisitions fluorescence), 45 cycles；Fluorescence leads to
Road selects FAM channels and HEX channels；
4) testing result judges：PCR result judgements are determined by each sense channel Ct values, are compareed in yin and yang attribute effective
In the case of, Ct value≤40 of Ct value≤40, Septin9 genes of internal reference ACTB genes, testing result is determined as the positive；It is interior right
According to the Ct values of Ct value≤40, Septin9 genes of ACTB genes>40, testing result is determined as feminine gender；Internal reference ACTB genes
Ct values>No matter what is worth the Ct values of 40, Septin9 genes, and testing result is judged in vain, to detect again.
The third aspect, a kind of method that the present invention provides described in kit or second aspect as described in relation to the first aspect are used
In the application of colorectal cancer extreme early lesion detection.
Compared with prior art, the device have the advantages that：
(1) kit of the present invention using PCR- fluorescence probes law technology detection Septin9 gene promoter regions whether first
Base not only with good sensitivity and specificity, but also also reduces detection time, improves detection efficiency；
(2) specific primer of kit unique design of the present invention, specificity fluorescent probe and specificity closing probe,
Compared to other detection in Gene Mutation reagents or kit on current domestic and international market, the screening range that methylates can be expanded, dropped
Low omission factor, as long as there is CpG islands at one to methylate, you can detect the positive, testing result have preferably specificity and
Accuracy；
(3) kit of the present invention optimizes detection method, using two-step method PCR, with respect to three-step approach PCR, shortens detection
The step of and the time, operation it is easier, clinical position is more efficient；
(4) kit of the present invention has the detection of colorectal cancer extreme early lesion high specificity, high sensitivity, contaminated
Small, the advantages that security performance is high, testing result have preferable accuracy and repeatability, can be real-time, noninvasive to Colon and rectum
Cancer patient is diagnosed, and recurrence monitoring and curative effect evaluation have important clinical value.
Description of the drawings
Fig. 1 quality-control product amplification curve diagrams of the present invention；
Fig. 2 positive sample amplification curve diagrams of the present invention；
Specific embodiment
The technological means and its effect taken further to illustrate the present invention, below in conjunction with the preferred implementation of the present invention
Example is come the technical solution that further illustrates the present invention, but the present invention is not limited in scope of embodiments.
In the examples where no specific technique or condition is specified, according to the described technology of document in the art or condition,
Or it is carried out according to product description.Reagents or instruments used without specified manufacturer, be can be by regular channel commercially available from
The conventional products of acquisition.
The preparation of 1 colorectal cancer extreme early detection kit of embodiment
PCR reagent：The MgCl of final concentration 0.1M Tris-HCl buffer solutions, 10mmol/L2, 0.5mmol/L KCl mixing
Liquid, 5U Taq archaeal dna polymerases, 2.5mmol/L dNTPs mixed liquor, by 3mL/ branch dispense store.
Specific primer, specificity fluorescent probe and specificity closing probe：The nucleotides sequence is classified as SEQ ID
NO.1-15 reacts for PCR, is dispensed by 0.25mL/ branch.
Negative quality-control product, the artificial synthesized non-Septin9 gene DNAs that methylate, using final concentration of 0.2mM, is pressed
The packing storage of 0.1mL/ branch.
Positive quality control product, the HeLa genomic DNAs to methylate using final concentration of 0.2mM, are dispensed by 0.1mL/ branch and stored up
It deposits.
Mentioned reagent pipe is separated and merged and is packaged in packing box.
2 colorectal cancer extreme early lesion detection of embodiment
Described method includes following steps：
1) take 5 μ L specific primers, specificity fluorescent probe and specificity closing probe mixed liquor, 30 μ LRT-PCR anti-
Reagent is answered, mixes shaken well on ice；
2) DNA sample after the conversion of 15 μ L sulphite, negative quality-control product, positive quality control product are taken respectively, add in step 1)
In reaction tube；
3) PCR reaction conditions are set：95 DEG C 5 minutes；95 DEG C 15 seconds, 60 DEG C 30 seconds (acquisitions fluorescence), 45 cycles；Fluorescence
Channel 1 selects FAM channels, and fluorescence channel 2 selects HEX channels；
4) reaction tube is put into fluorescence quantitative PCR instrument, clicks " beginning ", be detected；
5) PCR reaction results are exported, for analyzing.
3 testing result interpretation of embodiment
107 clinical research samples are detected using kit：12 I phases samples, 16 II phases samples, 45
III phases sample, 34 IV phases samples (having colonoscopy and pathological examination), and compared with colonoscopy testing result, determine Septin9
The best critical value of gene and ACTB genes.
Statistical result is analyzed using international SPSS softwares (20.0 version), it is as a result as follows：
1 data distribution of table is analyzed
It is calculated by SPSS softwares it can be seen that P>0.05, it is selected in data fit normal distribution.
2 Septin9 and ACTB reference values of table calculate analysis
The average value and standard deviation of the Septin9 and ACTB of 107 selected sample measures as can be drawn from Table 2, according to
Formula (1) calculates normal reference value.
According to formula (1) calculate, all 107 clinical research samples Septin9 gene references ranging from Ct≤≤
40.0；The term of reference of ACTB genes for Ct≤≤ 40.0 when, testing result accuracy is optimal, and (sensitivity and specificity are respectively
86.9% and 95.8%), omission factor is minimum.
Reference range
Table 3 and the comparison of colonoscopy result
4 authenticity of table calculates analysis
| Computational methods | Result of calculation |
| Sensitivity=A/ (A+C) * 100% | 86.9% |
| Specificity=D/ (B+D) * 100% | 95.8% |
| Positive predictive value=A/ (A+B) * 100% | 94.5% |
| Negative predictive value=D/ (C+D) * 100% | 89.1% |
| Thick consistent=(A+D)/(A+B+C+D) * 100% | 91.6% |
4 clinical sample of embodiment detects
As shown in Figure 1, negative quality-control product does not expand, no Ct values show that experimental implementation does not pollute；Positive quality control
Product have apparent amplification, show reliable experiment result.
The Ct values of sample Septin9 genes are 28.34 it can be seen from Fig. 2 and table 5, the Ct values of internal reference ACTB genes
It is 32.32, testing result is effective, represents that Septin9 gene promoter regions methylate, and testing result is the positive.
5 positive sample amplification of table
Applicant states that the present invention illustrates the method detailed of the present invention, but not office of the invention by above-described embodiment
It is limited to above-mentioned method detailed, that is, does not mean that the present invention has to rely on above-mentioned method detailed and could implement.Technical field
Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to each raw material of product of the present invention
Addition, selection of concrete mode etc., all fall within protection scope of the present invention and the open scope.
Sequence table
<110>Pu Maide（Beijing）Science and Technology Ltd.
<120>A kind of kit of colorectal cancer extreme early lesion detection, method and its application
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Claims (13)
1. a kind of kit of colorectal cancer extreme early lesion detection, which is characterized in that the kit is directed to including three Duis
Specific primer, specificity fluorescent probe and the specificity closing of Septin9 gene promoter region CpG islands joint-detection are visited
Needle；RT-PCR reacts buffer；RT-PCR reacts Taq enzyme；Negative quality-control product；Positive quality control product and separation simultaneously pack reagent bottle
Or the packing box of pipe.
2. kit according to claim 1, which is characterized in that the specific primer, specificity fluorescent probe and
The nucleotide sequence of specificity closing probe includes the segment as shown in SEQ ID NO.1-15；The SEQ ID NO：1-4,
SEQ ID NO：5-8, SEQ ID NO：Sequence shown in 9-12 is respectively used at detection Septin9 gene promoter regions three not
Tong CpG islands methylation；The SEQ ID NO：Sequence shown in 13-15 is used for for detecting internal reference ACTB genes
Whether sample DNA amount meets detection demand in assessment detection process.
3. kit according to claim 1 or 2, which is characterized in that the specificity fluorescent probe energy specific recognition
Septin9 gene methylation sequences；The specificity closing non-methylated DNA fragments of probe energy specific recognition Septin9 genes.
4. kit according to claim 1 or 2, which is characterized in that the specificity fluorescent probe and specificity are closed
Probe has modification.
5. kit according to claim 4, which is characterized in that the end of specificity fluorescent probe 5 ' is at least modified with
One kind in FAM, HEX, JOE, Texas Red, Cy5 group.
6. kit according to claim 4, which is characterized in that the end of specificity fluorescent probe 3 ' is at least modified with
One kind in BHQ1, BHQ2, BHQ3, DABCYL, TAMRA group.
7. kit according to claim 6, which is characterized in that the specificity closing probe 3 ' is terminal modified to be had between C3
Arm.
8. kit according to claim 1, it is characterised in that the specific primer concentration is 0.1~0.5 μm of ol/
L, specificity fluorescent probe and specificity closing concentration and probe concentration are 0.1~0.4 μm of ol/L.
9. kit according to claim 8, which is characterized in that RT-PCR reaction buffer pH value for 7.5~
8.5, by the Tris-HCl of 20~100mmol/L, the MgCl of 5~20mmol/L2It is formed with the KCl of 0.1~0.5mmol/L.
10. kit according to claim 1, which is characterized in that the RT-PCR reaction enzymes by hot start Taq polymerase and
DNTPs is formed.
11. kit according to claim 10, which is characterized in that the dosage of hot start Taq polymerase is 2~10U；dNTPs
For 2~5mmol/L.
12. kit according to claim 1, which is characterized in that the feminine gender quality-control product is artificial synthesized non-methyl
Change Septin9 gene DNAs, use final concentration of 0.2mM；The positive quality control product for the HeLa genomic DNAs to methylate, makes
With final concentration of 0.2mM.
13. a kind of kit utilized as described in claim 1,2,8, any one of 10 is used to detect the side of colorectal cancer lesion
Method, which is characterized in that include the following steps：
1) using plasma dna as detection sample, sulphite conversion is carried out；
2) each component is taken to mix, adds in the DNA sample after 15 μ L conversions, carry out pcr amplification reaction；
3) PCR amplification：Condition for 95 DEG C 5 minutes；95 DEG C 15 seconds, 60 DEG C 30 seconds (acquisitions fluorescence), 45 cycles；Fluorescence channel selects
Select FAM channels and HEX channels；
4) testing result judges：PCR result judgements are determined by each sense channel Ct values, and effective situation is compareed in yin and yang attribute
Under, Ct value≤40 of Ct value≤40, Septin9 genes of internal reference ACTB genes, testing result is determined as the positive；Internal reference
The Ct values ＞ 40 of Ct value≤40, Septin9 genes of ACTB genes, testing result is determined as feminine gender；Internal reference ACTB genes
Ct values ＞ 40, no matter what is worth the Ct values of Septin9 genes, and testing result is judged in vain, to detect again.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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