CN112391475B - Fluorescent quantitative PCR detection kit for SHOX2 gene methylation detection and application thereof - Google Patents
Fluorescent quantitative PCR detection kit for SHOX2 gene methylation detection and application thereof Download PDFInfo
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Abstract
The invention relates to a fluorescent quantitative PCR detection kit for SHOX2 gene methylation detection, which comprises a pair of methylation amplification primers and two methylation detection probes, wherein the pair of methylation amplification primers are used for amplifying a CpG sequence enrichment region from a target gene promoter to a sequence from 1 st exon to 3 rd exon, the CpG sequence enrichment region is a sequence amplified by a PCR reaction, and the CpG sequence enrichment region has at least 8 methylation CpG sequences; the two probes are used for detecting the positive strand and the negative strand of the PCR amplification product respectively, and the two probes use the same fluorescent reporter group and the same quenching group. In the invention, the double strand of the PCR product obtained by amplifying the methylation fragments is fully utilized, and each strand can generate a reaction signal, so that the sensitivity of detecting the methylation 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 SHOX2 gene methylation detection and application thereof.
Background
Gene methylation is an early event in tumors, and by detecting methylation markers on genes that are not methylated in normal tissue and other diseased tissue that are not cancerous, but are methylated only in tumor tissue, can aid in the diagnosis of tumors, and in particular play a role in early diagnosis of tumors. The auxiliary diagnosis of tumors by a noninvasive method is currently in clinical application stage, for example, early diagnosis of colorectal cancer by methylation fragments on free DNA in peripheral blood plasma, early diagnosis of bladder cancer by methylation fragments on free DNA in urine, and the like. In these methylation noninvasive early diagnosis methods, the vast majority of body fluid samples are free DNA released by normal tissue cells, and the free DNA released by tumor cells only occupies a very small proportion, so that the improvement of methylation methods for detecting free tumor cell DNA has been the direction of efforts of researchers.
Lung cancer is the first cancer of incidence rate in China, and although the lung cancer can be diagnosed by histological biopsy and cytological detection, the detection of the lung cancer is more advanced and the detection rate is not ideal. The SHOX2 gene methylation detection provides a more effective option for solving the problem, and a plurality of clinical experiments prove that the diagnosis rate is improved by combining the SHOX2 gene methylation detection with histological or cytological detection, so that the SHOX2 gene methylation detection is an effective tool for assisting in diagnosing lung cancer.
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. The current fluorescence quantitative method is mainly used for detecting the methylation fragments, wherein the DNA sequence after sulfite treatment is mainly used for maintaining the methylated C base as the C base, and the non-methylated C base is converted into the U base, so that a pair of primers and a probe can be designed according to the sequence difference after sulfite conversion to specifically detect the methylation. The exponential amplification of two DNA strands occurs after PCR amplification, but current fluorescent quantitative PCR detection methylation is only detected by designing a probe for one of the DNA strands, while the other amplified DNA strand is not utilized.
Disclosure of Invention
In order to solve the problems, the invention provides a fluorescent quantitative PCR detection kit for detecting the methylation of a SHOX2 gene, which 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 CpG sequence enrichment region in the sequence from a SHOX2 gene promoter to exons 1 to 3, the CpG sequence enrichment region is a sequence amplified by a PCR reaction, and the CpG sequence enrichment region has at least 8 methylation CpG sequences; the two probes are used for detecting the positive strand and the negative strand of the PCR amplification product respectively, 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 to detect the positive strand probe of a PCR amplification product and at least 2 of the at least 8 methylated CpG sequences are used to detect the negative strand probe of a PCR amplification product within the sequence amplified by one PCR reaction amplified by the pair of methylated amplification primers.
In one embodiment, the sequence from the SHOX2 gene promoter to exons 1-3 has a CpG sequence enrichment of a region within 200 bases.
In one embodiment, the pair of methylation amplification primers is used to amplify a CpG sequence enrichment region of 2415 to 2575 bases downstream of the transcriptional start base of the SHOX2 gene, which region has the sequence of SEQ ID NO:
wherein the following sequences of drawing straight lines are primer sequences, and the drawing line sequences and the dotted lines are probe sequences; there are 2 and 2 CpG sequences in this region for two methylation amplification primers, respectively, and 3 CpG sequences in this region for two methylation detection probes, respectively.
In one embodiment, the upstream primer of the pair of methylation amplified primers is SEQ ID No. 2: TGTTGTGTCGTATAGGGAGTC, the downstream primer is SEQ ID NO. 3: TAAACAACCAACATAACGTAAACG; the forward probes in the two methylation detection probes are SEQ ID NO. 4: ATTCGTAGACGTTTTTCGTT, and the reverse probe is SEQ ID NO. 5: CCTATACTCGTACGACCCCG.
In one embodiment, the invention provides the use of the above-described kit for detecting lung cancer.
In the invention, the double strand of the PCR product amplified for the SHOX2 gene methylation fragment is fully utilized, and each strand can generate a reaction signal, so that the sensitivity of detecting the SHOX2 gene methylation fragment can be improved.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed 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 other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of the reaction of a fluorescent quantitative PCR detection kit for methylation detection of a target gene according to the present invention, wherein the dark spots of the two entities represent methylated CpG islands;
FIG. 2 is a graph showing the results of methylation fluorescent quantitative PCR reactions performed on DNA extracted from alveolar lavage fluid of a lung cancer patient, as confirmed by lung biopsy puncture pathology.
Detailed Description
In order that those skilled in the art will better understand the technical solutions of the present application, the present invention will be further described with reference to the following examples, and it is apparent that the described examples are only some of the examples of the present application, not all the examples. All other embodiments, which can be made by one of ordinary skill in the art based on the embodiments herein without making any inventive effort, shall fall within the scope of the present application.
As shown in FIG. 1, the present invention finds a relatively enriched region of CpG sequences, which is a sequence amplified by a PCR reaction, by analyzing the sequence from the promoter of the target gene to exons 1 to 3, and has at least 8 methylated CpG sequences; the two probes are used for detecting the positive strand and the negative strand of the PCR amplification product respectively, and the two probes use the same fluorescent reporter group and the same quenching group.
The length of the sequence in the CpG sequence enrichment region is generally within 200 base sequences, so as to ensure higher amplification efficiency. If one PCR reaction amplifies a sequence greater than 200 bases, amplification efficiency is significantly reduced.
Both probes use the same fluorescent reporter and quencher, so that both probes fully utilize PCR to amplify each strand of the duplex, theoretically doubling the sensitivity of methylation fluorescent quantitation. The probes in the present invention may be, for example, taqman probes, beacon molecular Beacon probes, and fluorescent DNA probes based on fluorescence resonance energy transfer.
1. The reaction system of the present invention
1. Reaction system
| Composition of the components | Final concentration |
| GoTaq buffer | 1Х |
| dNTP (including dUTP) | 0.5mM each |
| MgCl2 | 3mM |
| Upstream primer | 0.4μM |
| Downstream primer | 0.4μM |
| Target gene positive strand probe | 0.15μM |
| Target gene negative strand probe | |
| ACTB upstream primer | 0.1μM |
| ACTB downstream primer | 0.1M |
| ACTB positive strand probe | 0.075μM |
| ACTB negative strand probe | 0.075μM |
| GoTaq hot start enzyme | 0.5μl |
| Sulfite-treated DNA templates | 6μl |
| Moisturizing to | 20μl |
Wherein ACTB is a reference gene.
2. Fluorescent quantitative PCR reaction conditions
2. SHOX2 gene methylation to detect lung cancer from alveolar lavage fluid
The SHOX2 gene is located in the negative strand of chr3:158,096,011-158,106,050. The initial position of the amplified SHOX2 gene is a PCR product of 160 bases in total from 2415 bases downstream to 2575 bases downstream of the transcription initiation base of the SHOX2 gene. The following sequences, which are drawn in straight lines, are primer sequences, and the drawn-line sequences and the broken lines are probe sequences.
In example one of the present invention (invention 1), the designed primer and probe sequences are as follows:
the forward probe Up and the reverse probe Lp are used simultaneously, wherein the Up contains 3 CG bases; lp contains 3 CG bases.
In the second embodiment (invention 2) of this example, the forward probe Up and the reverse probe Lp are used together as above, and 2 CG bases are contained on Up; lp contains 2 CG bases.
In example two (invention 2) of the present invention, the designed primer and probe sequences were as follows:
| SHOX 2-upstream primer | TGTTGTGTCGTATAGGGAGTC(SEQ ID NO:6) | Containing 2 CG bases |
| SHOX 2-downstream primer | TAAACAACCAACATAACGTAAACG(SEQ ID NO:7) | Containing 2 CG bases |
| SHOX 2-plus strand probe | FAM-ATTCGTAGACGTTTTTtGTT-BHQ1(SEQ ID NO:8) | Containing 2 CG bases |
| SHOX 2-minus strand probe | FAM-CCTATACTCGTACGACCCtG-BHQ1(SEQ ID NO:9) | Containing 2 CG bases |
As one of the comparisons (comparison 1), the primer was used as above, and the forward Up probe was not used, and only the reverse probe Lp was used;
| SHOX2-Lp | FAM-CCTATACTCGTACGACCCCG-BHQ1(SEQ ID NO:10) | containing 3 CG bases |
In contrast, the second (comparative 2), the primer was used as above, and the reverse Lp probe was not used, but only the forward probe Up was used;
| SHOX2-Up | FAM-ATTCGTAGACGTTTTTCGTT-BHQ1(SEQ ID NO:11) | containing 3 CG bases |
In comparison with this, three (comparison 3), the primers were used simultaneously for the forward Up probe and the reverse Lp probe, but the total CG base number was less than 4 CG bases.
| SHOX2-Up | FAM-ATTtGTAGACGTTTTTtGTT-BHQ1(SEQ ID NO:12) | Containing 1 CG base |
| SHOX2-Lp | FAM-CCTATACTCGTACGACCCtG-BHQ1(SEQ ID NO:13) | Containing 2 CG bases |
When the forward probe and the reverse probe are designed at the same time, the PCR amplification can be fully utilized to generate double chains, and higher sensitivity is generated; when 1 probe contains 2 CG sequences or more, methylated and unmethylated DNA can be sufficiently distinguished, resulting in higher specificity.
Clinical application cases. Taking 20 patients of lung cancer patients and non-cancer patients with infection confirmed by biopsy histopathology, taking 5ml of alveolar lavage fluid from each patient, extracting DNA in the alveolar lavage fluid by a magnetic bead method, converting the DNA by sulfite, and performing methylation fluorescent quantitative PCR reaction. The fluorescence quantification Ct is less than or equal to 40, and is determined as positive in methylation reaction; ct >40, was determined to be methylation negative. The sensitivity and specificity of the inventive protocol and 3 other protocols in diagnosing lung cancer by alveolar lavage are compared and the results are shown in Table 5.
It can be seen that the inventive group has improved sensitivity and specificity over the other comparative groups.
Taking 2ml of alveolar lavage fluid of 1 lung cancer patient confirmed by pathology, extracting DNA in alveolar lavage fluid cells by a magnetic bead method, converting the DNA by sulfite, performing methylation fluorescent quantitative PCR reaction, and comparing the performance of fluorescent quantitative reaction curves of double probes and single probes on sensitivity, wherein the result is shown in figure 2. The results of the comparison of the inventive 2 double probe (solid line) with the comparative 2 conventional single probe (dashed line) were used. Although the invention 1 and the traditional single probe detect positive results, the Ct value is advanced by 0.5 cycle compared with the single probe reaction through double-probe reaction, which is equivalent to the improvement of sensitivity by 0.5 power of 2 and is equivalent to the improvement of sensitivity by 1.4 times. In addition, in the comparison of the results of the invention 1 and the invention 2, the two have similar sensitivity, and the sensitivity of the invention 1 is improved by 1.8 times compared with that of the comparison 1.
It is to be understood that this invention 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 also encompassed by the appended claims.
Sequence listing
<110> Guangdong Huihuangjin Chuang biomedical science and technology Co., ltd
<120> fluorescent quantitative PCR detection kit for SHOX2 gene methylation detection and application thereof
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Claims (1)
1. A fluorescent quantitative PCR assay kit for the methylation detection of a SHOX2 gene, said kit comprising a pair of methylation amplification primers for amplifying a CpG sequence enrichment region of the sequence from the SHOX2 gene promoter to exons 1-3, said CpG sequence enrichment region being a sequence amplified by a PCR reaction, said CpG sequence enrichment region having at least 8 methylated CpG sequences, and two methylation detection probes; the two probes are respectively used for detecting a positive strand and a negative strand of a PCR amplification product, and the two probes use the same fluorescent reporter group and quenching group;
the pair of methylation amplification primers are used for amplifying a CpG sequence enrichment region of 2415 to 2575 bases downstream of a transcription initiation base of the SHOX2 gene, and the sequence of the CpG sequence enrichment region is SEQ ID NO. 1: TGTTGTGTCGTATAGGGAGTCGTATTCGTAGACGTTTTTCGTTGTTTTTGGGTTCGGGTTAAATTTTGTATAAGGTTTTTTGGATAGTTAGGTAATTTTCGTTTCGTTTGTTCGATCGGGGTCGTACGAGTATAGGCGTTTACGTTATGTTGGTTGTTTA; having 2 and 2 CpG sequences in the region can be used for two methylation amplification primers, respectively, and having 3 and 3 CpG sequences in the region can be used for two methylation detection probes, respectively;
the upstream primer of the pair of methylation amplified primers is SEQ ID NO. 2: TGTTGTGTCGTATAGGGAGTC, the downstream primer is SEQ ID NO. 3: TAAACAACCAACATAACGTAAACG; the forward probes in the two methylation detection probes are SEQ ID NO. 4: ATTCGTAGACGTTTTTCGTT, and the reverse probe is SEQ ID NO. 5: CCTATACTCGTACGACCCCG.
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