WO2024016461A1 - 一种双特异性探针捕获磁珠及其制备方法和应用 - Google Patents

一种双特异性探针捕获磁珠及其制备方法和应用 Download PDF

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WO2024016461A1
WO2024016461A1 PCT/CN2022/119600 CN2022119600W WO2024016461A1 WO 2024016461 A1 WO2024016461 A1 WO 2024016461A1 CN 2022119600 W CN2022119600 W CN 2022119600W WO 2024016461 A1 WO2024016461 A1 WO 2024016461A1
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probe
capture
magnetic beads
bispecific
gene
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French (fr)
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戴立忠
周镕
纪博知
刘佳
郭俊
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圣湘生物科技股份有限公司
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • the invention belongs to the field of biological detection, and specifically relates to a preparation method of capturing magnetic beads. More specifically, it relates to a method for preparing bispecific probe capture magnetic beads.
  • detecting patients' stool samples has higher sensitivity and accuracy.
  • the components contained in the samples are complex, mostly various types of bacteria and food residues, which cause greater background interference and suppression of target detection, and the content of cancer cells is low, making the target The detection rate is low and unstable.
  • the ordinary magnetic bead extraction kits on the market can meet the subsequent detection needs for the extraction and purification of samples such as plasma, urine, or thoracic and ascites fluid.
  • the corresponding target fragments must be enriched. There are certain difficulties in extraction and purification.
  • the conventional magnetic bead capture probe currently used is a specific nucleotide sequence, about 30-50 bp in length. The longer the length, the higher the capture specificity. However, a sequence that is too long will cause it to form a folding dimer. Or hairpin structure, which affects subsequent magnetic bead capture efficiency.
  • the specific detection sites are mainly differences in CpG sites. Due to the particularity of this site, methyl groups may appear during the detection process. There is only a 1-3 base difference between methylated and unmethylated targets. Ordinary capture probes can still bind and capture normally when a single or even 3 bases are mismatched. This non-specific capture will lead to subsequent capture. The amount of nucleic acid is insufficient or the target fragment detection is inaccurate.
  • the present invention provides a method for preparing dual-specific probe capture magnetic beads, which is characterized in that the method includes:
  • the 5' end of the bispecific probe is modified with a primary amino group, and hypoxanthine is introduced into the 15-25 bp region of the 5' end of the probe;
  • the length of the bispecific probe is 20-70 bp.
  • the amount of hypoxanthine is 1-8.
  • the amount of hypoxanthine is 3-6.
  • the amount of hypoxanthine can be 1, 2, 3, 4, 5, 6, 7, or 8.
  • the capture magnetic beads are carboxyl magnetic beads, and the 5' end of the bispecific probe is modified with a primary amino group.
  • the carboxyl groups of the carboxyl magnetic beads are activated, including:
  • the carboxyl group of the activated magnetic beads includes: repeating step S2.
  • coupling carboxyl magnetic beads with bispecific probes includes:
  • the bispecific probe, the activated magnetic beads, 2-morpholinoethanesulfonic acid, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were mixed, shaken, and incubated.
  • the preparation method further includes: blocking of bispecific capture magnetic beads.
  • the blocking includes: 1) After coupling is completed, place it on a magnetic stand for adsorption, add PBST solution (phosphate Tween buffer) containing 1% BSA (bovine serum albumin), and mix with shaking Evenly, seal.
  • PBST solution phosphate Tween buffer
  • BSA bovine serum albumin
  • the target of the bispecific probe is SDC2 gene, NDRG4 gene, Septin9 gene, ⁇ -actin gene and/or E. coli.
  • the target of the bispecific probe is the SDC2 gene.
  • probe sequence is:
  • the target of the bispecific probe is the NDRG4 gene.
  • probe sequence is:
  • the target of the bispecific probe is the Septin9 gene.
  • probe sequence is:
  • the target of the bispecific probe is the ⁇ -actin gene.
  • probe sequence is:
  • the probe can also target E. coli, and the sequence is
  • bispecific capture magnetic beads can better capture target fragments, ensure capture stability, and have higher sensitivity.
  • low-concentration nucleic acids can significantly increase the detection rate, without the need for multi-step sample purification, reducing operating steps, and quickly and efficiently extracting target nucleic acids. At the same time, it can effectively reduce false captures caused by mismatches.
  • the present invention provides bispecific capture magnetic beads, which are characterized in that they are prepared by the above preparation method.
  • the target of the bispecific probe is SDC2 gene, NDRG4 gene, Septin9 gene, ⁇ -actin gene and/or E. coli.
  • the target of the bispecific probe is the SDC2 gene.
  • probe sequence is:
  • the target of the bispecific probe is the NDRG4 gene.
  • probe sequence is:
  • the target of the bispecific probe is the Septin9 gene.
  • probe sequence is:
  • the target of the bispecific probe is the ⁇ -actin gene.
  • probe sequence is:
  • the probe can also target E. coli, and the sequence is
  • the present invention provides a nucleic acid extraction kit, which is characterized in that it includes the bispecific capture magnetic beads as described above.
  • the present invention provides a kit for extracting nucleic acid from stool samples, which is characterized in that it includes the bispecific capture magnetic beads as described above.
  • the kit includes polyethylene glycol.
  • the concentration of polyethylene glycol is 15% to 35%.
  • the concentration of polyethylene glycol is 20% to 30%.
  • the concentration of polyethylene glycol is 20%.
  • Using an appropriate concentration of polyethylene glycol can increase the capture efficiency of the bispecific capture magnetic beads and further improve the capture sensitivity of the bispecific capture magnetic beads.
  • the kit further includes positive controls and negative controls.
  • the present invention provides the use of specific capture magnetic beads as described above for preparing a nucleic acid extraction kit.
  • the present invention provides a nucleic acid extraction method, which includes extracting a sample using bispecific capture magnetic beads as described above.
  • sample is feces, urine, plasma, or thoracic and ascites fluid.
  • the sample is preferably feces.
  • Figure 1 is a comparison chart of the nucleic acid extraction effects of high-concentration stool samples with different types of SDC2 gene-modified magnetic beads
  • Figure 2 is a comparison chart of the nucleic acid extraction effects of low-concentration stool samples with different types of SDC2 gene-modified magnetic beads
  • Figure 3 is a comparison chart of the nucleic acid extraction effects of high-concentration stool samples with different types of ⁇ -actin gene modified magnetic beads
  • Figure 4 is a comparison chart of the nucleic acid extraction effects of low-concentration stool samples with different types of ⁇ -actin gene modified magnetic beads
  • Figure 5 is a comparison chart of the nucleic acid extraction effects of high-concentration plasma samples with different types of SDC2 gene-modified magnetic beads
  • Figure 6 is a comparison chart of the nucleic acid extraction effects of low-concentration plasma samples with different types of SDC2 gene-modified magnetic beads
  • Figure 7 is a comparison chart of the nucleic acid extraction effects of high-concentration plasma samples with different types of ⁇ -actin gene modified magnetic beads
  • Figure 8 is a comparison chart of the nucleic acid extraction effects of low-concentration plasma samples with different types of ⁇ -actin gene modified magnetic beads
  • Figure 9 is a comparison chart of the nucleic acid extraction effects of high-concentration urine samples with different types of SDC2 gene-modified magnetic beads
  • Figure 10 is a comparison chart of the nucleic acid extraction effects of low-concentration urine samples with different types of SDC2 gene-modified magnetic beads
  • Figure 11 is a comparison chart of the nucleic acid extraction effects of high-concentration urine samples with different types of ⁇ -actin gene modified magnetic beads
  • Figure 12 is a comparison chart of the nucleic acid extraction effects of low-concentration urine samples using different types of ⁇ -actin gene modified magnetic beads
  • Figure 13 is a comparison chart of the nucleic acid extraction effects of different types of SDC2 gene-modified magnetic beads in high-concentration thoracic and ascites fluid samples;
  • Figure 14 is a comparison chart of the nucleic acid extraction effects of low-concentration thoracic and ascites fluid samples with different types of SDC2 gene-modified magnetic beads;
  • Figure 15 is a comparison chart of the nucleic acid extraction effects of different types of magnetic beads modified with ⁇ -actin gene in high-concentration thoracic and ascites fluid samples;
  • Figure 16 is a comparison chart of the nucleic acid extraction effects of low-concentration thoracic and ascites fluid samples with different types of ⁇ -actin gene modified magnetic beads;
  • FIG. 17 shows the effects of PEG extraction at different concentrations
  • Figure 18 is a diagram showing the nucleic acid extraction effects of capture probes at different positions of the SDC2 gene
  • Figure 19 shows the nucleic acid extraction effect of capture probes at different positions of the NDRG4 gene
  • Figure 20 is a diagram showing the nucleic acid extraction effects of capture probes at different positions of the Septin9 gene
  • Figure 21 is a diagram of the nucleic acid extraction effect of different capture probes introducing a mismatched base into Septin9;
  • Figure 22 is a diagram of the nucleic acid extraction effect of different capture probes introduced by Septin9 with three mismatched bases.
  • capture probes were prepared for the methylated target gene SDC2 and Escherichia coli, and human-derived internal standard ⁇ -actin was added as an internal reference.
  • Capture sequence 5’-TTGTAATTTTTAAGGGAGGAGIIIITTTTATTGGTT-3’ (SEQ ID NO: 4).
  • methylated fragments are human genome fully methylated standards.
  • the methylated standards after sulfite conversion are put into the negative feces supernatant ( Figures 1 to 4) and plasma.
  • Simulated samples containing exogenous interfering substances were prepared from ( Figures 5-8), urine ( Figures 9-12) or thoracic and ascites fluid ( Figures 13-16).
  • the content of high-concentration methylated fragments in the simulated samples was 100ng/ ⁇ L.
  • the content of low-concentration methylated fragments is 0.1ng/ ⁇ L
  • unmethylated nucleic acid fragments are added as background signals
  • the content of unmethylated fragments is 100ng/ ⁇ L.
  • the bispecific probe capture magnetic beads, ordinary probe capture magnetic beads and carboxyl magnetic beads prepared in Example 1 were used to extract high and low concentration simulated samples respectively.
  • the extraction steps were carried out according to the operation of Example 2, and finally washed with 50 ⁇ L TE buffer. DNA removal.
  • the nucleic acid extracted in Example 2 was subjected to sulfite conversion.
  • the conversion kit was EZ DNA Methylation-lingthting Kit. The conversion step was carried out strictly in accordance with the operating instructions. Finally, the converted nucleic acid was eluted with 50 ⁇ L TE buffer.
  • the extracted nucleic acid was detected by fluorescence PCR, and a methylation primer probe was designed based on the SDC2 promoter region for testing and verification.
  • the human internal standard ⁇ -actin was also designed as an internal reference gene.
  • Reverse primer 5’-ACAATATAACTCCCAAATAAACCCG-3’(SEQ ID NO:10)
  • Fluorescent probe 5’-TTCGGAGTTGTTAATCGGCGTG-3’ (SEQ ID NO: 11)
  • Fluorescent probe 5’-TCTAACTAGGACCGCAGAGGAAAGAAGGAATT-3’(SEQ ID NO:14)
  • Reverse primer 5’-CCAAAAAAAAAAAACTACTTATTCCAATTCAC-3’(SEQ ID NO:16)
  • Fluorescent probe 5’-TTAAGGGAGGAGTAGGTT-3’ (SEQ ID NO: 17).
  • the target nucleic acid in the dual-specific capture magnetic beads is higher than that of ordinary probe capture magnetic beads and carboxyl magnetic beads, and the Ct value is higher.
  • specific capture magnetic beads are less affected by inhibitors in complex sample types and have higher capture efficiency, especially for the detection of low-concentration nucleic acid samples.
  • the modified capture magnetic beads The beads can effectively bind the target fragment and eliminate the influence of interfering substances. Therefore, both bispecific capture magnetic beads and ordinary capture magnetic beads can effectively distinguish the target fragment and interfering substances.
  • the extraction effect of carboxyl magnetic beads is the worst. Due to the interference of unmethylated nucleic acid fragments, carboxyl magnetic beads cannot effectively and selectively bind the target fragments.
  • the extraction concentration of the target fragments in feces, urine, plasma or ascites samples is relatively low. Low.
  • the dual-specific probe capture magnetic beads have a better capture effect on E. coli.
  • Example 5 Effect of introducing hypoxanthine at different positions of the capture probe on capture efficiency
  • the methylated target genes SDC2, NDRG4 and Septin9 were used as capture probes, and at the same time, at the 5' end, 3' end and Hypoxanthine was introduced into the middle region to compare the nucleic acid extraction effect of the capture magnetic beads.
  • the results are shown in Figures 18 to 20. It can be seen from the figures that the introduction of hypoxanthine in the region 15 to 25 bp from the 5’ end of the bispecific probe can greatly improve the sensitivity of the capture probe.
  • the target gene Septin9 after methylation in colorectal cancer was used as the capture target fragment, and 1 to 3 mismatched bases were introduced into the synthesized methylated fragment. .
  • the mismatched fragments were put into the negative stool supernatant to prepare a simulated sample.
  • the content of mismatched methylated fragments in the simulated sample was 100ng/ ⁇ L.
  • Bispecific magnetic beads, ordinary magnetic beads and carboxyl magnetic beads were used at the same time. Bead comparison nucleic acid extraction effect:
  • carboxyl magnetic beads cannot distinguish the accuracy of nucleic acid sequences. They can bind as long as there is nucleic acid present, so the nucleic acid concentration in the detection result is the highest; ordinary capture magnetic beads only have 1 to 3 bases in the sequence. When the base is different, there is still a small amount of mismatched sequence that binds to the capture probe, making the test result positive; due to the introduction of hypoxanthine in the bispecific capture magnetic beads, the specificity of the capture sequence is enhanced, and for mismatched bases in the sequence It cannot bind to the magnetic beads normally, so the test result is negative, and its capture accuracy is the highest. Therefore, it can be seen that the capture accuracy of the bispecific probe of the present invention is much higher than that of ordinary probes and carboxyl magnetic beads. Especially when there is only a mismatch of 1 base, the capture accuracy is significantly improved.

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Abstract

本发明提供了一种双特异探针捕获磁珠的制备方法，包括：将捕获磁珠与双特异性探针进行耦合，所述双特异性探针5'端15bp-25bp区域位置引入次黄嘌呤，所述双特异性探针的长度为20~70bp。

Description

一种双特异性探针捕获磁珠及其制备方法和应用
相关申请的交叉引用
本申请要求申请日为2022年07月20日，申请号为202210861620.9，题名为“一种双特异性探针捕获磁珠及其制备方法和应用”的优先权，以上中国专利申请的全部内容在此以引入方式并入本申请。
技术领域
本发明属于生物检测领域，具体地，涉及一种捕获磁珠的制备方法。更具体地，涉及一种双特异性探针捕获磁珠制备方法。
背景技术
目前，基于复杂样本(如粪便样本)DNA的筛查技术，由于其同时具备非侵入性和高敏感性的优点，因此，被用来当做肠道微生物或癌症筛查的手段，多款基于DNA的肠癌筛查产品已获得国家药监局的批准，其检测靶标主要包括：Septin9、SDC2、BMP3、NDRG4、miRNA等。
与检测患者血浆、尿液或胸腹水中游离DNA的癌症早期筛查方法相比，检测患者的粪便样本具有更高的敏感度和准确性。然而，对于粪便样本而言，其样本中所含成分复杂，大多为各种类型的细菌和食物残渣，对靶标检测造成较大的背景干扰和抑制，并且癌细胞的含量较低，使靶标的检出率低且不稳定。目前，市面上的普通磁珠法提取试剂盒对于血浆、尿液或胸腹水这类样本的提取和纯化可以满足其后续检测需求，但对于粪便样本等复杂类样本，将对应的目的片段富集提取和纯化存在一定难度。
基于此，有研究将DNA/RNA捕获探针与羧基磁珠的羧基基团结合形成捕获磁珠，在复杂类型的样本中对特定序列进行抓取。然而，目前使用 的常规磁珠捕获探针为一段特定的核苷酸序列，长度30-50bp左右，长度越长其捕获特异性越高，但过长的序列会导致其自身形成折叠二聚体或发夹结构，影响后续磁珠捕获效率。尤其在检测类似于肠癌甲基化与非甲基化的靶标时，其特异性检测位点主要是CpG位点的差异，由于该位点的特殊性，在检测过程中有可能出现甲基化与非甲基化靶标仅存在1-3个碱基的差异，普通捕获探针在单个甚至3个的碱基发生错配时仍然能正常结合捕获，这种非特异性的捕获会导致后续捕获核酸量不足或目的片段检测不准确。
因此，本领域需要一种用于从复杂样本中特异提取靶标核酸的方法，其准确性高，灵敏度好。
发明内容
有鉴于此，第一方面，本发明提供一种双特异探针捕获磁珠的制备方法，其特征在于，所述方法包括：
将捕获磁珠与双特异性探针进行耦合，
其中，所述双特异性探针5’端采用伯氨基修饰，且所述探针5’端15～25bp区域位置引入次黄嘌呤；
其中，所述双特异性探针的长度为20～70bp。
在一些具体的实施方案中，所述次黄嘌呤的数量为1～8。
在一些具体的实施方案中，所述次黄嘌呤的数量为3～6。
在一些具体的实施方案中，所述次黄嘌呤的数量可以为1、2、3、4、5、6、7，或8。
在一些具体的实施方案中，所述捕获磁珠为羧基磁珠，所述双特异性探针5’端采用伯氨基修饰。
在一些具体的实施方案中，对羧基磁珠的羧基基团进行活化，包括：
S1、将磁珠置于磁力架上吸附；
S2、加入2-吗啉乙磺酸后振荡混匀，置于磁力架吸附。
更优选地，所述活化磁珠的羧基基团包括：重复S2步骤。
在一些具体的实施方案中，所述将羧基磁珠与双特异性探针进行耦合包括：
将双特异性探针与所述经活化的磁珠、2-吗啉乙磺酸、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐混合震荡孵育。
在一些具体的实施方案中，所述制备方法进一步包括：双特异性捕获磁珠的封闭。
在一些具体的实施方案中，所述封闭包括：1)耦合完成之后置于磁力架上吸附，并加入PBST溶液(磷酸盐吐温缓冲液)，含1％BSA(牛血清蛋白)，振荡混匀，进行封闭。
在一些具体的实施方案中，所述双特异性探针的靶标是SDC2基因、NDRG4基因、Septin9基因、β-actin基因和/或大肠杆菌(E.coli)。
在一些具体的实施方案中，所述双特异性探针的靶标是SDC2基因。
进一步地，该探针序列为：
5’-CGGCGTTTATTGGTTTTCGGAGTTGIIIITCGGCGTGTAA-3’(SEQ ID NO:1)。
在一些具体的实施方案中，所述双特异性探针的靶标是NDRG4基因。
进一步地，该探针序列为：
5’-TTTATCGGGTATTTTAGTCIIIIAGAAGGCGGAAGTTACG-3’(SEQ ID NO:2)。
在一些具体的实施方案中，所述双特异性探针的靶标是Septin9基因。
进一步地，该探针序列为：
5’-TTAGTTATTATGTCGGATTTIIIIGTTAACGCGTAGTTGG-3’(SEQ ID NO:3)。
在一些具体的实施方案中，所述双特异性探针的靶标是β-actin基因。
进一步地，该探针序列为：
5’-TTGTAATTTTTAAGGGAGGAGIIIITTTTATTGGTT-3’(SEQ ID NO:4)。
进一步地，该探针还可以靶标大肠杆菌，序列为
5’-AGTTTATCTGCAAGGTGAIIIITTAATTCCTCTCTTTCCT-3’(SEQ ID NO:5)。
使用本发明的磁珠制备方法，获得双特异性捕获磁珠能够更好的捕获靶标片段，保证捕获的稳定性，其灵敏度更高。对于复杂样本类型中低浓度核酸可显著提高检出率，且不需要多步样本纯化，减少操作步骤，对目标核酸进行快速高效的提取，同时，可以有效降低错配带来的错误捕获。
第二方面，本发明提供一种双特异性捕获磁珠，其特征在于，由上述的制备方法制备而成。
在一些具体的实施方案中，所述双特异性探针的靶标是SDC2基因、NDRG4基因、Septin9基因、β-actin基因和/或大肠杆菌。
在一些具体的实施方案中，所述双特异性探针的靶标是SDC2基因。
进一步地，该探针序列为：
5’-CGGCGTTTATTGGTTTTCGGAGTTGIIIITCGGCGTGTAA-3’(SEQ ID NO:1)。
在一些具体的实施方案中，所述双特异性探针的靶标是NDRG4基因。
进一步地，该探针序列为：
5’-TTTATCGGGTATTTTAGTCIIIIAGAAGGCGGAAGTTACG-3’(SEQ ID NO:2)。
在一些具体的实施方案中，所述双特异性探针的靶标是Septin9基因。
进一步地，该探针序列为：
5’-TTAGTTATTATGTCGGATTTIIIIGTTAACGCGTAGTTGG-3’(SEQ  ID NO:3)。
在一些具体的实施方案中，所述双特异性探针的靶标是β-actin基因。
进一步地，该探针序列为：
5’-TTGTAATTTTTAAGGGAGGAGIIIITTTTATTGGTT-3’(SEQ ID NO:4)。
进一步地，该探针还可以靶标大肠杆菌，序列为
5’-AGTTTATCTGCAAGGTGAIIIITTAATTCCTCTCTTTCCT-3’(SEQ ID NO:5)。
第三方面，本发明提供一种提取核酸试剂盒，其特征在于，包括如上所述的双特异性捕获磁珠。
更进一步地，本发明提供一种提取粪便样本核酸的试剂盒，其特征在于，包括如上所述的双特异性捕获磁珠。
在一些具体的实施方案中，所述试剂盒包括聚乙二醇。
优选地，所述聚乙二醇的浓度为15％～35％。
更优选地，所述聚乙二醇的浓度为20％～30％。
最优选地，所述聚乙二醇的浓度为20％。
使用合适浓度的聚乙二醇，能够增加所述双特异性捕获磁珠的捕获效率，进一步提升双特异性捕获磁珠捕获的灵敏度。
在一些具体的实施方案中，所述试剂盒进一步包括阳性对照和阴性对照。
第四方面，本发明提供一种如上所述特异性捕获磁珠用于制备提取核酸试剂盒的用途。
第五方面，本发明提供一种核酸提取方法，其包括，使用如上所述的双特异性捕获磁珠对样本进行提取。
进一步地，所述样本为粪便、尿液、血浆，或胸腹水。
所述样本优选为粪便。
附图说明
图1为SDC2基因不同类型修饰磁珠高浓度粪便样本核酸提取效果对比图；
图2为SDC2基因不同类型修饰磁珠低浓度粪便样本核酸提取效果对比图；
图3为β-actin基因不同类型修饰磁珠高浓度粪便样本核酸提取效果对比图；
图4为β-actin基因不同类型修饰磁珠低浓度粪便样本核酸提取效果对比图；
图5为SDC2基因不同类型修饰磁珠高浓度血浆样本核酸提取效果对比图；
图6为SDC2基因不同类型修饰磁珠低浓度血浆样本核酸提取效果对比图；
图7为β-actin基因不同类型修饰磁珠高浓度血浆样本核酸提取效果对比图；
图8为β-actin基因不同类型修饰磁珠低浓度血浆样本核酸提取效果对比图；
图9为SDC2基因不同类型修饰磁珠高浓度尿液样本核酸提取效果对比图；
图10为SDC2基因不同类型修饰磁珠低浓度尿液样本核酸提取效果对比图；
图11为β-actin基因不同类型修饰磁珠高浓度尿液样本核酸提取效果对比图；
图12为β-actin基因不同类型修饰磁珠低浓度尿液样本核酸提取效果对 比图；
图13为SDC2基因不同类型修饰磁珠高浓度胸腹水样本核酸提取效果对比图；
图14为SDC2基因不同类型修饰磁珠低浓度胸腹水样本核酸提取效果对比图；
图15为β-actin基因不同类型修饰磁珠高浓度胸腹水样本核酸提取效果对比图；
图16为β-actin基因不同类型修饰磁珠低浓度胸腹水样本核酸提取效果对比图；
图17为不同浓度PEG提取效果图；
图18为SDC2基因不同位置捕获探针核酸提取效果图；
图19为NDRG4基因不同位置捕获探针核酸提取效果图；
图20为Septin9基因不同位置捕获探针核酸提取效果图；
图21为Septin9引入1个错配碱基不同捕获探针核酸提取效果图；
图22为Septin9引入3个错配碱基不同捕获探针核酸提取效果图。
具体实施方式
下文将结合具体实施方案和实施例，具体阐述本发明，本发明的优点和各种效果将由此更加清楚地呈现。本领域技术人员应理解，这些具体实施方案和实施例是用于说明本发明，而非限制本发明。
实施例1、制备双特异性探针捕获磁珠
1.1羧基基团活化：
1)从4℃冰箱内取出1mL羧基磁珠置于磁力架上吸附2min，待溶液变澄清之后弃上清；
2)加入1mL 2-吗啉乙磺酸(MES，浓度0.05～0.1M，pH＝5.0～6.0)后振荡混匀5min，置于磁力架吸附2min，弃上清；
3)重复步骤2一次；
1.2羧基磁珠与伯氨基修饰的双特异性探针耦合：
1)从磁力架上取下离心管，分别加入10μL伯氨基修饰的双特异性探针(100μM，5’端采用伯氨基修饰，长度为20～70bp)、100μL 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(10-20mg/mL)、1mL 2-吗啉乙磺酸，其体积比为1:10:100；
2)将耦合混合液置于恒温金属震荡浴上，室温(20～31℃)振荡孵育1～2小时后，放置于4℃振荡6～10h(保持磁珠悬浮状态)；
1.3封闭与保存：
1)耦合完成之后置于磁力架上吸附2min，去掉上清液，加入1mL PBST溶液(磷酸盐吐温缓冲液)，pH＝7.0，含1％BSA(牛血清蛋白)，25℃振荡混匀1.5h，封闭磁珠表面未活化的羧基基团；
2)将封闭后的磁珠置于磁力架上，去上清，加入1mL 1％SDS洗涤液(pH＝7.0～7.5)去除未能耦合的探针,轻轻振荡混匀，室温静置1min后置于磁力架上吸附2min，弃上清；
3)重复步骤2一次；
4)向离心管加入1mL TE buffer，置于2～8℃保存。
实施例2、双特异性探针捕获磁珠提取样本核酸
2.1细胞裂解：
1)在10～15mL离心管中加入3mL细胞裂解液(10-20mmol/L EDTA,100mmol/L Tris,pH＝7.5-8.0,0.8％(W/V)SDS)和2-4mL样本(粪便样本离心后取上清液，尿液、腹水样本离心后取沉淀)；
2.2磁珠捕获：
1)95℃加热10-15min，加入50-100μL捕获磁珠和2mL 10％-40％聚乙二醇(pH＝6.0-7.0)，盖上管盖，振荡混匀2min，室温静置1h；
2)将离心管置于磁力架上，5min后缓慢吸弃废液；
2.3洗涤及保存：
1)加入2mL洗涤液1(2.0-3.0M盐酸胍，异丙醇，pH＝6.5-7.5)，震荡混匀30s，将离心管置于磁性分离器。磁吸3min，将液体完全吸弃；
2)重复步骤1一次；
3)加入2mL洗涤液2(70％-75％乙醇)，震荡混匀30s，将离心管置于磁性分离器。磁吸3min，将液体完全吸弃；
4)重复步骤3一次；
5)室温(20～30℃)开盖静置5min；
6)加入50μL预热至50℃的TE缓冲液(0.05-0.1M Tris-HCl，50mM EDTA，PH＝7.5-8.0)，震荡混匀30s，将离心管壁上的磁珠洗脱到管底，室温静置5 min洗脱，置于2～8℃保存。
实施例3、双特异性探针捕获探针提取靶标核酸
为验证双特异性探针捕获磁珠与普通捕获磁珠的区别，针对甲基化后的目的基因SDC2以及大肠杆菌制备捕获探针进行测试验证，同时加入人源性内标β-actin作为内参基因：
捕获SDC2基因甲基化的探针：
捕获序列：
5’-CGGCGTTTATTGGTTTTCGGAGTTGTTAATCGGCGTGTAA-3’(SEQ ID NO:6)；
捕获SDC2基因甲基化的双特异性探针：
捕获序列：
5’-CGGCGTTTATTGGTTTTCGGAGTTGIIIITCGGCGTGTAA-3’(SEQ ID NO:1)；
捕获大肠杆菌的探针：
捕获序列：
5’-AGTTTATCTGCAAGGTGATTCCTTAATTCCTCTCTTTCCT-3’(SEQ ID NO:7)；
捕获大肠杆菌的双特异性探针：
捕获序列：
5’-AGTTTATCTGCAAGGTGAIIIITTAATTCCTCTCTTTCCT-3’(SEQ ID NO:5)。
捕获β-actin基因甲基化的探针：
捕获序列：
5’-TTGTAATTTTTAAGGGAGGAGTAGGTTTTATTGGTT-3’(SEQ ID NO:8)；
捕获β-actin基因甲基化的双特异性探针：
捕获序列：5’-TTGTAATTTTTAAGGGAGGAGIIIITTTTATTGGTT-3’(SEQ ID NO:4)。
为验证特异性捕获探针对于甲基化阳性样本的提取效果，本实验需采用高低两个浓度核酸量的样本进行测试，由于无法在提取过程中对核酸量进行控制，因此通过人为定量加入特异性甲基化片段的方法进行测试，甲基化片段为人基因组全甲基化标准品，将亚硫酸盐转化后的甲基化标准品投入到阴性粪便上清液(图1～4)、血浆(图5～8)、尿液(图9～12)或胸腹水(图13～16)中制备成含有外源性干扰物的模拟样本，模拟样本中高浓度甲基化片段含量为100ng/μL，低浓度甲基化片段含量为0.1ng/μL，同时加入未甲基化的核酸片段作为背景信号，未甲基化片段含量为100ng/μL。
特异性捕获：
采用实施例1中制备的双特异性探针捕获磁珠、普通探针捕获磁珠以及羧基磁珠分别对高低浓度模拟样本进行提取，提取步骤按照实施例2操作进行，最后用50μL TE buffer洗脱核酸。
亚硫酸盐转化：
将实施例2中提取后的核酸进行亚硫酸盐转化，转化试剂盒为EZ DNA Methylation-lingthting Kit，转化步骤严格按照操作说明书进行，最后用50μL TE buffer洗脱转化后的核酸。
PCR扩增检测：
通过荧光PCR对提取核酸进行检测，以SDC2启动子区域设计甲基化引物探针进行测试验证，为保证检测准确性，同时设计人源性内标β-actin作为内参基因。
检测SDC2基因甲基化的引物：
正向引物：5’-CGTAGGAGTTTTGGTTTGTCG-3’(SEQ ID NO:9)
反向引物：5’-ACAATATAACTCCCAAATAAACCCG-3’(SEQ ID NO:10)
检测SDC2基因甲基化的探针：
荧光探针：5’-TTCGGAGTTGTTAATCGGCGTG-3’(SEQ ID NO:11)
检测大肠杆菌的引物：
正向引物：5’-TGTACAAGTCCACAAGGAAAGTAAAGAT-3’(SEQ ID NO:12)
反向引物：5’-TGTTTCGATGAGTTTATCTGCAAGGT-3’(SEQ ID NO:13)
检测大肠杆菌的探针：
荧光探针：5’-TCTAACTAGGACCGCAGAGGAAAGAGAGGAATT-3’(SEQ ID NO:14)
检测β-actin基因甲基化的引物：
正向引物：5’-GTGTGTTGGGTGGTGGTTATTT-3’(SEQ ID NO:15)
反向引物：5’-CCAAAAAAAAAACTACTTATTCCAATTCAC-3’(SEQ  ID NO:16)
检测β-actin基因甲基化的探针：
荧光探针：5’-TTAAGGGAGGAGTAGGTT-3’(SEQ ID NO:17)。
PCR反应体系和扩增体系如下表1所示：
表1、PCR反应体系表
组分 体积(μL/人份)
PCR buffer 补齐至50μL
MgCl 2(1mol/L) 0.2
dNTPs(100mM) 1
靶标正向引物(50μM) 0.2
靶标反向引物(50μM) 0.2
靶标荧光探针(50μM) 0.1
内标正向引物(50μM) 0.2
内标反向引物(50μM) 0.2
内标荧光探针(50μM) 0.1
DNA聚合酶(5U) 1
模板 10
表2、PCR荧光扩增程序表
Figure PCTCN2022119600-appb-000001
不同类型的磁珠提取不同样本核酸的效果如下表3～6所示。
表3、不同类型修饰磁珠扩增Ct值对比表(粪便样本)
Figure PCTCN2022119600-appb-000002
Figure PCTCN2022119600-appb-000003
表4、不同类型修饰磁珠扩增Ct值对比表(血浆样本)
Figure PCTCN2022119600-appb-000004
表5、不同类型修饰磁珠扩增Ct值对比表(尿液样本)
Figure PCTCN2022119600-appb-000005
表6、不同类型修饰磁珠扩增Ct值对比表(腹水样本)
Figure PCTCN2022119600-appb-000006
对于粪便样本，双特异性捕获磁珠中的靶标核酸均高于普通探针捕获磁珠以及羧基磁珠，Ct值更靠前。同时，特异性捕获磁珠相较于羧基磁珠 在复杂的样本类型中受抑制物影响较小，捕获效率更高，尤其是对低浓度的核酸样本检测更为明显，经修饰后的捕获磁珠能有效的结合目的片段，排除干扰物的影响，所以双特异性捕获磁珠与普通捕获磁珠均能有效区分靶标片段与干扰物。羧基磁珠提取效果最差，由于存在未甲基化的核酸片段干扰，羧基磁珠无法有效的选择性结合靶标片段，不管是在粪便、尿液、血浆或腹水样本中目的片段提取浓度均较低。
在尿液、血浆及腹水样本中，外源性干扰物较少，样本环境纯度明显高于粪便样本，所以双特异性捕获磁珠与普通探针捕获磁珠捕获效率无明显区别，均能捕获特异性靶标片段。
而对于大肠杆菌的捕获，其结果如表7所示。
表7、不同类型修饰磁珠扩增Ct值对比表(粪便样本)
Figure PCTCN2022119600-appb-000007
从表中可以看出，双特异性探针捕获磁珠对于大肠杆菌的捕获效果更好。
实施例4、双特异性探针捕获探针结合PEG提取靶标核酸
在双特异性捕获磁珠提取过程中，我们发现加入一定浓度的聚乙二醇可以增加捕获序列之间的互补结合率。通过在提取过程中加入不同浓度的聚乙二醇进行对比测试，结果如图17所示。从图中可以看出，加入20％和30％的PEG的提取效果要优于其余浓度的PEG。
实施例5、捕获探针不同位置引入次黄嘌呤对捕获效率的影响
为验证本发明捕获探针不同位置引入次黄嘌呤对捕获效率的影响，以 甲基化后的目的基因SDC2、NDRG4和Septin9作为捕获探针，同时在捕获序列的5’端、3’端及中间区域位置引入次黄嘌呤，对比捕获磁珠核酸提取效果.。其结果如图18～20所示，从图中可以看出，距离双特异性探针5’端15～25bp区域位置引入次黄嘌呤能够极大的提高捕获探针的灵敏度。
捕获SDC2基因甲基化的探针：
捕获序列：
5’-CGGCGTTTATTGGTTTTCGGAGTTGTTAATCGGCGTGTAA-3’(SEQ ID NO:6)；
捕获SDC2基因甲基化的双特异性探针1：
捕获序列：
5’-CGGCGTTTATTGGTTTTCGGAGTTGIIIITCGGCGTGTAA-3’(SEQ ID NO:1)；
捕获SDC2基因甲基化的双特异性探针2：
捕获序列：
5’-CIIIITTTATTGGTTTTCGGAGTTGTTAATCGGCGTGTAA-3’(SEQ ID NO:18)；
捕获SDC2基因甲基化的探针3：
捕获序列：
5’-CGGCGTTTATTGGTTTTCGGAGTTGTTAATCGGCGIIIIA-3’(SEQ ID NO:19)。
捕获NDRG4基因甲基化的探针：
捕获序列：
5’-TTTATCGGGTATTTTAGTCGCGTAGAAGGCGGAAGTTACG-3’(SEQ ID NO:20)；
捕获NDRG4基因甲基化的双特异性探针1：
捕获序列：
5’-TTTATCGGGTATTTTAGTCIIIIAGAAGGCGGAAGTTACG-3’(SEQ ID NO:2)；
捕获NDRG4基因甲基化的双特异性探针2：
捕获序列：
5’-TTIIIIGGGTATTTTAGTCGCGTAGAAGGCGGAAGTTACG-3’(SEQ ID NO:21)；
捕获NDRG4基因甲基化的双特异性探针3：
捕获序列：
5’-TTTATCGGGTATTTTAGTCGCGTAGAAGGCGGAAIIIICG-3’(SEQ ID NO:22)。
捕获Septin9基因甲基化的探针：
捕获序列：
5’-TTAGTTATTATGTCGGATTTCGCGGTTAACGCGTAGTTGG-3’(SEQ ID NO:23)；
捕获Septin9基因甲基化的双特异性探针1：
捕获序列：
5’-TTAGTTATTATGTCGGATTTIIIIGTTAACGCGTAGTTGG-3’(SEQ ID NO:3)；
捕获Septin9基因甲基化的双特异性探针2：
捕获序列：
5’-TTIIIIATTATGTCGGATTTCGCGGTTAACGCGTAGTTGG-3’(SEQ ID NO:24)；
捕获Septin9基因甲基化的双特异性探针3：
捕获序列：
5’-TTAGTTATTATGTCGGATTTCGCGGTTAACGCGTIIIIGG-3’(SEQ ID NO:25)。
对比例1、不同捕获探针的捕获准确性
为验证本发明捕获探针在粪便样本中的捕获准确性，以结直肠癌甲基化后的目的基因Septin9作为捕获目的片段，在合成的甲基化片段中引入1～3个错配碱基。将引入错配后的片段投入到阴性粪便上清液中制备成模拟样本，模拟样本中错配的甲基化片段含量为100ng/μL，同时采用双特异性磁珠、普通磁珠以及羧基磁珠对比核酸提取效果：
引入1个错配碱基的Septin9基因甲基化的序列：
错配序列：
5’-TTAGTTATTATGT TGGATTTCGCGGTTAACGCGTAGTTGG-3’(SEQ ID NO:26)；
引入3个错配碱基的Septin9基因甲基化的序列：
错配序列：
5’-TTAGTTATTATGT TGGATTTCGCGGTTAA TG TGTAGTTGG-3’(SEQ ID NO:27)。
表8、不同捕获磁珠提取Ct值对比表
Figure PCTCN2022119600-appb-000008
从图21～22和表8可以看出羧基磁珠无法区分核酸序列准确性，只要有核酸存在下即可结合，所以检测结果核酸浓度最高；普通捕获磁珠在序列仅存在1～3个碱基的区别时，仍有少量错配序列与捕获探针结合，使检测结果为阳性；双特异性捕获磁珠由于引入次黄嘌呤，使捕获序列特异性 增强，对于序列中存在错配碱基而无法正常与磁珠结合，所以检测结果为阴性，其捕获准确性最高。因此，可以看出，本发明的双特异性探针捕获准确性远高于普通探针和羧基磁珠，特别是当只有1个碱基的错配时，捕获准确性显著提高。

Claims (10)

  1. 一种双特异探针捕获磁珠的制备方法，所述方法包括：
    将捕获磁珠与双特异性探针进行耦合，
    其中，所述双特异性探针5’端15bp～25bp区域位置引入次黄嘌呤；
    其中，所述双特异性探针的长度为20～70bp。
  2. 根据权利要求1所述的方法，其中，所述双特异性探针的靶标是SDC2基因、NDRG4基因、Septin9基因、β-actin基因，和/或大肠杆菌。
  3. 根据权利要求2所述的方法，其中，所述双特异性探针的序列为SEQ ID NO:1～5所示序列中的一项或多项。
  4. 根据权利要求1～3中任一项所述的方法，其中，所述捕获磁珠为羧基磁珠，所述双特异性探针5’端采用伯氨基修饰。
  5. 根据权利要求4所述的方法，其中，将羧基磁珠与双特异性探针进行耦合包括：
    S1、将磁珠置于磁力架上吸附；
    S2、加入2-吗啉乙磺酸后振荡混匀，置于磁力架吸附进行活化；
    S3、将双特异性探针与步骤S2中经活化的羧基磁珠、2-吗啉乙磺酸、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐孵育。
  6. 一种双特异探针捕获磁珠，其中，由如权利要求1～5中任一项所述的制备方法制备而成。
  7. 一种提取核酸试剂盒，其中，包括权利要求6所述的双特异性探针捕获磁珠。
  8. 根据权利要求7所述的试剂盒，其中，所述试剂盒包括聚乙二醇。
  9. 根据权利要求7所述的试剂盒，其中，所述聚乙二醇的浓度为20％～30％。
  10. 一种如权利要求6所述的双特异性探针捕获磁珠用于制备提取核酸试剂盒的用途。
PCT/CN2022/119600 2022-07-20 2022-09-19 一种双特异性探针捕获磁珠及其制备方法和应用 WO2024016461A1 (zh)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108676878A (zh) * 2018-05-23 2018-10-19 杭州诺辉健康科技有限公司 基于ndrg4基因甲基化序列的结直肠癌早期检测方法
US20190010557A1 (en) * 2015-12-31 2019-01-10 Creative Biosciences (Guangzhou) Co., Ltd. Molecular detection/diagnosis reagent for tumor
CN110564831A (zh) * 2019-08-30 2019-12-13 北京优迅医学检验实验室有限公司 用于测序文库的封闭试剂及提高靶向捕获效率的方法
CN110699427A (zh) * 2019-10-25 2020-01-17 江苏为真生物医药技术股份有限公司 羧基磁珠偶联修饰的核酸探针及其制备方法和应用
CN114657232A (zh) * 2022-03-11 2022-06-24 上海英基生物科技有限公司 一种用于提高靶向捕获效率的通用封闭试剂及其应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190010557A1 (en) * 2015-12-31 2019-01-10 Creative Biosciences (Guangzhou) Co., Ltd. Molecular detection/diagnosis reagent for tumor
CN108676878A (zh) * 2018-05-23 2018-10-19 杭州诺辉健康科技有限公司 基于ndrg4基因甲基化序列的结直肠癌早期检测方法
CN110564831A (zh) * 2019-08-30 2019-12-13 北京优迅医学检验实验室有限公司 用于测序文库的封闭试剂及提高靶向捕获效率的方法
CN110699427A (zh) * 2019-10-25 2020-01-17 江苏为真生物医药技术股份有限公司 羧基磁珠偶联修饰的核酸探针及其制备方法和应用
CN114657232A (zh) * 2022-03-11 2022-06-24 上海英基生物科技有限公司 一种用于提高靶向捕获效率的通用封闭试剂及其应用

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