CN112391475A - Fluorescent quantitative PCR detection kit for detecting methylation of SHOX2 gene and application thereof - Google Patents
Fluorescent quantitative PCR detection kit for detecting methylation of SHOX2 gene and application thereof Download PDFInfo
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Abstract
The invention relates to a fluorescent quantitative PCR detection kit for detecting the methylation of SHOX2 gene, 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 section of CpG sequence enrichment region from a target gene promoter to the 1 st-3 rd exon sequence, 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 the methylated fragments are fully utilized, and each chain can generate a reaction signal, 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 the methylation of a SHOX2 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.
Although lung cancer can be diagnosed by histological biopsy and cytological detection, the lung cancer is detected in a more advanced stage and the detection rate is not ideal. The methylation detection of the SHOX2 gene provides a more effective option for solving the problem, and multiple clinical tests prove that the combined use of the methylation detection and the cytological detection improves the diagnosis rate and is an effective tool for assisting the diagnosis of the 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. 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 problems, the invention provides a fluorescent quantitative PCR detection kit for detecting the methylation of the 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 is used for amplifying a CpG sequence enrichment region from a promoter of the SHOX2 gene to the sequences of the 1 st exon to the 3 rd exon, the CpG sequence enrichment region is a sequence amplified by 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 detecting positive strand probes and at least 2 of the at least 8 methylated CpG sequences are used for detecting negative strand probes of PCR amplification products 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 SHOX2 gene to exon 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 2415 downstream to 2575 bases downstream of the transcription initiation base of the SHOX2 gene, and the sequence of the CpG sequence enriched region is SEQ ID NO: 1:
wherein the sequence of the lower straight line is a primer sequence, and the sequence of the lower wave line and the dotted line are probe sequences; having 2 and 2 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.
In one embodiment, the forward primer of the pair of methylated amplification primers is SEQ ID NO: 2: TGTTGTGTCGTATAGGGAGTC, the downstream primer is SEQ ID NO: 3: TAAACAACCAACATAACGTAAACG, respectively; the forward probes of the two methylation detection probes are SEQ ID NO: ATTCGTAGACGTTTTTCGTT, and the reverse probe is SEQ ID NO: 5: CCTATACTCGTACGACCCCG are provided.
In one embodiment, the present invention provides the use of the above kit for detecting lung cancer.
In the invention, double chains of PCR products obtained by amplifying the methylation fragments of the SHOX2 gene are fully utilized, and each chain can generate a reaction signal, so that the sensitivity for detecting the methylation fragments of the SHOX2 gene 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 a methylation-based fluorescence quantitative PCR reaction of DNA extracted from alveolar lavage fluid of a patient whose lung cancer was confirmed by the pathology of lung biopsy puncture.
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, detection of lung cancer from alveolar lavage fluid by SHOX2 gene methylation
The SHOX2 gene is located in the chr3:158,096,011 and 158,106,050 minus strand. The initial position of the amplified SHOX2 gene is a PCR product of 160 bases in total from 2415 to 2575 bases downstream of the transcription initial base of the SHOX2 gene. The following sequences marked with straight lines are primer sequences, and the sequences marked with wavy lines and dotted lines are probe sequences.
In the first embodiment (invention 1), the primer and probe sequences are designed as follows:
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 on Up; lp contains 2 CG bases.
In example two of the present invention (invention 2), the designed primer and probe sequences are as follows:
| SHOX 2-upstream primer | TGTTGTGTCGTATAGGGAGTC(SEQ ID NO:6) | Containing 2 CG bases |
| SHOX 2-downstream primer | TAAACAACCAACATAACGTAAACG(SEQ ID NO:7) | Comprises2 CG bases |
| SHOX 2-sense 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 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;
| SHOX2-Lp | FAM-CCTATACTCGTACGACCCCG-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;
| SHOX2-Up | FAM-ATTCGTAGACGTTTTTCGTT-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.
| 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 simultaneously, double chains can be generated by fully utilizing PCR amplification, and higher sensitivity is generated; when 1 probe contains more than or equal to 2 CG sequences, methylated DNA and unmethylated DNA can be fully distinguished, and high specificity is generated.
Clinical application cases. 20 persons of lung cancer patients and non-cancer infected patients confirmed by biopsy histopathology were taken, 5ml of alveolar lavage fluid was taken from each person, DNA in the alveolar lavage fluid was extracted by the magnetic bead method, and after the DNA was transformed with sulfite, a quantitative PCR reaction was carried out by methylation fluorescence. 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 present protocol was compared to 3 other design protocols in diagnosing lung cancer by alveolar lavage fluid, and the results are shown in Table 5.
It can be seen that the group of the present invention has improved sensitivity and specificity compared to the other comparative groups.
2ml of pathologically confirmed lung cancer patients were sampled from 1 lung cancer patient's alveolar lavage fluid, DNA in alveolar lavage fluid cells was extracted by the magnetic bead method, DNA was transformed with sulfite, and then a methylation fluorescent quantitative PCR reaction was performed to compare the performance of the fluorescent quantitative reaction curves of the double probe and the single probe in sensitivity, and the results are shown in FIG. 2. The results of a comparison between the double probe of the present invention 2 (solid line) and the conventional single probe of comparative 2 (dotted line) 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 0.5 cycle through double-probe reaction compared with single-probe reaction, which is equivalent to that the sensitivity is improved by 0.5 power of 2 and is equal to that the sensitivity is improved by 1.4 times. In addition, in comparison of the results of the invention 1 and the invention 2, the two have similar sensitivities, 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 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.
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Claims (6)
1. A fluorescent quantitative PCR detection kit for detecting the methylation of a SHOX2 gene is characterized by comprising a pair of methylation amplification primers and two methylation detection probes, wherein the pair of methylation amplification primers is used for amplifying a CpG sequence enrichment region from a promoter of the SHOX2 gene to a sequence from a 1 st exon to a 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 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 sequence from the promoter of SHOX2 gene to exons 1-3 is a CpG sequence-rich region within 200 bases of its sequence.
4. The kit of claim 3, wherein the pair of methylation amplification primers is used for amplifying a CpG sequence enriched region from 2415 downstream to 2575 downstream of the transcription initiation base of the SHOX2 gene, and the sequence of the CpG sequence enriched region is SEQ ID NO: 1:
wherein the sequence of the lower straight line is a primer sequence, and the sequence of the lower wave line and the dotted line are probe sequences; having 2 and 2 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: TGTTGTGTCGTATAGGGAGTC, the downstream primer is SEQ ID NO: 3: TAAACAACCAACATAACGTAAACG, respectively; the forward probes of the two methylation detection probes are SEQ ID NO: ATTCGTAGACGTTTTTCGTT, and the reverse probe is SEQ ID NO: 5: CCTATACTCGTACGACCCCG are provided.
6. Use of a kit according to any one of claims 1 to 5 for the detection of urothelial carcinoma of the bladder.
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