CN105543378A - NDRG4 gene methylation detection primers, probe and kit for early diagnosis of intestinal cancer - Google Patents
NDRG4 gene methylation detection primers, probe and kit for early diagnosis of intestinal cancer Download PDFInfo
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Abstract
The invention discloses a group of NDRG4 gene methylation detection primers, probe and kit for early diagnosis of an intestinal cancer. NDRG4-1FP and NDRG4-1RP are adopted as the primers, and the nucleotide sequences of the primers are shown as SEQ ID NO.1 and SEQ ID NO.2; NDRG4-2Pb is adopted as the probe, and the nucleotide sequence of the probe is shown as SEQ ID NO.3. A kit containing the primers and the probe is adopted as the kit. According to the NDRG4 gene methylation detection primers, probe and kit for early diagnosis of the intestinal cancer, a set of NDRG4 gene methylation detection systems and procedures are constructed through the primers and the probe, the primers and the probe form the kit, therefore, the high sensibility and specificity on NDRG4 gene methylation detection are achieved, and the early intestinal cancer can be accurately and specifically diagnosed; in addition, a detection method is easy, convenient and rapid to operate, and the very important significance and application prospect on early diagnosing and screening of the intestinal cancer are achieved.
Description
Technical field
The invention belongs to biomedicine technical field.More specifically, relate to one group of NDRG4 gene methylation for intestinal cancer early diagnosis and detect primer and probe and detection kit thereof.
Background technology
Large bowel cancer (colorectalcancer, CRC) be one of clinical modal malignant tumour, in recent years along with expanding economy, the change of the mode of life of people especially dietary structure, intestinal cancer has become one of the fastest malignant tumour of China's sickness rate rising, and the life and health of compatriots in serious threat.And patients with terminal prognosis is very poor, at present, early diagnosis early treatment is carried out to improve the curative ratio of intestinal cancer to intestinal cancer, reduce mortality ratio, become exigence.
DNA methylation is usually the earliest events in tumor development process, and therefore, methylating of specific gene can be used as the molecular marker of early diagnosis of tumor.NDRG4 (N-mycdownstream-reguiatedgene4) is one of cancer suppressor gene NDRG gene family member, NDRG4 gene length 32kb, be made up of 17 exons and 16 introns, large quantity research shows that the growth of this gene and tumour cell, differentiation and transfer have certain relation, CpG island is contained in its gene 5 ' end regulation and control region, usually methylated in colorectal cancer generation evolution, NDRG4 gene methylation is considered to the important biomolecule feature (Veerie etc.) of colorectal cancer.Therefore, NDRG4 gene is the potential source biomolecule mark of candidate tumor suppressor gene and the rectum cancer, and methylating of NDRG4 promotor can be used as biomarker, for early diagnosis colorectal cancer.
And for the detection of NDRG4 gene methylation, detection sensitivity and specificity are especially vital factors, the accurate judgement of intestinal cancer early diagnosis is had great importance.And detection sensitivity and specificity are subject to the impact of many factors, therefore, for ensureing and improving NDRG4 gene methylation detection sensitivity and specific correlative study, be the Focal point and difficult point utilizing this gene to carry out intestinal cancer early diagnosis.
Summary of the invention
The technical problem to be solved in the present invention overcomes deficiency and the defect that intestinal cancer early diagnosis technology is carried out in the existing NDRG4 of utilization gene test, the NDRG4 gene methylation of a kind of hypersensitivity, high specific is provided to detect primer and probe, and detection sample easy to usely can obtain the faecal samples processed, be highly suitable for Clinical screening, the early diagnosis of intestinal cancer is had great importance.
The object of this invention is to provide one group of NDRG4 gene methylation for intestinal cancer early diagnosis and detect primer and probe
Another object of the present invention is to provide a kind of NDRG4 gene methylation detection kit utilizing above-mentioned primer and probe preparation to go out.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
One group of NDRG4 gene methylation being used for intestinal cancer early diagnosis detects primer and probe, and described primer is NDRG4-1FP and NDRG4-1RP, and its nucleotide sequence is respectively as shown in SEQIDNO.1 and 2; Described probe is NDRG4-2Pb, and its nucleotide sequence is as shown in SEQIDNO.3.
For a NDRG4 gene methylation detection kit for intestinal cancer early diagnosis, include above-mentioned primer NDRG4-1FP, NDRG4-1RP and probe NDRG4-2Pb.
Further, mentioned reagent box also comprises when utilizing primer NDRG4-1FP, NDRG4-1RP and probe NDRG4-2Pb to carry out pcr amplification, obtain reagent needed for pcr template, required reagent comprises the capture probe NDRG4-CP catching object fragment when extracting testing sample DNA, and the nucleotide sequence of capture probe NDRG4-CP is as shown in SEQIDNO.4.
Preferably, the working concentration of above-mentioned primer NDRG4-1FP, NDRG4-1RP and probe NDRG4-2Pb is 50 ~ 500 μMs.
More preferably, the working concentration of above-mentioned primer NDRG4-1FP, NDRG4-1RP and probe NDRG4-2Pb is 100 μMs.
Further preferably, above-mentioned detection kit also comprises when utilizing primer NDRG4-1FP, NDRG4-1RP and probe NDRG4-2Pb to carry out pcr amplification, PCR amplification system reagent, comprises the water of dNTP, magnesium ion, reaction buffer (buffer), enzyme, nuclease free.
Preferably, described system reagent comprises the water of the dNTP of 5 ~ 20mM, the magnesium ion of 10 ~ 40mM, 1 × ~ 10 × reaction buffer, enzyme, nuclease free.
More preferably, described system reagent comprises the water of the dNTP of 5 ~ 15mM, the magnesium ion of 15 ~ 35mM, 1 × ~ 10 × reaction buffer, enzyme, nuclease free.
Most preferably, described system reagent comprises the water of the magnesium ion of dNTP, 25mM of 10mM, 5 × reaction buffer (5 × buffer), enzyme, nuclease free.
Preferably, when utilizing mentioned reagent box to carry out the detection of NDRG4 gene methylation, in PCR system, the reaction final concentration of each component is as follows:
More preferably, when utilizing mentioned reagent box to carry out the detection of NDRG4 gene methylation, PCR system is carried out according to following ratio:
More preferably, described enzyme is warm start polysaccharase.As Promega company
warm start enzyme or SIGMA company
warm start enzyme.
In addition, preferably, when utilizing mentioned reagent box to carry out the detection of NDRG4 gene methylation, PCR program is as follows:
95 DEG C of 300s; 1 circulation;
95 DEG C of 20s, 62 DEG C of 30s, 70 DEG C of 30s; 10 circulations;
95 DEG C of 20s, 58 DEG C of 60s, 72 DEG C of 30s; 30 ~ 40 circulations;
37 DEG C of 30s; 1 circulation.
More preferably, when utilizing mentioned reagent box to carry out the detection of NDRG4 gene methylation, PCR program is as follows:
95 DEG C of 300s; 1 circulation;
95 DEG C of 20s, 62 DEG C of 30s, 70 DEG C of 30s; 10 circulations;
95 DEG C of 20s, 58 DEG C of 60s, 72 DEG C of 30s; 35 ~ 40 circulations;
37 DEG C of 30s; 1 circulation.
In addition, preferably, above-mentioned testing sample is ight soil, and mentioned reagent box also comprises ight soil pretreating reagent, and pretreating reagent is grinding buffer solution.
Further preferably, described grinding buffer solution is specially: one or more in the water of nuclease free, physiological saline, TE or PBS.
The above-mentioned primer of the present invention and probe and test kit thereof have good application prospect in the early diagnosis of intestinal cancer.
The present invention has following beneficial effect:
The present invention is directed to the early diagnosis of intestinal cancer, design obtains one group to the extraordinary primer of NDRG4 gene methylation Detection results and probe, and utilize above-mentioned primer and probe, successfully construct a set of NDRG4 gene methylation detection system and program, and set up into test kit, for the detection of NDRG4 gene methylation, there is hypersensitivity and specificity, can the accurate early stage intestinal cancer of specific diagnostic, to the early diagnosis of intestinal cancer with examination has great importance and application prospect.
In addition, primer, probe and detection kit that the present invention is above-mentioned, ight soil can be used as sample, sample process simply, is easily got, and detection method is regular-PCR method, easy and simple to handle, quick, the time of about 75 ~ 90min is only needed to complete detection, without the need to high instrument and testing cost, be highly suitable for detection and basic unit's examination of a large amount of sample.
Accompanying drawing explanation
Fig. 1 is the amplification of primer sets NDRG4-0FP/NDRG4-0RP.
Fig. 2 is the amplification of primer sets NDRG4-1FP/NDRG4-1RP.
Fig. 3 is the amplification of primer sets NDRG4-2FP/NDRG4-2RP.
Fig. 4 is the amplification of primer sets NDRG4-3FP/NDRG4-3RP.
Fig. 5 is the amplification of primer sets NDRG4-4FP/NDRG4-4RP.
Fig. 6 is the amplification of probe NDRG4-1Pb.
Fig. 7 is the amplification of probe NDRG4-2Pb.
Fig. 8 is the amplification of probe NDRG4-3Pb.
Fig. 9 is the detected result of faecal samples.
Embodiment
Further illustrate the present invention below in conjunction with Figure of description and specific embodiment, but embodiment does not limit in any form to the present invention.Unless stated otherwise, the present invention adopts reagent, method and apparatus are the art conventional reagent, method and apparatus.
Unless stated otherwise, agents useful for same of the present invention and material are commercial.
Embodiment 1 primed probe designs
1, for the detection of NDRG4 gene methylation, devise a large amount of primer, after screening, obtain 5 groups of primers as shown in table 1 below.
Table 1 primer sequence
2, probe design
For the detection of NDRG4 gene methylation, devise a large amount of probe, after screening, obtain 3 probes as shown in table 2 below.
Table 2
| Probe title | Sequence (5 ' → 3 ') |
| NDRG4-1Pb | CGGAAGTTACGCGCGAGG |
| NDRG4-2Pb | TCGTTTATCGGGTATTTTAGTCGCGT |
| NDRG4-3Pb | TCGTTCGTTTATCGGGTATTTT |
Embodiment 2 sample detection methods
1, sample process and DNA extraction:
(1) fecal sample and damping fluid are according to 1g ight soil: after the ratio mixed grinding of 4ml damping fluid, centrifugally leave and take supernatant liquor, abandon throw out.
(2) supernatant 5ml is shifted after getting the centrifugal 5min of 8ml supernatant 4000rpm in the new 15ml centrifuge tube that 3ml cell pyrolysis liquid is housed in advance.
(3) in each centrifuge tube, add 100ul and catch magnetic bead, water-bath 92 DEG C hatches 10min; Room temperature shaker 100rpm hatches 1h, and brief centrifugation is placed on magnetic frame 5min, abandons supernatant.
(4) utilize following capture probe, object fragment can be caught.
Capture probe 1:TCCCTCGCGCGTGGCTTCCGCCTTCTGCGCGGCTGGGGTGCCCGGTGG
(5) in 15ml centrifuge tube, add 500ul washings, vibration shakes up, and makes tube wall magnetic bead hang completely, transfers in new 2ml centrifuge tube after brief centrifugation.The dry bath 900rpm of room temperature hatches 1min, is placed in 1min on magnetic frame, abandons supernatant.Repeat 4 times.
(6) add 55 μ l elutriants, brief centrifugation, dry bath 92 DEG C of 900rpm hatch 10min.Brief centrifugation, is placed on magnetic frame, and within 3min, transferase 45 0ul elutriant is in new EP pipe.
(7) to methylate process to the DNA fragmentation in the 6th step with EZmethylationkit (ZymoResearch Products), last sample carries out PCR detection.
2, PCR system and program are respectively as shown in table 3, table 4.
Table 3PCR system
| Composition | Add-on (μ l) |
| Forward primer (FP) (100 μMs) | 0.125 |
| Reverse primer (RP) (100 μMs) | 0.125 |
| Probe (100 μMs) | 0.05 |
| dNTP(10mM) | 1 |
| Magnesium ion (25mM) | 5 |
| 5*buffer | 5 |
| Reaction enzymes | 0.5 |
| The water of nuclease free | 8.2 |
| DNA to be measured | 5 |
| Add up to | 25 |
Table 4PCR program
The optimization of embodiment 3 primed probe
According to the method for embodiment 2, each group of primer utilizing embodiment 1 to design respectively and probe detect, and optimize primer and probe.Result is as follows:
1, primer optimization
As shown in table 5 and accompanying drawing 1 ~ 5, the amplification display of 5 groups of primer sets, optimum primer sets is NDRG4-1FP/NDRG4-1RP.
Table 5
2, probe optimization
As shown in table 6 and accompanying drawing 6 ~ 8, the amplification display of 3 probe groups, optimum probe is NDRG4-2Pb.
Table 6
| Probe title | Sequence (5 ' → 3 ') | Amplification |
| NDRG4-1Pb | CGGAAGTTACGCGCGAGG | As shown in Figure 6 |
| NDRG4-2Pb | TCGTTTATCGGGTATTTTAGTCGCGT | As shown in Figure 7 |
| NDRG4-3Pb | TCGTTCGTTTATCGGGTATTTT | As shown in Figure 8 |
Embodiment 4 test kit is assembled
1, research and analyse through above-mentioned, finally obtain optimum detection primer, probe, and DNA catches
Probe is as shown in table 7 below:
Table 7
2, test kit is assembled
Detection kit comprises following component:
(1) primer NDRG4-1FP and NDRG4-1RP, its nucleotide sequence is respectively as shown in SEQIDNO.1 and 2.
(2) probe NDRG4-2Pb, its nucleotide sequence is as shown in SEQIDNO.3.
(3) capture probe NDRG4-CP, its nucleotide sequence is as shown in SEQIDNO.4.Object fragment is caught when the effect of capture probe NDRG4-CP is and extracts testing sample DNA.
(4) PCR amplification system reagent, the water of the magnesium ion of dNTP, 25mM of 10mM, 5 × reaction buffer (5 × buffer), enzyme, nuclease free.
(5) sample (as ight soil) pretreating reagent: grinding buffer solution.Described grinding buffer solution is specially: one or more in the water of nuclease free, physiological saline, TE or PBS.
3, test kit using method
(1) through a large amount of experiments and research display, when utilizing mentioned reagent box to carry out the detection of NDRG4 gene methylation, best PCR system is carried out according to following ratio:
(2) through a large amount of experiments and research display, when utilizing mentioned reagent box to carry out the detection of NDRG4 gene methylation, best PCR program is as follows:
95 DEG C of 300s; 1 circulation;
95 DEG C of 20s, 62 DEG C of 30s, 70 DEG C of 30s; 10 circulations;
95 DEG C of 20s, 58 DEG C of 60s, 72 DEG C of 30s; 35 ~ 40 circulations;
37 DEG C of 30s; 1 circulation.
Embodiment 5 sample detection
1, three kinds of fecal samples below clinical collection:
(1) 46 routine intestines mirror and pathologic finding are diagnosed as the fecal sample (intestinal cancer, Ca) of patients with bowel cancer;
(2) 46 routine intestines mirrors and pathologic finding are diagnosed as the fecal sample (before cancer adenoma, LA) of the patient of adenoma (polyp diameter >=1cm) before cancer;
(3) 46 routine enteroscopies are diagnosed as the fecal sample (normal, Norm) of normal patient.
2, the test kit utilizing embodiment 4 to set up detects above faecal samples, uses MedCalc software to carry out Treatment Analysis to data.
3, before NDRG4 gene test colorectal cancer and cancer, the result of adenoma is as shown in Figure 9.Wherein, Fig. 9 A is the ROC curve of NDRG4 gene test colorectal cancer; Fig. 9 B is the ROC curve of adenoma before NDRG4 gene test cancer (polyp diameter >=1cm).
Result shows, and for colorectal cancer, mentioned reagent box of the present invention is 65.2% to the detection sensitivity of NDRG4 gene, and specificity is 97.8%, and experimenter's area under curve is 0.826.
For adenoma before cancer (polyp diameter >=1cm), mentioned reagent box of the present invention is 45.7% to NDRG4 gene test susceptibility, and specificity is 97.8%, and experimenter's area under curve is 0.694.
In sum, primer of the present invention and probe and detection kit thereof can not only sensitiveer, checkout and diagnosis colorectal cancers more accurately, especially early stage colorectal cancer, and for adenoma before cancer, also there is good detection sensitivity and very high specificity, before cancer, can just realize the object of checkout and diagnosis by adenoma stage.
SEQUENCELISTING
<110> Guangzhou Kang Li open-birth thing science and technology limited Company
The NDRG4 gene methylation that <120> mono-group is used for intestinal cancer early diagnosis detects primer and probe and test kit thereof
<160>4
<170>PatentInversion3.3
<210>1
<211>20
<212>DNA
<213>PCR forward direction primer NDRG4-1FP
<400>1
cggttttcgttcgttttttc20
<210>2
<211>18
<212>DNA
To primer NDRG4-1RP after <213>PCR
<400>2
cctcgagcgtaacttccg18
<210>3
<211>26
<212>DNA
<213>PCR reacts probe NDRG4-2Pb
<400>3
tcgtttatcgggtattttagtcgcgt26
<210>4
<211>48
<212>DNA
<213> capture probe NDRG4-CP
<400>4
tccctcgcgcgtggcttccgccttctgcgcggctggggtgcccggtgg48
Claims (10)
1. one group of NDRG4 gene methylation being used for intestinal cancer early diagnosis detects primer and probe, and it is characterized in that, described primer is NDRG4-1FP and NDRG4-1RP, and its nucleotide sequence is respectively as shown in SEQIDNO.1 and 2; Described probe is NDRG4-2Pb, and its nucleotide sequence is as shown in SEQIDNO.3.
2. for a NDRG4 gene methylation detection kit for intestinal cancer early diagnosis, it is characterized in that, described detection kit includes primer NDRG4-1FP, NDRG4-1RP described in claim 1 and probe NDRG4-2Pb.
3. detection kit according to claim 2, it is characterized in that, also comprise when utilizing primer NDRG4-1FP, NDRG4-1RP and probe NDRG4-2Pb to carry out pcr amplification, obtain reagent needed for pcr template, required reagent comprises the capture probe NDRG4-CP catching object fragment when extracting testing sample DNA, and the nucleotide sequence of capture probe NDRG4-CP is as shown in SEQIDNO.4.
4. detection kit according to claim 2, it is characterized in that, the working concentration of described primer NDRG4-1FP, NDRG4-1RP and probe NDRG4-2Pb is 50 ~ 500 μMs.
5. detection kit according to claim 2, it is characterized in that, also comprise when utilizing primer NDRG4-1FP, NDRG4-1RP and probe NDRG4-2Pb to carry out pcr amplification, PCR amplification system reagent, comprises the water of dNTP, magnesium ion, reaction buffer, enzyme, nuclease free.
6. detection kit according to claim 5, is characterized in that, described system reagent comprises the water of the dNTP of 5 ~ 20mM, the magnesium ion of 10 ~ 40mM, 1 × ~ 10 × reaction buffer, enzyme, nuclease free.
7. detection kit according to claim 5, is characterized in that, utilize this test kit to carry out NDRG4 gene methylation when detecting, in PCR system, the reaction final concentration of each component is as follows:
Forward primer 0.1 ~ 2 μM
Reverse primer 0.1 ~ 2 μM
Probe 0.1 ~ 1 μM
dNTP0.1～2mM
Magnesium ion 2 ~ 10mM
Reaction buffer 1 × reaction system cumulative volume
Enzyme 0.1U ~ 0.6U/ μ L
Water 1 × reaction system cumulative volume of nuclease free.
8. detection kit according to claim 5, it is characterized in that, described enzyme is warm start polysaccharase.
9. detection kit according to claim 2, is characterized in that, utilize this test kit to carry out NDRG4 gene methylation when detecting, PCR program is as follows:
95 DEG C of 300s; 1 circulation;
95 DEG C of 20s, 62 DEG C of 30s, 70 DEG C of 30s; 10 circulations;
95 DEG C of 20s, 58 DEG C of 60s, 72 DEG C of 30s; 30 ~ 40 circulations;
37 DEG C of 30s; 1 circulation.
10. detection kit according to claim 3, it is characterized in that, described testing sample is ight soil, and required reagent also comprises ight soil pretreating reagent, and pretreating reagent is grinding buffer solution.
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| CN201610048731.2A CN105543378A (en) | 2016-01-23 | 2016-01-23 | NDRG4 gene methylation detection primers, probe and kit for early diagnosis of intestinal cancer |
| PCT/CN2016/109629 WO2017124854A1 (en) | 2016-01-23 | 2016-12-13 | Primers, probe and kit for detecting ndrg4 gene methylation for early diagnosis of intestinal cancer |
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