WO2023236037A1 - 一种hpv核酸检测试剂盒及其制备方法与应用 - Google Patents

一种hpv核酸检测试剂盒及其制备方法与应用 Download PDF

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WO2023236037A1
WO2023236037A1 PCT/CN2022/097378 CN2022097378W WO2023236037A1 WO 2023236037 A1 WO2023236037 A1 WO 2023236037A1 CN 2022097378 W CN2022097378 W CN 2022097378W WO 2023236037 A1 WO2023236037 A1 WO 2023236037A1
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probe
sequence
seq
nucleic acid
end region
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PCT/CN2022/097378
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French (fr)
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陈嘉昌
张源明
王辉芳
刘向东
李楚明
唐海辉
王维世
胡朝晖
柳俊
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广州市金圻睿生物科技有限责任公司
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    • C—CHEMISTRY; METALLURGY
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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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
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    • C12Q1/686—Polymerase chain reaction [PCR]
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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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes

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  • the invention relates to the field of biomedicine, and in particular to an HPV nucleic acid detection kit and its preparation method and application.
  • HPV Human papillomavirus
  • the 18 medium- and high-risk types of human papillomavirus include: 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56 , 58, 59, 66, 68, 73, 82; low-risk human papillomavirus (HPV) includes 6, 11, 40, 42, 43, 81, 83, etc.
  • HPV pathogens There are many techniques for detecting HPV pathogens. The main methods include acetic acid staining screening, iodine test screening, Pap smear examination, liquid-based cytology, colposcopy, histological testing, molecular testing, etc.
  • clinical features and routine laboratory tests require interpretation by professional doctors and take a long time.
  • the CN111020061B patent discloses a kit and method for detecting human papillomavirus using high-throughput sequencing, which can detect up to 40 types of HPV simultaneously.
  • high-throughput sequencing By interpreting the read number of the detection results, it is possible to avoid the missed detection of low-abundance types due to the suppression of low-abundance by high abundance, but this requires a lot of manpower and material resources, and the operator needs a rich experimental foundation.
  • the entire experimental process takes a long time, and the results also require professional bioinformatics analysis and other limitations.
  • the CN101487042A patent discloses a method and kit for multiplex detection of HPV using a solid-phase carrier substrate and oligonucleotide probes of each subtype of HPV to prepare a DNA microarray chip.
  • a solid-phase carrier substrate By designing specific types of primers and probes and combining them with chips to form an array, high-throughput detection of HPV types can be achieved.
  • its method is limited by limitations such as high cost and high process requirements caused by the use of chips.
  • real-time detection mode target sequence detection and PCR amplification are performed simultaneously without additional steps. Therefore, the real-time detection mode is simple and straightforward. However, the maximum number of target sequences that this mode can detect in a single-tube detection is limited by the number of fluorescence detection channels of the real-time PCR instrument, which generally does not exceed 6. Therefore, based on the advantages of simple and direct methodology, it is necessary to improve the real-time fluorescence quantitative PCR method in order to detect more target genes in a single tube detection.
  • a dual probe combination for multiplex nucleic acid detection which includes a U probe and a B probe targeting a target gene sequence.
  • the composition of the U probe from the 5' end to the 3' end is: 5' end region, Stem loop region, 3' end region; the composition of the B probe from the 5' end to the 3' end is: 5' end region, 3' end region; the 5' end region sequence of the U probe is a specific target
  • the sequence is complementary to the gene sequence.
  • the 3' end region sequence is the only sequence in nature.
  • the length of the stem loop region sequence is 5bp-45bp.
  • the 5' end of the stem loop region sequence is modified with a fluorescent quenching group, and the 3' end of the stem loop region sequence is modified with fluorescence.
  • Another object of the present invention is to provide a multiplex nucleic acid detection kit, which includes an amplification primer set targeting the target nucleic acid sequence and the above-mentioned dual probe combination.
  • Another object of the present invention is to provide the application of the above-mentioned multiplex nucleic acid detection kit in the detection of human papillomavirus.
  • Another object of the present invention is to provide a human papillomavirus detection method.
  • the inventor of the present invention conducted in-depth research on the genetic information of human papillomavirus (HPV) and designed a dual detection probe combination of U probe and B probe targeting the HPV gene sequence.
  • HPV human papillomavirus
  • the precise design of the U probe and B probe structures makes the 3' end of the U probe and the 5' end of the B probe reversely complementary when detecting specific HPV types.
  • the 5' end of the U probe needs to be Matching the template
  • the 3' end of the B probe needs to match the template for successful detection, which is sufficient to ensure detection specificity.
  • the 3' end of the 5' end region sequence of the U probe was 5
  • the probe detection performance is best.
  • the relative position of the quenching group and the fluorescent group on the U probe is 10 bp to 45 bp, it can also It effectively avoids the poor quenching effect caused by the relative position of the quenching group and the fluorescent group being too far, that is, the background fluorescence becomes stronger, resulting in poorer actual detection results.
  • Figure 8 is a schematic diagram of the detection of HPV16 type samples in Example 4 when the relative position of the U probe and the B probe is 10 bp.
  • Figure 10 is a schematic diagram of the detection of HPV16 type samples in Example 5 when the relative position of the quenching group and the fluorescent group on the U probe is 5 bp.
  • Figure 11 is a schematic diagram of the detection of HPV16 type samples in Example 5 when the relative position of the quenching group and the fluorescent group on the U probe is 10 bp.
  • Figure 12 is a schematic diagram of the detection of HPV16 type samples in Example 5 when the relative position of the quenching group and the fluorescent group on the U probe is 25 bp.
  • Figure 13 is a schematic diagram of the detection of HPV16 type samples in Example 5 when the relative position of the quenching group and the fluorescent group on the U probe is 45 bp.
  • Some embodiments of the present invention provide a dual probe combination for multiplex nucleic acid detection, which includes a U probe and a B probe targeting the target gene sequence, and the U probe is composed from the 5' end to the 3' end.
  • the sequence is: 5' end region, stem-loop region, 3' end region; the composition of the B probe from the 5' end to the 3' end is: the 5' end region, the 3' end region; the U probe 5
  • the 'end region sequence is a sequence complementary to the specific target gene sequence, the 3' end region sequence is the only sequence in nature, the length of the stem loop region sequence is 10-45bp, the 5' end of the stem loop region sequence is modified with a fluorescence quenching group, and the stem loop region sequence is modified with a fluorescence quenching group.
  • the 3' end of the loop region sequence is modified with a fluorescent reporter group;
  • the 3' end region sequence of the B probe is a sequence complementary to the target gene sequence, and the 5' end region sequence is a natural sequence that is reverse complementary to the 3' end region sequence of the U probe.
  • the only sequence in the sequence; when the U probe and the B probe are combined with the target fragment at the same time, the distance between the 3' end of the 5' end region sequence of the U probe and the 5' end of the 5' end region sequence of the B probe The distance is 5 bp to 15 bp; the unique sequence in nature is a sequence that does not hybridize with the target gene sequence.
  • the above-mentioned U probe and B probe are combined with the target fragment at the same time, there is a gap between the 3' end of the 5' end region sequence of the above mentioned U probe and the 5' end of the 5' end region sequence of the B probe.
  • the distance is 5bp ⁇ 10bp.
  • the distance between the 5' end and the 3' end of the stem-loop region sequence of the above-mentioned U probe is 5 bp to 45 bp. It is further preferably 10 bp to 45 bp.
  • the distance between the fluorescent quenching group modified at the 5' end of the stem-loop region sequence of the above-mentioned U probe and the fluorescent reporter group modified at the 3' end is most appropriate, thereby effectively avoiding quenching
  • the quenching effect caused by the relative position of the group and the fluorescent group being too far is poor, that is, the background fluorescence becomes stronger, resulting in a worse actual detection effect.
  • Some embodiments of the present invention also provide the application of the above-mentioned dual probe combination in preparing a kit or a kit detection reagent.
  • Some embodiments of the present invention also provide a multiplex nucleic acid detection kit, which includes an amplification primer set targeting the target nucleic acid sequence and the above-mentioned dual probe combination.
  • the above-mentioned amplification primer set includes an L primer pair, and the composition of each L primer in the L primer pair from the 5' end to the 3' end is: 5' end region, 3' end region; The sequence length of the 5' end region is 15-25 bp; the 3' end region is a sequence complementary to the specific target gene sequence.
  • the above-mentioned amplification primer set also includes a universal primer pair, and further, each universal primer in the universal primer pair is reverse to the 5' end region sequence of each L primer in the L primer pair. complementary.
  • the above-mentioned multiplex nucleic acid detection kit includes at least one of the following components: The first group: L primer pair, U probe and B probe for HPV16, wherein the L primer pair sequence is such as SEQ ID NO.1 ⁇ SEQ ID NO.2 is shown, the U probe sequence is shown in SEQ ID NO.3, the B probe sequence is shown in SEQ ID NO.4; second group: L primer pair, U for HPV18 Probe and B probe, among which, the sequence of L primer pair is as shown in SEQ ID NO.5 ⁇ SEQ ID NO.6, the sequence of U probe is as shown in SEQ ID NO.7, and the sequence of B probe is as shown in SEQ ID NO.
  • the third group L primer pair, U probe and B probe for HPV58, among which, the sequence of L primer pair is as shown in SEQ ID NO.9 ⁇ SEQ ID NO.10, and the sequence of U probe is as shown in SEQ ID NO.11 is shown, and the B probe sequence is as SEQ ID NO.12;
  • Group 4 L primer pair, U probe and B probe for HPV52, among which, the L primer pair sequence is as SEQ ID NO. 13 ⁇ SEQ ID NO.14 is shown, the U probe sequence is shown in SEQ ID NO.15, and the B probe sequence is shown in SEQ ID NO.16.
  • the above-mentioned multiplex nucleic acid detection kit also includes a universal primer pair. Further, the sequences of the universal primer pairs are as shown in SEQ ID NO. 47 ⁇ SEQ ID NO. 48.
  • Some embodiments of the present invention also provide the application of the above multiplex nucleic acid detection kit in human papillomavirus detection.
  • Some embodiments of the present invention also provide a human papillomavirus detection method.
  • nucleic acid from the biological sample to be tested
  • multiplex nucleic acid detection is achieved by performing melting curve analysis on the reaction product obtained by the above-mentioned PCR detection to determine the type of human papillomavirus nucleic acid sequence present in the reaction system.
  • the reaction procedure of the above-mentioned PCR detection is landing PCR.
  • the biological sample is selected from serum, plasma, whole blood, fresh tissue, formalin-fixed paraffin-embedded tissue, urine, bacterial culture, viral culture, cell line culture, synthetic of plasmids.
  • the above-mentioned biological sample nucleic acid is ribonucleic acid
  • the reaction system also includes reverse transcriptase
  • the reaction program also includes reverse transcription PCR.
  • LF and LR respectively contain sequences complementary to the specific target gene sequence and unique sequences in nature (universal primers).
  • the U probe is divided into two parts: the specific target sequence and the unique sequence in nature.
  • the target gene region of the U probe It is complementary to the specific target sequence, and its 3-terminal end is connected to the artificially introduced sequence (stem-loop region and 3' end).
  • the B probe is divided into two parts: the specific target sequence and the unique sequence in nature.
  • the target gene region of the B probe is the same as
  • the specific target sequence is complementary, and its 5-terminal end is connected to an artificially introduced sequence, and the unique sequence in nature in the B probe is complementary to the unique sequence in nature at the 3-terminal end of the U probe.
  • L primer consists of 2 parts.
  • the 5’ end is a sequence of 15 to 25 bases that is unique in nature and serves as a universal primer, and the 3’ end is a sequence complementary to the specific target gene sequence.
  • B probe consists of 2 parts.
  • the 5’ end is a unique sequence in nature, which is complementary to some of the unique sequences in nature in the U probe; the 3’ end is a sequence complementary to the specific target gene sequence.
  • U probe and B probe the relative position of the two probes is 5-10bp.
  • the U probe is a fluorescent probe.
  • the 5' end is a sequence complementary to the specific target gene sequence.
  • the 3' end is a unique sequence in nature. It is then modified with C3 to block non-specific extension.
  • the 5' end is the only sequence in nature.
  • a fluorescent quenching group is used to provide the quenching function, and a fluorescent reporter group is used in the middle position to perform fluorescence analysis. Under natural conditions or when matched with a template, the fluorescence signal emitted by the reporter group is absorbed by the quenching group due to the principle of energy resonance transfer, and no fluorescence signal is generated.
  • the 5-terminal region of the B probe is complementary to the partial sequence of the U probe's unique sequence in nature, and an extension reaction is performed under the action of nucleic acid polymerase.
  • step (3) Since the PCR product produced in step (3) is in a double-stranded state, the fluorescent group and the quenching group are separated to generate fluorescence, so melting curve analysis can be performed, and according to the results of the melting curve analysis, each Whether the target nucleotide sequences to be tested are present in the sample, and then it is determined whether the human papillomavirus corresponding to each target nucleic acid sequence is present in the sample to be tested.
  • Example 2 An HPV typing detection kit
  • PCR primer-probe sequence is shown in Table 2-1 below to determine the Tm
  • the amplified sequence information of the value is shown in Table 2-2 below:
  • serial number Components volume 1 5 ⁇ PCR Buffer 5 ⁇ L 2 DNA polymerase (5U/ ⁇ L) 5 ⁇ L 3 dNTPs(20mM) 1 ⁇ L 4 UDG enzyme (1U/ ⁇ L) 1 ⁇ L 5 MFR(1pmol) 1 ⁇ L 6 MUB(1pmol) 1 ⁇ L
  • the PCR reaction program is set according to the following table 2-4:
  • the primer and probe design of the detection system for the detection of HPV16 type alone was taken as an example.
  • the PCR primer and probe sequences are as follows As shown in Table 4-1 (the primer sequence is the same as that in Example 2):
  • the “-” in the table indicates the position of the modifying group and the name of the modifying group.
  • the detection is taken as an example of the primer and probe design of the detection system for detecting HPV16 type alone.
  • the PCR primer and probe sequences are shown in Table 5-1 below (the primer sequences are the same as those in Example 2):
  • the "-" in the table indicates the position of the modifying group and the name of the modifying group, and the font in the sequence list is tilted to indicate the base where the quenching group is connected, and the font in the sequence list is tilted and bold to indicate the base. is the location where the fluorophore is attached.

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Abstract

本发明公开了针对HPV基因序列设计的U探针和B探针的双探针组合及其应用，含有针对HPV基因序列设计的U探针和B探针的双探针组合以及L性引物和通用引物体系的多重核酸检测试剂盒及其应用。还公开了基于上述试剂盒的检测HPV的方法。

Description

一种HPV核酸检测试剂盒及其制备方法与应用 技术领域
本发明涉及生物医药领域，特别是涉及一种HPV核酸检测试剂盒及其制备方法与应用。
背景技术
人乳头瘤病毒(Human papillomavirus，HPV)是一种诱发外生殖器良性病变和女性子宫颈癌的主要病原体。依据不同型别与宫颈癌发生危险性的高低可将其分为低危型和中高危型HPV。高危型人乳头瘤病毒的持续感染或反复感染是造成宫颈癌及宫颈上皮内高级别病变(CIN2/3)的主要原因。
根据CFDA对人乳头瘤病毒(HPV)的划分，18种中高危型人乳头瘤病毒(HPV)包括：16、18、26、31、33、35、39、45、51、52、53、56、58、59、66、68、73、82；低危型人乳头瘤病毒(HPV)包括6、11、40、42、43、81、83等。HPV病原体检测技术有多种，主要方法有醋酸染色筛查法、碘试验筛查法、巴氏涂片检查、液基细胞学、阴道镜检查、组织学检测、分子检测等。然而，临床特征和常规的实验室检测需要专业的医生进行判读，且花费时间较长。
CN111020061B专利中公开了一种使用高通量测序检测人乳头瘤病毒的试剂盒和方法，其可以同时检测多达40种HPV。通过对检测结果的reads数进行判读，能避免由于高丰度对低丰度抑制而造成的低丰度型别漏检，但需要花费大量的人力物力操作，且操作者需要丰富的实验基础，整个实验流程耗时长，对于结果也需要专业的生物信息学分析等局限性。
CN103184294A专利中公开了一种使用PCR法同时检测HPV16和HPV18型别的多重检测方法和试剂盒。其针对HPV16，18的DNA序列分别设计了2对特异性引物和2条Taqman引物探针，利用不同的荧光基团组合进行标记探针，从而在同一扩增管中准确的检测并区分出HPV16，18感染。然而其具有极大地受限于实时荧光PCR仪检测荧光通道数量的限制，而导致通量低的局限性，难 以满足种类繁多的HPV检测需求。
CN101487042A专利中公开了一种采用固相载体基片，配合HPV各亚型的寡核苷酸探针，制备成DNA微阵列芯片，对HPV进行多重检测的方法和试剂盒。其通过设计特异型别的引物探针与芯片结合组成阵列，可以实现高通量检测HPV型别的效果。但其方法受限于芯片的使用导致的高成本、高工艺要求等局限性。
国内外大量文献和专利均已报道，采用分子检测技术，特别是PCR检测技术，具有检测快速、操作简捷、特异性好等显著优点。实时荧光定量PCR方法是一种分子生物学中常用的核酸检测方法，与普通PCR方法相比，其操作简便，应用广泛。对待测样本的靶基因进行PCR扩增过程中，通过仪器实时监测反应体系产生的荧光信号，对PCR进程进行实时检测。普通PCR扩增技术需要在完成扩增之后，对产物进行电泳分析，分析过程繁琐费时，同时PCR产物的开盖可能导致实验室环境的污染。
实现荧光信号的方法主要分为探针法和染料法两类，探针法通过在反应体系中添加荧光基团标记的寡核苷酸探针，通过探针的信号释放，实现对靶序列的特异性检测；染料法则是添加双链DNA荧光染料在反应体系中，通过荧光染料结合到DNA双链小沟中，实现对靶序列的检测；
在实时检测模式中，靶序列的检测和PCR扩增同时进行，无需额外的步骤。因此，实时检测模式简便直接。但是，这种模式在单管检测中能检测的最大靶序列的数目受限于实时PCR仪器的荧光检测通道的数目，一般不超过6个。因此，基于简便直接的方法学优势，需要对实时荧光定量PCR法进行改进，以期实现单管检测中检测更多的靶基因数目。
综述所述，虽然已经报道了多种人乳头瘤病毒检测方法，但是都有各自的局限性。因此，仍然需要开发一种更为快速简便、灵敏特异、成本低的高通量人乳头瘤病毒核酸检测方法。
发明内容
基于此，本发明的目的之一在于提供一种用于多重核酸检测的双探针组合。
包括如下技术方案：
一种用于多重核酸检测的双探针组合，其包括针对靶基因序列的U探针和B探针，所述U探针从5’端到3’端的组成依次为：5’端区、茎环区、3’端区；所述B探针从5’端到3’端的组成依次为：5’端区、3’端区；所述U探针5’端区序列为特异性靶基因序列互补的序列，3’端区序列为自然界中唯一序列，茎环区序列长度为5bp-45bp，茎环区序列5’末端修饰荧光淬灭基团，茎环区序列3’末端修饰荧光报告基团；所述B探针3’端区序列为靶基因序列互补的序列，5’端区序列为与U探针3’端区序列反向互补的自然界中唯一序列；所述U探针和B探针同时与目的片段结合时，所述U探针5’端区序列的3’端与B探针5’端区序列的5’端之间的距离为5bp～15bp；所述自然界中唯一序列为不与靶基因序列杂交的序列。
本发明的另一目的还在于提供上述双探针组合在制备试剂盒或试剂盒检测试剂中的应用。
本发明的另一目的还在于提供一种多重核酸检测试剂盒，其包括针对目标核酸序列的扩增引物组和上述双探针组合。
本发明的另一目的还在于提供上述多重核酸检测试剂盒在人乳头瘤病毒检测中的应用。
本发明的另一目的还在于提供一种人乳头瘤病毒检测方法。
包括如下技术方案：
获取待测生物样本核酸；
采用上述多重核酸检测试剂盒对上述生物样本核酸进行PCR检测。
本发明的发明人基于对人乳头瘤病毒(Human papillomavirus，HPV)的遗传信息进行了深入研究，设计了一种针对HPV基因序列的U探针和B探针的双检测探针组合，通过对U探针和B探针结构的精密设计，使得在针对特定HPV型别检测时，U探针的3’端和B探针的5’端反向互补，同时U探针的5’端需与 模板匹配、B探针的3’端需与模板匹配方可进行成功检测，从而足以保证检测特异性。并结合特异针对U探针和B探针相关位置距离的优化筛选发现，当U探针和B探针同时与目的片段结合，U探针5’端区序列的3’端与B探针5’端区序列的5’端之间的距离为5bp～10bp时，探针检测的性能最佳，同时当U探针上淬灭基团与荧光基团的相对位置为10bp～45bp时还能有效避免因淬灭基团与荧光基团相对位置太远而导致的淬灭效果较差，即本底荧光变强，导致真实检测效果变差。通过上述检测体系有效克服传统实时荧光定量PCR分型局限性，通过特殊信号和熔解曲线分析实现单管多重分型，并且通过各自与模板的匹配以及U探针与B探针之间的互补，极大地提高了检测特异性。
并基于此，开发了一种多重核酸检测试剂盒和单管检测多靶标的PCR熔解曲线方法，通过引入L型引物和通用引物体系：保证扩增的均一性；消除了由于特异性引物之间扩增效率的差异，导致的扩增产物中不同扩增子的差异。此方法操作时间短，3小时内即可完成检测，检测过程中不需要开盖，可减少PCR产物污染，且检测特异性高，可以减少PCR反应中非特异性扩增，提高方法学检测灵敏度，结果易判读。
附图说明
图1为本发明HPV核酸检测试剂盒中引物探针结构设计原理图。
图2为实施例2中采用本发明试剂盒检测HPV16型别样本的结果图。
图3为实施例2中采用本发明试剂盒检测HPV18型别样本的结果图。
图4为实施例2中采用本发明试剂盒检测HPV52型别样本的结果图。
图5为实施例2中采用本发明试剂盒检测HPV58型别样本的结果图。
图6为实施例2中采用本发明试剂盒检测多重样本的结果图，即在FAM通道，能同时检出特异的HPV16、HPV18、HPV58、HPV52型别熔解峰。
图7为实施例4中针对U探针和B探针的相对位置5bp时，针对HPV16型别样本检测示意图。
图8为实施例4中针对U探针和B探针的相对位置10bp时，针对HPV16 型别样本检测示意图。
图9为实施例4中针对U探针和B探针的相对位置15bp时，针对HPV16型别样本检测示意图。
图10为实施例5中针对U探针上淬灭基团与荧光基团的相对位置5bp时，针对HPV16型别样本检测示意图。
图11为实施例5中针对U探针上淬灭基团与荧光基团的相对位置10bp时，针对HPV16型别样本检测示意图。
图12为实施例5中针对U探针上淬灭基团与荧光基团的相对位置25bp时，针对HPV16型别样本检测示意图。
图13为实施例5中针对U探针上淬灭基团与荧光基团的相对位置45bp时，针对HPV16型别样本检测示意图。
具体实施方式
下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。实施例中所用到的各种常用化学试剂，均为市售产品。
除非另有定义，本发明所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不用于限制本发明。本发明所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
此外，如本发明所使用的，术语“或”是包含性的“或”符号，并且等同于术语“和/或”，除非上下文另有明确规定。
为了便于理解本发明，下面将对本发明进行更全面的描述。本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使对本发明公开内容的理解更加透彻全面。
以下结合具体实施例对本发明作进一步详细的说明。
本发明的一些实施例提供了一种用于多重核酸检测的双探针组合，其包括针对靶基因序列的U探针和B探针，所述U探针从5’端到3’端的组成依次为：5’端区、茎环区、3’端区；所述B探针从5’端到3’端的组成依次为：5’端区、3’端区；所述U探针5’端区序列为特异性靶基因序列互补的序列，3’端区序列为自然界中唯一序列，茎环区序列长度为10-45bp，茎环区序列5’末端修饰荧光淬灭基团，茎环区序列3’末端修饰荧光报告基团；所述B探针3’端区序列为靶基因序列互补的序列，5’端区序列为与U探针3’端区序列反向互补的自然界中唯一序列；所述U探针和B探针同时与目的片段结合时，所述U探针5’端区序列的3’端与B探针5’端区序列的5’端之间的距离为5bp～15bp；所述自然界中唯一序列为不与靶基因序列杂交的序列。
在其中一些实施例中，上述U探针和B探针同时与目的片段结合时，上述U探针5’端区序列的3’端与B探针5’端区序列的5’端之间的距离为5bp～10bp。
在其中一些实施例中，上述U探针的茎环区序列5’末端与3’末端之间的距离为5bp～45bp。进一步优选为10bp～45bp，此时上述U探针的茎环区序列5’末端修饰的荧光淬灭基团与3’末端修饰荧光报告基团之间的距离最为合适，从而有效避免因淬灭基团与荧光基团相对位置太远而导致的淬灭效果较差，即本底荧光变强，导致真实检测效果变差。
本发明的一些实施例还提供了上述双探针组合在制备试剂盒或试剂盒检测试剂中的应用。
本发明的一些实施例还提供了一种多重核酸检测试剂盒，其包括针对目标核酸序列的扩增引物组和上述双探针组合。
在其中一些实施例中，上述扩增引物组包括L引物对，所述L引物对中的每一条L引物从5’端到3’端的组成依次为：5’端区、3’端区；所述5’端区序列长度为15～25bp；所述3’端区为特异性靶基因序列互补的序列。
在其中一些实施例中，上述扩增引物组还包括通用引物对，进一步地，所述通用引物对中的每一条通用引物与L引物对中的每一条L引物的5’端区序列反向互补。
在其中一些实施例中，上述多重核酸检测试剂盒包括以下任意至少一组组分：第一组：针对HPV16的L引物对、U探针和B探针，其中，L引物对序列如SEQ ID NO.1～SEQ ID NO.2所示，U探针序列如SEQ ID NO.3所示，B探针序列如SEQ ID NO.4所示；第二组：针对HPV18的L引物对、U探针和B探针，其中，L引物对序列如SEQ ID NO.5～SEQ ID NO.6所示，U探针序列如SEQ ID NO.7所示，B探针序列如SEQ ID NO.8所示；第三组：针对HPV58的L引物对、U探针和B探针，其中，L引物对序列如SEQ ID NO.9～SEQ ID NO.10所示，U探针序列如SEQ ID NO.11所示，B探针序列如SEQ ID NO.12所示；第四组：针对HPV52的L引物对、U探针和B探针，其中，L引物对序列如SEQ ID NO.13～SEQ ID NO.14所示，U探针序列如SEQ ID NO.15所示，B探针序列如SEQ ID NO.16所示。在其中一些实施例中，上述多重核酸检测试剂盒还包括通用引物对，进一步地，所述通用引物对序列如SEQ ID NO.47～SEQ ID NO.48所示。
本发明的一些实施例还提供了上述多重核酸检测试剂盒在人乳头瘤病毒检测中的应用。
本发明的一些实施例还提供了一种人乳头瘤病毒检测方法。
包括如下技术方案：
获取待测生物样本核酸；
采用上述多重核酸检测试剂盒对上述生物样本核酸进行PCR检测。
在其中的一些实施例中，通过对上述PCR检测得到的反应产物进行熔解曲线分析，从而判断反应体系中所存在人乳头瘤病毒核酸序列的种类，实现多重核酸检测。
在其中一些实施例中，上述PCR检测的反应程序为降落PCR。
在其中一些实施例中，上述生物样本选自血清、血浆、全血、新鲜组织、福尔马林固定石蜡包埋组织、尿液、细菌培养物、病毒培养物、细胞系培养物、人工合成的质粒。
在其中一些实施例中，上述生物样本核酸为核糖核酸，反应体系还包括逆 转录酶，反应程序还包括逆转录PCR。
实施例1针对HPV检测的引物探针组成
针对待检测的HPV，查阅相关专业文献，根据文献研究确定病毒保守区段，选择至少1段特异性的靶基因序列，使用引物设计软件Primer Premier 5.0，基于选择的特异性靶基因序列，设计上游寡核苷酸引物L型引物-F(LF引物)、下游寡核苷酸引物L型引物-R(LR引物)、U型探针(U探针)序列靶基因区、Bridge探针(B探针)序列靶基因区，以及通用引物。
其中，LF和LR分别包含与特异性靶基因序列互补的序列以及自然界中唯一序列(通用引物)，U探针分为特异性靶序列与自然界中唯一序列两部分，U探针的靶基因区与特异性靶序列互补，其3端接上人工引入序列(茎环区和3’端)，B探针分为特异性靶序列与自然界中唯一序列两部分，B探针的靶基因区与特异性靶序列互补，其5端接上人工引入序列，且B探针中自然界中唯一序列与U探针中位于3端的部分自然界中唯一序列互补。通过对U探针和B探针中的特异性靶标序列，进行不同核酸的检测，通过对U探针和B探针中自然界中唯一序列的设计进行多重熔解曲线的分析。
L引物：由2部分构成。5’端为一段自然界中唯一序列15～25个碱基组成，并作为通用引物，3’端为特异性靶基因序列互补的序列。
U探针：提供荧光信号释放体系，实现熔解曲线分析。由2部分组成，5’端为特异性靶基因序列互补的序列，3’为自然界中唯一序列(茎环区和3’端)，再修饰C3封闭，阻碍非特异性延伸，自然界中唯一序列的5端位置使用荧光淬灭基团，提供淬灭功能，中间位置使用荧光报告基团，起到荧光分析的作用。淬灭基团与荧光基团的相对位置为10bp-45bp，且本发明中，荧光报告基团包括但不仅限于FAM、HEX、ROX、VIC、CY5、TAMRA、TET、CY3和JOE，荧光淬灭基团包括但不限于BHQ1、BHQ2。
B探针：由2部分组成。5’端为自然界中唯一序列，与U探针中部分自然界中唯一序列互补；3’端为特异性靶基因序列互补的序列。
U探针和B探针：两种探针相对位置为5-10bp。
通用引物：在降落PCR扩增的第3个循环之后，可以对前2个循环产生的所有PCR产物进行扩增，确保各靶标的扩增均一性。
上述引物探针在进行待测样本检测时反应过程，具体如下：
(1)将待测样品的核酸与(1)所述的上下游引物、U探针、B探针与具有5’-3’外切酶活性的DNA聚合酶接触，置于进行PCR运行体系中，U探针在DNA聚合酶的作用下酶切，产生游离的3端区，B探针在DNA聚合酶的作用下酶切，留下与U探针互补结合5端区；
(2)U探针为荧光探针，5’端为特异性靶基因序列互补的序列，3’为自然界中唯一序列，再修饰C3封闭，阻碍非特异性延伸，自然界中唯一序列的5端位置使用荧光淬灭基团，提供淬灭功能，中间位置使用荧光报告基团，起到荧光分析的作用。在自然条件或与模板匹配时，报告基团发出的荧光信号，因能量共振转移原理，被淬灭基团吸收，不产生荧光信号。
(3)B探针5端区在酶切后，与U探针自然界中唯一序列的部分序列互补，在核酸聚合酶的作用下进行延伸反应。
(4)对步骤(3)产生的PCR产物由于处在双链状态，荧光基团与淬灭基团分开而产生荧光，因此可以进行熔解曲线分析，并根据熔解曲线分析的结果，确认每一种待测靶核苷酸序列是否存在于所述样品中，并进而确定与每一种靶核酸序列对应的人乳头瘤病毒是否存在于待测样品中。
实施例2一种HPV分型检测试剂盒
根据实施例1所述针对HPV检测的引物探针体系，制备一种可同时检测HPV16、HPV18、HPV58、HPV52型别的试剂盒，其中PCR引物探针序列如下表2-1所示，决定Tm值的扩增序列信息如下表2-2所示：
表2-1
Figure PCTCN2022097378-appb-000001
Figure PCTCN2022097378-appb-000002
Figure PCTCN2022097378-appb-000003
其中“LF”表示上游引物，“LR”表示下游引物，“TF”表示上游通用引物，“TR”表示下游通用引物，“U”表示带荧光基团的探针，“B”表示连接模板与“U”探针的探针。
表2-2
SEQ ID NO. 型别 序列5’-3’ Tm
17 16 tcgactcgtacttccgttggacgtagaaccataacaggtagtg 83.94
18 18 cgtccacttcaggttacgtaatgttagctcgttcaccgttggttgatgtttag 83.98
19 58 tcgcaaagtgtcataacgctagagctacaaggatcgggtgcatcgtataagagtt 83.99
20 52 tcgcaggatctttggccctcgactgcccaatccgtgtctggtattacg 83.86
实时荧光定量PCR反应体系如下表2-3所示进行配制：
表2-3
序号 组分 体积
1 5×PCR Buffer 5μL
2 DNA聚合酶(5U/μL) 5μL
3 dNTPs(20mM) 1μL
4 UDG酶(1U/μL) 1μL
5 MFR(1pmol) 1μL
6 MUB(1pmol) 1μL
7 纯化水 补水至反应体系20μL
配制完反应体系后，振荡混匀，离心后，使用宏石(SLAN-96S)上机。
PCR反应程序按照如下表2-4设置：
表2-4
Figure PCTCN2022097378-appb-000004
结果判读标准：
检测Tm值结果由宏石(SLAN-96S)软件自动判读，不同HPV型别的检测取决于对应的荧光及特异的Tm值。
结果分析：
采用上述试剂盒，通过上述检测方法，对30例样本进行检测，检测结果图如图2-5可知，本发明试剂盒可以准确检测出HPV16、HPV18、HPV58、HPV52型别的样本。
实施例3探针序列优化
为进一步研究探针序列对试剂盒检测性能的影响，分别针对检测HPV16、HPV18、HPV58、HPV52型别设计U探针和B探针序列，具体如下表3-1所示(其中引物序列与实施例2中的相同)：
3-1
Figure PCTCN2022097378-appb-000005
Figure PCTCN2022097378-appb-000006
采用上述试剂盒，通过实施例2所述检测方法，对30例样本进行检测，检测结果图如图6可知，在FAM通道，能单管同时检出特异的HPV16、HPV18、HPV58、HPV52型别熔解峰，说明本发明试剂盒可有效进行HPV多重样本检测。针对其进行进一步分析发现，决定Tm值的扩增序列信息如下表3-2所示：
表3-2
SEQ ID NO. 型别 序列5’-3’ Tm
29 16 tcgactctacttg ttggacgtagaaccataacaggtagtg 71.97
30 18 cg tacttcagttagtaa tgttactgttcacgttggttgatgtttag 76.21
31 58 tgcgcaaagtgtcataacgctagagc tacaaggatcgggtgcatcgtataagagtt 81.11
32 52 tcgcagcatctttcctcgagctgcccaatccgtgtctggtattacg 85.31
结合上表3-2可知，上述检测结果中能单管同时检出特异的HPV16、HPV18、HPV58、HPV52型别熔解峰。因此在多重样本检测体系中，本实施例中设计的探针序列更有利于多重样本检测。
实施例4在单样本测试U探针和B探针的相对位置
为进一步探究针对单一型别的检测体系中U探针和B探针的相对位置对检测性能的影响，以单独检测HPV16型别的检测体系引物探针设计为例，其中PCR引物探针序列如下表4-1所示(其中引物序列与实施例2中的相同)：
表4-1
Figure PCTCN2022097378-appb-000007
表格中“-”后表示修饰基团的位置以及修饰基团的名称。
采用上述试剂盒，通过实施例2所述检测方法，分别对30例样本进行检测，检测结果图如图7-9所示，可知在FAM通道均能产生HPV16型别特异性熔解峰，但相对位置为5bp和10bp时的熔解峰，会较相对位置15bp的熔解峰高。
实施例5在单样本测试U探针上淬灭基团与荧光基团的相对位置
为进一步探究针对单一型别的检测体系中U探针上淬灭基团与荧光基团的相对位置(即U探针的茎环区序列5’末端与3’末端之间的距离)对检测性能的影响，以单独检测HPV16型别的检测体系引物探针设计为例，其中PCR引物探 针序列如下表5-1所示(其中引物序列与实施例2中的相同)：
表5-1
Figure PCTCN2022097378-appb-000008
Figure PCTCN2022097378-appb-000009
注：表格中“-”后表示修饰基团的位置以及修饰基团的名称，并且序列表中的字体为倾斜的碱基表示为淬灭基团连接位置，字体为倾斜加粗的碱基表示为荧光基团连接位置。
采用上述试剂盒，通过实施例4所述检测方法，分别对30例样本进行检测，检测结果图如图10-13所示，可知在FAM通道均能产生HPV16型别特异性熔解峰，但相对位置为10bp、25bp和45bp的熔解峰峰高较高且一致，并会较相对位置5bp的熔解峰高。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (11)

  1. 一种用于多重核酸检测的双探针组合，其特征在于，包括针对靶基因序列的U探针和B探针，所述U探针从5’端到3’端的组成依次为：5’端区、茎环区、3’端区；所述B探针从5’端到3’端的组成依次为：5’端区、3’端区；
    所述U探针5’端区序列为特异性靶基因序列互补的序列，3’端区序列为自然界中唯一序列，茎环区序列长度为5bp-45bp，茎环区序列5’末端修饰荧光淬灭基团，茎环区序列3’末端修饰荧光报告基团；
    所述B探针3’端区序列为靶基因序列互补的序列，5’端区序列为与U探针3’端区序列反向互补的自然界中唯一序列；
    所述U探针和B探针同时与目的片段结合时，所述U探针5’端区序列的3’端与B探针5’端区序列的5’端之间的距离为5bp～15bp；
    所述自然界中唯一序列为不与靶基因序列杂交的序列。
  2. 根据权利要求1所述双探针组合，其特征在于，所述U探针和B探针同时与目的片段结合时，所述U探针5’端区序列的3’端与B探针5’端区序列的5’端之间的距离为5bp～10bp。
  3. 根据权利要求1-2任一项所述双探针组合，其特征在于，所述U探针的茎环区序列5’末端与3’末端之间的距离为10bp～45bp。
  4. 权利要求1-3任一项所述双探针组合在制备试剂盒或试剂盒检测试剂中的应用。
  5. 一种多重核酸检测试剂盒，其特征在于，包括针对目标核酸序列的扩增引物组和权利要求1-3任一项所述双探针组合。
  6. 根据权利要求5所述检测试剂盒，其特征在于，所述扩增引物组包括L引物对，所述L引物对中的每一条L引物从5’端到3’端的组成依次为：5’端区、3’端区；
    所述5’端区序列长度为15bp～25bp；所述3’端区为特异性靶基因序列互补的序列。
  7. 根据权利要求5-6任一项所述多重核酸检测试剂盒，其特征在于，所述扩增引物组还包括通用引物对，进一步地，所述通用引物对中的每一条通用引 物与L引物对中的每一条L引物的5’端区序列为相同序列。
  8. 根据权利要求5-6任一项所述多重核酸检测试剂盒，其特征在于，包括以下任意至少一组组分：
    第一组：针对HPV16的L引物对、U探针和B探针，其中，L引物对序列如SEQ ID NO.1～SEQ ID NO.2所示，U探针序列如SEQ ID NO.3所示，B探针序列如SEQ ID NO.4所示；
    第二组：针对HPV18的L引物对、U探针和B探针，其中，L引物对序列如SEQ ID NO.5～SEQ ID NO.6所示，U探针序列如SEQ ID NO.7所示，B探针序列如SEQ ID NO.8所示；
    第三组：针对HPV58的L引物对、U探针和B探针，其中，L引物对序列如SEQ ID NO.9～SEQ ID NO.10所示，U探针序列如SEQ ID NO.11所示，B探针序列如SEQ ID NO.12所示；
    第四组：针对HPV52的L引物对、U探针和B探针，其中，L引物对序列如SEQ ID NO.13～SEQ ID NO.14所示，U探针序列如SEQ ID NO.15所示，B探针序列如SEQ ID NO.16所示。
  9. 根据权利要求5-6任一项所述多重核酸检测试剂盒，其特征在于，还包括通用引物对，进一步地，所述通用引物对序列如SEQ ID NO.47～SEQ ID NO.48所示。
  10. 权利要求5-9任一项所述多重核酸检测试剂盒在人乳头瘤病毒检测中的应用。
  11. 一种人乳头瘤病毒检测方法，其特征在于，包括如下步骤：
    获取待测生物样本核酸；
    采用权利要求5-9任一项所述多重核酸检测试剂盒对上述生物样本核酸进行PCR检测。
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