CN112501297A - Fluorescent quantitative PCR detection kit for detecting methylation of Septin9 gene and application thereof - Google Patents
Fluorescent quantitative PCR detection kit for detecting methylation of Septin9 gene and application thereof Download PDFInfo
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Abstract
The invention relates to a fluorescent quantitative PCR detection kit for detecting methylation of Septin9 gene and application thereof, wherein the kit comprises a pair of methylation amplification primers and two methylation detection probes, the pair of methylation amplification primers is used for amplifying a section of CpG sequence enrichment region from Septin9 gene promoter to 1-3 exon sequences, the section of CpG sequence enrichment region is a sequence amplified by PCR reaction, and the section of CpG sequence enrichment region has at least 8 methylated CpG sequences; the two probes are respectively used for detecting the positive strand and the negative strand of a PCR amplification product, and the two probes use the same fluorescent reporter group and the same quenching group. In the invention, double chains of PCR products obtained by amplifying methylated fragments of Septin9 gene are fully utilized, and each chain can generate reaction signals, so that the sensitivity of detecting the methylated fragments can be improved.
Description
Technical Field
The invention relates to the field of molecular diagnosis, in particular to a fluorescent quantitative PCR detection kit for detecting methylation of Septin9 gene and application thereof.
Background
Gene methylation is an early event of tumors, and can assist in diagnosing tumors, particularly in early diagnosis of tumors, by detecting methylation markers on genes that are not methylated in normal tissues and other diseased tissues that are not cancerous, but are methylated only in tumor tissues. The auxiliary diagnosis of tumor by non-invasive method is now in clinical application, such as early diagnosis of carcinoma of large intestine by methylation fragment on free DNA in peripheral blood plasma, early diagnosis of bladder cancer by methylation fragment on free DNA in urine, etc. In these methods for the noninvasive and early diagnosis of tumor by methylation, most of the body fluid samples to be detected are free DNA released by normal tissue cells, while the free DNA released by tumor cells only accounts for a very small proportion, so that the improvement of the methylation method for detecting free tumor cell DNA has been the direction of the research and development efforts.
Septin is a conservative skeleton protein gene family with GTPas activity, at least 14 human family members are respectively named as Septin 1-14, wherein Septin9 is positioned at 17q25, and Septin9 protein is coded. Researches show that the transcription product of Septin9 gene has multiple translation initiation sites and has variant splicing, 18 different protein products can be generated, Septin9 is used as a family member related to cytoplasmic division, the expression of Septin9 is highly related to the occurrence and development of colorectal cancer, Septin9 has obvious expression difference in normal human colorectal tissue and colorectal cancer tissue, and DNA methylation is a main mechanism for regulating Septin9 gene expression. Wherein, the CpG island hypermethylation of the Septin9 promoter region can be detected in samples such as peripheral blood and the like, so that the CpG island hypermethylation becomes a biomarker of colorectal cancer.
The specificity of diagnosis of colorectal cancer by methylation detection of Septin9 is good, the comprehensive sensitivity of colorectal cancer screening in stage I-IV can reach 40-70%, and the diagnosis kit is a colorectal cancer molecular marker with specificity and sensitivity.
The fluorescent quantitative method plays an important role in detecting gene methylation due to the simplicity of the method and the popularization of fluorescent quantitative PCR instruments. At present, a fluorescence quantitative method is used for detecting methylated fragments, mainly by using DNA sequences treated by sulfite, methylated C bases are kept as C bases, and unmethylated C bases are converted into U bases, so that a pair of primers and a probe can be designed according to sequence differences after sulfite conversion to specifically detect methylation. After PCR amplification, exponential amplification of two DNA strands is generated, but the current fluorescence quantitative PCR detection methylation only detects one DNA strand by designing a probe, and the other amplified DNA strand is not utilized.
Disclosure of Invention
In order to solve the above problems, the present invention provides a fluorescent quantitative PCR detection kit for methylation detection of the Septin9 gene, the kit comprises a pair of methylation amplification primers and two methylation detection probes, the pair of methylation amplification primers is used for amplifying a CpG sequence enrichment region from the promoter of the Septin9 gene to the 1 st-3 rd exon sequences, the CpG sequence enrichment region is a sequence amplified by a PCR reaction, and the CpG sequence enrichment region has at least 8 methylated CpG sequences; the two probes are respectively used for detecting the positive strand and the negative strand of a PCR amplification product, and the two probes use the same fluorescent reporter group and the same quenching group.
In one embodiment, at least 2 of the at least 8 methylated CpG sequences are used for positive strand probes and at least 2 of the at least 8 methylated CpG sequences are used for negative strand probes of the PCR amplification product within the sequences amplified by one PCR reaction using the pair of methylated amplification primers.
In one embodiment, the CpG sequence rich region from the promoter of the Septin9 gene to the sequence of exons 1-3 is within 200 bases in length.
In one embodiment, the pair of methylation amplification primers is used for amplifying a CpG sequence enriched region from 2847 to 2745 bases upstream of the transcription initiation base of Septin9 gene, and the sequence of the region is SEQ ID NO: 1:
wherein the sequence of the following straight line is a primer sequence, and the sequence of the wave-cutting line and the dotted line are probe sequences; in this region there are 3And 3 CpG sequences can be used for two methylation amplification primers, respectively, and 3 CpG sequences in the region can be used for two methylation detection probes, respectively.
In one embodiment, the forward primer of the pair of methylated amplification primers is SEQ ID NO: 2: GGGAGGTCGGTGCGGGTGC, the downstream primer is SEQ ID NO: 3: ATAAAAAAAAAAAACGAACGCC, respectively; the forward probes of the two methylation detection probes are SEQ ID NO: TGATTCGTTCGGGAGGCGGG, and the reverse probe is SEQ ID NO: 5: CGCTACGCCCCCGCC are provided.
In one embodiment, the present invention provides the use of the above kit for detecting colorectal cancer.
In the invention, double chains of PCR products obtained by amplifying methylated fragments of Septin9 gene are fully utilized, and each chain can generate reaction signals, so that the sensitivity of detecting the methylated fragments can be improved.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present application, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a reaction diagram of the fluorescent quantitative PCR detection kit for methylation detection of a target gene according to the present invention, in which two solid black dots represent methylated CpG islands;
FIG. 2 is a graph showing the results of methylation fluorescent quantitative PCR reaction performed on DNA extracted from peripheral blood plasma of a colorectal cancer patient confirmed by enteroscopy.
Detailed Description
In order to make the technical solutions in the present application better understood by those skilled in the art, the present invention will be further described with reference to the following examples, and it is apparent that the described examples are only a part of the examples of the present application, and not all examples. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
As shown in FIG. 1, the present invention finds a region relatively rich in CpG sequences by analyzing the sequence from the promoter of the target gene to exons 1-3, wherein the region rich in CpG sequences is a sequence amplified by a PCR reaction, and the region rich in CpG sequences has at least 8 methylated CpG sequences; the two probes are respectively used for detecting the positive strand and the negative strand of a PCR amplification product, and the two probes use the same fluorescent reporter group and the same quenching group.
The sequence length of the enriched region of the CpG sequence is usually within 200 base sequences so as to ensure higher amplification efficiency. If a sequence amplified by one PCR reaction is more than 200 bases, the amplification efficiency is significantly reduced.
The same fluorescent reporter group and quencher group are used for both probes, so that the two probes fully utilize each chain of a PCR amplified double chain, and the sensitivity of methylation fluorescence quantification is theoretically doubled. The probe in the invention can be, for example, a Taqman probe, a Beacon molecular Beacon probe, and a fluorescent DNA probe based on fluorescence resonance energy transfer.
First, the reaction system of the invention
1. Reaction system
| Composition (I) | Final concentration |
| GoTaq buffer solution | 1Х |
| dNTP (including dUTP) | 0.5mM each |
| MgCl2 | 3mM |
| Upstream primer | 0.4μM |
| Downstream primer | 0.4μM |
| Positive strand probe of target gene | 0.15μM |
| Negative strand probe for target gene | |
| ACTB upstream primer | 0.1μM |
| ACTB downstream primer | 0.1M |
| ACTB plus strand probe | 0.075μM |
| ACTB minus strand probe | 0.075μM |
| GoTaq hot start enzyme | 0.5μl |
| Sulfite-treated DNA template | 6μl |
| Make up water to | 20μl |
Wherein ACTB is an internal reference gene.
2. Fluorescent quantitative PCR reaction condition
Secondly, Septin9 gene methylation detection of colorectal cancer from blood plasma
The following sequence is a sulfite-converted sequence, methylated CG remains as a CG base, while the C base alone becomes a T base (indicated by lower case T)
The Septin9 gene is located on the positive chain of chr17: 77376083-77500596. Through analysis, PCR products with the initial position of amplified Septin9 gene being Septin9 gene transcription initial base upstream 2847 to upstream 2745 bases totaling 102 bases are selected, and the specific sequence is as follows, wherein the sequence with the following straight line is a primer sequence, and the sequence with the following wave line and the dotted line are probe sequences.
In embodiment one (invention 1) of this example, the primer and probe sequences were designed as follows:
| Septin9-F | GGGAGGTCGGTGCGGGTGC(SEQ ID NO:2) | containing 3 CG bases |
| Septin9-R | ATAAAAAAAAAAAACGAACGCC(SEQ ID NO:3) | Containing 3 CG bases |
| Septin9-Up | FAM-TGATTCGTTCGGGAGGCGGG-BHQ1(SEQ ID NO:4) | Containing 3 CG bases |
| Septin9-Lp | FAM-CGCTACGCCCCCGCC-BHQ1(SEQ ID NO:5) | Containing 3 CG bases |
The forward probe Up and the reverse probe Lp are used simultaneously, and the Up contains 3 CG basic groups; lp contains 3 CG bases.
In the second embodiment (invention 2) of this example, the forward probe Up and the reverse probe Lp were used together as above, with 2 CG bases in Up; lp contains 2 CG bases.
In the second embodiment of the present invention (the present invention 2), the designed primer and probe sequences are as follows:
| Septin9-F | GGGAGGTCGGTGCGGGTGC(SEQ ID NO:6) | containing 3 CG bases |
| Septin9-R | ATAAAAAAAAAAAACGAACGCC(SEQ ID NO:7) | Containing 3 CG bases |
| Septin9-Up | FAM-TGATTCGTTCGGGAGGtGGG-BHQ1(SEQ ID NO:8) | Containing 3 CG bases |
| Septin9-Lp | FAM-tGCTACGCCCCCGCC-BHQ1(SEQ ID NO:9) | Containing 3 CG bases |
As a comparison (comparative 1), the primer was the same as above, the forward Up probe was not used, and only the reverse probe Lp was used;
| Septin9-Lp | FAM-CGCTACGCCCCCGCC-BHQ1(SEQ ID NO:10) | containing 3 CG bases |
As a second comparison (comparison 2), primers were as above, with no reverse Lp probe and only the forward probe Up;
| Septin9-Up | FAM-TGATTCGTTCGGGAGGCGGG-BHQ1(SEQ ID NO:11) | containing 3 CG bases |
In the third comparison (comparison 3), the forward Up probe and the reverse Lp probe were used together with the above primers, but the total number of CG bases was less than 4 CG bases.
| Septin9-Up | FAM-TGATTCGTTCGGGAGGtGGG-BHQ1(SEQ ID NO:12) | Containing 2 CG bases |
| Septin9-Lp | FAM-tGCTACGCCCCtGCC-BHQ1(SEQ ID NO:13) | Containing 1 CG base |
When the forward probe and the reverse probe are designed simultaneously, double chains can be generated by fully utilizing PCR amplification, and higher sensitivity is generated; when each probe contains 2 CG sequences or more, methylated and unmethylated DNAs can be sufficiently distinguished, resulting in high specificity.
Clinical application cases. 20 cases of patients with colorectal cancer confirmed by enteroscopy and 20 cases of non-cancer patients were collected, 5ml of peripheral blood was collected from each patient, plasma was obtained by centrifugation, free DNA in the plasma was extracted by the magnetic bead method, DNA was transformed with sulfite, and then, quantitative PCR reaction by methylation fluorescence was carried out. The fluorescence quantitative Ct is less than or equal to 40, and the methylation reaction is positive; ct >40, negative for methylation reaction. The sensitivity and specificity of the protocol of the invention and 3 other design protocols were compared in the detection of colorectal cancer by plasma and the results are shown in table 1.
It can be seen that the group of the present invention has improved sensitivity and specificity compared to the other comparative groups.
As shown in FIG. 2, the methylation fluorescence quantitative PCR reaction of DNA extracted from peripheral blood plasma of one example of a colorectal cancer patient was confirmed by enteroscopy. The results of the comparison of the double probe of the present invention 1 (solid line) with the conventional single probe (dotted line) comparative 1 were used. Although the positive results are detected by the single probe and the traditional probe in the invention 1, the Ct value is advanced by 1.5 cycles through the double-probe reaction compared with the single-probe reaction, which is equivalent to that the sensitivity is improved by 1.5 times of 2 and is equal to that the sensitivity is improved by 2.8 times. In addition, in comparison of the results of the invention 1 and the invention 2, the two have similar sensitivity, and the sensitivity of the invention 2 is improved by 2.6 times compared with that of the comparison 1.
It is to be understood that the invention disclosed is not limited to the particular methodology, protocols, and materials described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
Sequence listing
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<120> fluorescent quantitative PCR detection kit for Septin9 gene methylation detection and application thereof
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Claims (6)
1. A fluorescence quantitative PCR detection kit for methylation detection of Septin9 gene is characterized by comprising a pair of methylation amplification primers and two methylation detection probes, wherein the pair of methylation amplification primers are used for amplifying a section of CpG sequence enrichment region from Septin9 gene promoter to 1-3 exon sequences, the section of CpG sequence enrichment region is a sequence amplified by PCR reaction, and the section of CpG sequence enrichment region has at least 8 methylated CpG sequences; the two probes are respectively used for detecting the positive strand and the negative strand of a PCR amplification product, and the two probes use the same fluorescent reporter group and the same quenching group.
2. The quantitative fluorescence detection kit of claim 1, wherein within the sequence amplified by one PCR reaction of the pair of methylated amplification primers, at least 2 methylated CpG sequences of the at least 8 methylated CpG sequences are used for detecting a positive strand probe of a PCR amplification product, and at least 2 methylated CpG sequences are used for detecting a negative strand probe of the PCR amplification product.
3. The kit of claim 1, wherein the CpG sequence rich region from the promoter of the Septin9 gene to the 1 st-3 rd exon is within 200 bp sequence in length.
4. The kit of claim 3, wherein the pair of methylation amplification primers is used for amplifying a CpG sequence enriched region from 2847 to 2745 bases upstream of the transcription initiation base of Septin9 gene, and the sequence of the region is SEQ ID NO: 1:GGGAGGtCGGTGCGGGTGCGGGAAttTGATtCGttCGGGAGGCGGGGGCGGGGCGGGGGCGt AGCGCGCGGGGAGGGGtCGGCGttCGttTTttTtttttAT，wherein the sequence of the following straight line is a primer sequence, and the sequence of the wave-cutting line and the dotted line are probe sequences; having 3 and 3 CpG sequences in this region can be used for two methylation amplification primers, respectively, and having 3 and 3 CpG sequences in this region can be used for two methylation detection probes, respectively.
5. The kit of claim 4, wherein the upstream primer of the pair of methylation amplification primers is SEQ ID NO: 2: GGGAGGTCGGTGCGGGTGC, the downstream primer is SEQ ID NO: 3: ATAAAAAAAAAAAACGAACGCC, respectively; the forward probes of the two methylation detection probes are SEQ ID NO: TGATTCGTTCGGGAGGCGGG, and the reverse probe is SEQ ID NO: 5: CGCTACGCCCCCGCC are provided.
6. Use of a kit according to any one of claims 1 to 5 for the detection of colorectal cancer.
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