CN113564269A - 阻碍细菌保守区域逆转录的探针组合物及其应用 - Google Patents
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Abstract
本发明提供一种阻碍细菌保守区域逆转录的探针组合物,包括针对16S rRNA和23SrRNA序列保守性区域的96条探针,序列如SEQ ID NO.1‑96所示,每条探针能够结合不低于10%的常见细菌rRNA,能够特异高效地识别并结合这些靶细菌rRNA的保守序列区域,并阻碍这些区域RNA的逆转录,同时保留非保守区域rRNA的正常逆转录。使用本发明公布的细菌rRNA逆转录探针进行RNA mNGS检测具有操作简单(一步操作)、耗时短(2min)、损失小和成本低等优点,并显著提升了RNA mNGS检测技术的有效数据占比、检出率、灵敏度和准确性,非常适用于RNA mNGS的自动化检测。
Description
技术领域
本发明专利涉及一种阻碍细菌保守区域逆转录的探针组合物及其在RNA建库中的应用,属于生物技术领域。
背景技术
病原微生物检测在感染性疾病的诊断中极为重要,而传统的检测方法主要有分离培养和生化鉴定、涂片镜检、免疫学方法、PCR检测、基因芯片技术,这些只能对已知的病原微生物进行检测,而无法对未知的症状进行有效的诊断。随着肠道微生物和病原微生物领域的兴起,许多疾病或症状是多种微生物共同作用的结果。因此,高通量基因组学技术和高通量转录组学技术被用于系统鉴定病理学样本中的微生物种类,为病原微生物致使疾病的诊断提供了强大的诊断工具,被称为宏基因组新一代测序技术(metagenomics nextgeneration sequencing,mNGS)。这种技术是一种无需培养,无偏好性,直接提取临床样本中DNA/RNA,采用高通量测序技术,经过数据库比对与生信分析,一次性完成细菌,真菌,病毒和寄生虫等病原体的检测。但是对于病原微生物中细菌核糖体RNA所占比例较大,并且在这些rRNA在细菌中高度保守,这就导致很难区分这些相同的区域是来源哪一种病原菌同时也占据了测序数据,使得有效的数据较少。这不仅提高了测序的数据量和测序成本,也降低了病原微生物检出的效率和准确性。
发明内容
本发明的目的是提供一种阻碍细菌保守区域逆转录的探针组合物,每条探针能够结合不低于10%的常见细菌rRNA,能够特异高效地识别并结合这些靶细菌rRNA的保守序列区域,并阻碍这些区域RNA的逆转录,同时保留非保守区域rRNA的正常逆转录。
本发明采用的技术方案为:一种细菌16S rRNA保守区域逆转录的探针组合物,其特征在于,为下表所示的探针混合物
优选的,所述探针序列中斜体下划线部分的碱基为LNA修饰碱基,3’端-NH 2C 6封闭。
本发明还公开了一种细菌23S rRNA保守区域逆转录的探针组合物,其特征在于,为下表所示的探针混合物
优选的,所述探针序列中斜体下划线部分的碱基为LNA修饰碱基,3’端-NH 2C 6封闭。
本发明还公开了上述的探针组合物在RNA建库中的应用。
本发明的作用机理:在逆转录缓冲液中,在高温条件下让样本总RNA分子接触探针组合物和随机引物,以使探针与其中的rRNA分子优选形成RNA:DNA的杂交双链,获得高温杂交混合物。
将获得的高温杂交混合物降温,在低温条件下让总RNA分子接触探针组合物和随机引物,以使随机引物与其它RNA分子形成RNA:DNA的杂交双链,获得低温杂交混合物;探针组合物经锁核酸修饰后Tm值和结合稳定性远高于随机引物,因此探针组合物可以在高温条件下与rRNA结合。而随机引物Tm值很低,只能在低温条件下与其它RNA结合。
将上述步骤中生成低温杂交混合物接触逆转录酶,生成一链cDNA。生成一链cDNA过程中rRNA无法被逆转录酶作为模板延伸而被去除;
常规手段完成下游RNA NGS文库的构建。
根据SILVA数据库中收录的2万多种常见细菌的16S rRNA和23S rRNA序列保守性区域,本发明设计了96条探针(SEQ ID NO.1-96),包括针对16S rRNA的42条探针序列及针对23S rRNA的54条探针序列,每条探针能够结合不低于10%的常见细菌rRNA,能够特异高效地识别并结合这些靶细菌rRNA的保守序列区域,并阻碍这些区域RNA的逆转录,同时保留非保守区域rRNA的正常逆转录。5S rRNA由于长度太短通常不参与RNA NGS建库,所以不需要额外使用探针去除。使用本发明设计的细菌rRNA逆转录探针进行RNA mNGS检测,能够在逆转录过程中有效去除细菌中大部分重叠部分的rRNA的序列,提高RNA mNGS检测过程中有效数据的占比,降低了测序的成本以及提高病原微生物的检出率,具有操作简单(一步操作)、耗时短(2min)、损失小和成本低等优点,并显著提升了RNA mNGS检测技术的有效数据占比、检出率、灵敏度和准确性,非常适用于RNA mNGS的自动化检测。
附图说明
图1细菌rRNA保守区域逆转录阻碍探针作用原理。
图2三种条件下RNA标准品测序数据中各RNA的占比。
图3RNA标准品在不同测序深度下细菌的检测数比较。
图4三种条件下细菌检测率比较。
图5不同RNA标准品投入量情况下细菌的检测数比较。
图6三种条件下病原样本RNA的细菌检出数比较。
图7不同病原样本RNA投入量的细菌检出数比较。
具体实施方式
实施例1细菌rRNA逆转录阻碍去除探针的设计和制备
本发明实施例中提供的细菌rRNA逆通过以下详细说明结合附图可以进一步理解本发明的特点和优点。所提供的实施例仅是对本发明方法的说明,而不以任何方式限制本发明揭示的其余内容。本实施例所使用的探针和引物序列及修饰如表1所示。
表1
探针及引物序列转录阻碍去除探针组合物,可以适用于多种高通量测序平台,如Illumina、华大MGI-seq、Nanopore或者Pacbio等,应用于转录组学和表观转录组学领域研究等检测范围,用于有效的去除病原微生物样本RNA中的rRNA,提高病原微生物的检出率。
探针组合物包括针对SILVA数据库中收录的2万多种细菌rRNA分子保守性区域设计合成的单链DNA探针中的一种或多种的组合,探针序列及修饰见表1,细菌rRNA包括16srRNA和23s rRNA的一种或者多种的组合。
每条单链DNA探针的长度为20nt-25nt;
每条探针3’端被NH2C6修饰封闭,中间含有多个位点的锁核酸修饰,50%的锁核酸位点位于探针5’端前三分之一的区域;
每条探针自身互补值小于5,Tm值大于80℃。
根据以上条件,设计的细菌rRNA探针组合物包含96条探针,探针序列及修饰如表1所示。
表1所有探针用DEPC水溶解成20μM终浓度,等体积混合且混合物中每条单链DNA探针的浓度为0.5-2μM。
实施例2:含细菌RNA的人类病源RNA标准品制备与检测。
在本实施例中,我们制备了含细菌RNA的人类病源RNA标准品,并利用RNA建库测序验证标准品中细菌的含量比例及细菌rRNA保守区域逆转录阻碍探针对rRNA保守区域的去除效果。具体实施方式如下:
1)RNA标准品混合物的制备:使用翊圣生物的Cell/Tissue Total RNAKit(Cat#19211)提取293F细胞的RNA。使用翊圣生物的Bacterial RNA Kit(Cat#19301)提取大肠杆菌和嗜热脂肪芽孢杆菌的RNA。其他10种细菌RNA来源于菁良基因科技,包括铜绿假单胞菌、化脓性链球菌、淋球菌、产气荚膜杆菌、流感杆菌、沙门菌、消化链球菌、脑膜炎奈瑟菌、金黄色葡萄球菌、肺炎克雷伯菌。
使用Nanodrop测定标准品浓度。将以上RNA按照下表的比例进行混合:
表2
Nanodrop测定RNA标准品浓度,并用DEPC水稀释成100ng/μL的RNA标准品。
2)RNA标准品文库构建
取100ng RNA标准品,使用翌圣生物的HieffUltima Dual-mode RNALibrary Prep Kit for Illumina(Cat#12252)进行RNA NGS文库的构建。并在IlluminaNovaSeq 6000平台进行测序。
表3
组分 | 用量 |
制备的人类病源RNA标准品 | 0.1-1000ng |
2μM human rRNA probe mix(202110257924.X) | 1μL |
1μM bacteria rRNA probe mix(实施例1) | 1μL |
2×Frag/Prime buffer | 8.5μL |
补加DEPC水至 | 17μL |
吹打混匀后瞬离。95℃ 5min,75℃ 1min,55℃ 1min,室温放置。
表4
组分 | 用量 |
上述反应体系 | 17μL |
Strand Specificity Reagent | 6μL |
1st Strand Enzyme Mix | 2μL |
总体积 | 25μL |
按照翌圣生物的HieffUltima Dual-mode RNA Library Prep Kit forIllumina(Cat#12252)的说明书进行二链合成、接头连接和PCR扩增。回收的文库用Qubit定量后,用Qsep检测文库的大小分布。获得的文库在Illumina的NovaSeq 6000平台上进行测序。将测序结果分别比对到人类和上述病源细菌转录组上进行分析。
使用细菌rRNA保守区域逆转录阻碍探针进行RNA mNGS检测示意图见图1。对RNA标准品的mNGS检测结果见图2-图5,加入人rRNA逆转录阻碍探针可以显著增加细菌RNA的占比(约10倍),加入细菌rRNA保守区域逆转录阻碍探针可以显著增加细菌rRNA非保守区域和其他非rRNA数据的占比(约4倍)(图2)。我们检测了不同测序深度下细菌检测效率,发现加入人rRNA逆转录阻碍探针和细菌rRNA保守区域逆转录阻碍探针能够有效降低细菌检测所需的测序深度,提高检测的效率(图3和图4)。三组数据呈现很好的线性相关,说明建库过程中加入快速去除探针不会造成明显的损失和偏好性(图4)。这些说明逆转录阻碍探针可以有效提高细菌RNA的有效数据占比,降低细菌检测的成本。此外,我们还测试了逆转录阻碍探针对不同RNA标准品投入量条件下的细菌检测结果,发现加入了细菌rRNA保守区域逆转录阻碍探针可以显著提高RNA mNGS检测的灵敏度,可以做到低至0.1ng的RNA的有效细菌检出(图5)。
实施例3:细菌rRNA保守区域逆转录阻碍探针在病原RNA样本检测上的作用。
在本实施例中,我们细菌rRNA保守区域逆转录阻碍探针对病原样本RNA进行了mRNA NGS检测,病原样本RNA来源于金匙医学。实施方式如实施例2。
结果如图6和图7所示,加入人rRNA逆转录阻碍探针和细菌rRNA保守区域逆转录阻碍探针病源样本中细菌的检测准确性、效率和灵敏度。
综上,本发明公布了一组阻碍细菌rRNA保守区域逆转录的DNA探针组合物,这组探针的设计根据SILVA数据库中收录的2万多种常见细菌的16S rRNA和23S rRNA序列保守性区域进行设计,包含96条探针(SEQ ID NO.1-96),每条探针能够结合不低于10%的常见细菌rRNA,能够特异高效地识别并结合这些靶细菌rRNA的保守序列区域,并阻碍这些区域RNA的逆转录,同时保留非保守区域rRNA的正常逆转录。使用本发明公布的细菌rRNA逆转录探针进行RNA mNGS检测具有操作简单(一步操作)、耗时短(2min)、损失小和成本低等优点,并显著提升了RNA mNGS检测技术的有效数据占比、检出率、灵敏度和准确性,非常适用于RNAmNGS的自动化检测。
序列表
<110> 翌圣生物科技(上海)股份有限公司
<120> 阻碍细菌保守区域逆转录的探针组合物及其应用
<141> 2021-07-21
<160> 96
<170> SIPOSequenceListing 1.0
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<213> 人工序列(Artificial Sequence)
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<210> 3
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
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<210> 4
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
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<210> 5
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
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<210> 6
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
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<210> 7
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ctcagtccca gtgtggctga 20
<210> 8
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ctgctgcctc ccgtaggagt 20
<210> 9
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
ccattgtgca atattcccca 20
<210> 10
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
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<210> 11
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
tttacaaccc gaaggccttc 20
<210> 12
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
gtattaccgc ggctgctggc 20
<210> 13
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
ctttacgccc agtaattccg 20
<210> 14
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
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<210> 15
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
ttcccaggtt gagcccgggg 20
<210> 16
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
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<210> 17
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
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<210> 18
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
cgccttcgcc actggtgttc 20
<210> 19
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
gctttcgcac ctcagcgtca 20
<210> 20
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
actaccaggg tatctaatcc 20
<210> 21
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
atcgtttacg gcgtggacta 20
<210> 22
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
ccgtactccc caggcggaat 20
<210> 23
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
cccgtcaatt cctttgagtt 20
<210> 24
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
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<210> 25
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
ggtaaggttc ttcgcgttgc 20
<210> 26
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
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<210> 27
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
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<210> 28
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
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<210> 29
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
gctcgttgcg ggacttaacc 20
<210> 30
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
taaggacaag ggttgcgctc 20
<210> 31
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
accggcagtc tccttagagt 20
<210> 32
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
tgacgtcatc cccaccttcc 20
<210> 33
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
taaggggcat gatgacttga 20
<210> 34
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
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<210> 35
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
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<210> 36
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
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<210> 37
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
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<210> 38
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
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<210> 39
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
cccgggaacg tattcaccgc 20
<210> 40
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
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<210> 41
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 41
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<210> 42
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 42
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<210> 43
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 43
aggcatccac cgtgcgccct 20
<210> 44
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 44
ttcatcgcct ctgactgcca 20
<210> 45
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 45
ggtttcccca ttcggaaatc 20
<210> 46
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 46
ggtacttaga tgtttcagtt 20
<210> 47
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 47
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<210> 48
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 48
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<210> 49
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 49
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<210> 50
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 50
gtatttagcc ttggaggatg 20
<210> 51
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 51
ctttccctca cggtactggt 20
<210> 52
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 52
tttcaggttc tatttcactc 20
<210> 53
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 53
tcattctaca aaaggcacgc 20
<210> 54
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 54
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<210> 55
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 55
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<210> 56
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 56
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<210> 57
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 57
tttcggggag aaccagctat 20
<210> 58
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 58
caaacagtgc tctacctcca 20
<210> 59
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 59
ttggtaagtc gggatgaccc 20
<210> 60
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 60
ttagcacccg ccgtgtgtct 20
<210> 61
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 61
ctgggctgtt tccctttcga 20
<210> 62
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 62
gggaccttag ctggcggtct 20
<210> 63
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 63
tgcttctaag ccaacctcct 20
<210> 64
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 64
gtgagctatt acgcactctt 20
<210> 65
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 65
agccccggta cattttcggc 20
<210> 66
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 66
ttacagaacg ctcccctacc 20
<210> 67
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 67
ttatcgttac ttatgtcagc 20
<210> 68
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 68
gaacccttgg tcttccggcg 20
<210> 69
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 69
tcgactacgc ctttcggcct 20
<210> 70
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 70
gtacaggaat attaacctgt 20
<210> 71
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 71
ccgggacaac cgtcgcccgg 20
<210> 72
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 72
cttagaggct tttcctggaa 20
<210> 73
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 73
cctgtgtcgg tttgcggtac 20
<210> 74
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 74
cgagttcctt aacgagagtt 20
<210> 75
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 75
cccttctccc gaagttacgg 20
<210> 76
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 76
ctgtgttttt gataaacagt 20
<210> 77
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 77
accttccagc accgggcagg 20
<210> 78
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 78
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<210> 79
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 79
ggtcggaact tacccgacaa 20
<210> 80
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 80
ttacgccatt cgtgcaggtc 20
<210> 81
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 81
tgagtctcgg gtggagacag 20
<210> 82
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 82
gatttcaatt tcactgagtc 20
<210> 83
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 83
ggggtctttc cgtcctgtcg 20
<210> 84
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 84
agtaaaggtt cacggggtct 20
<210> 85
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 85
cctcccacct atcctacaca 20
<210> 86
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 86
ttaaagggtg gtatttcaag 20
<210> 87
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 87
ggcgaccgcc ccagtcaaac 20
<210> 88
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 88
ctccgttact ctttaggagg 20
<210> 89
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 89
tctcgcagtc aagctccctt 20
<210> 90
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 90
cttttatccg ttgagcgatg 20
<210> 91
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 91
ccgacatcga ggtgccaaac 20
<210> 92
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 92
ggcgaacagc ccaacccttg 20
<210> 93
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 93
gaactgtctc acgacgttct 20
<210> 94
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 94
catcccggtc ctctcgtact 20
<210> 95
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 95
agatgctttc agcggttatc 20
<210> 96
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 96
tgggaaatct catcttgagg 20
Claims (6)
2.根据权利要求1所述的阻碍细菌16S rRNA保守区域逆转录的探针组合物,其特征为:所述探针序列中斜体下划线部分的碱基为LNA修饰碱基,3’端-NH2C6封闭。
4.根据权利要求3所述的阻碍细菌23S rRNA保守区域逆转录的探针组合物,其特征为:所述探针序列中斜体下划线部分的碱基为LNA修饰碱基,3’端-NH2C6封闭。
5.一种阻碍细菌保守区域逆转录的探针组合物,其特征为:包括权利要求1或2所述的阻碍细菌16S rRNA保守区域逆转录的探针组合物,以及权利要求3或4所述的阻碍细菌23SrRNA保守区域逆转录的探针组合物。
6.权利要求1-5中任一项所述的探针组合物在RNA建库中的应用。
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