CN115747317A - A detection method and kit for Shigella nucleic acid molecules - Google Patents

A detection method and kit for Shigella nucleic acid molecules Download PDF

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CN115747317A
CN115747317A CN202211549907.4A CN202211549907A CN115747317A CN 115747317 A CN115747317 A CN 115747317A CN 202211549907 A CN202211549907 A CN 202211549907A CN 115747317 A CN115747317 A CN 115747317A
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nucleic acid
target dna
detection method
primary guide
ssdna
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沈鹤霄
李国龙
吕永玲
张帆
任泉
方凯
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Maintain Biomedical Wuhan Co ltd
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Abstract

本发明公开了一种志贺菌属核酸分子的检测方法,其包括利用Argonaute蛋白的特性,初级向导ssDNAs介导Argonaute蛋白剪切靶标DNA后,断裂产生的次级向导ssDNA再次被Argonaute蛋白利用去剪切与次级向导ssDNA互补的荧光报告核酸;靶标DNA的序列如SEQ ID NO.1所示。检测结果表明,该检测方法在现有技术的基础上有更好的灵敏性和重复性。

Figure 202211549907

The invention discloses a method for detecting nucleic acid molecules of the genus Shigella, which comprises utilizing the characteristics of the Argonaute protein, and after the primary guide ssDNAs mediate the cutting of the target DNA by the Argonaute protein, the secondary guide ssDNA produced by the breakage is used again by the Argonaute protein to remove Cut the fluorescent reporter nucleic acid complementary to the secondary guide ssDNA; the sequence of the target DNA is shown in SEQ ID NO.1. The detection results show that the detection method has better sensitivity and repeatability on the basis of the existing technology.

Figure 202211549907

Description

一种志贺菌属核酸分子的检测方法及试剂盒A detection method and kit for Shigella nucleic acid molecules

技术领域technical field

本发明涉及生物技术领域,具体而言,涉及一种志贺菌属核酸分子的检测方法及试剂盒。The invention relates to the field of biotechnology, in particular to a detection method and a kit for nucleic acid molecules of the genus Shigella.

背景技术Background technique

Argonaute(Ago)蛋白质是一种可编程核酸酶,广泛分布于真核生物和原核生物。真核生物Ago蛋白质在RNA干扰途径中起关键作用,能够在小RNA或者小DNA的引导下识别甚至切割与之互补的RNA。原核生物Ago蛋白质可能参与宿主防御和DNA复制,偏爱在小DNA的引导下切割与之互补的DNA。基于这些特性,Ago蛋白质能够在核酸检测和遗传操作等方面得以应用。Argonaute (Ago) protein is a programmable nuclease widely distributed in eukaryotes and prokaryotes. Eukaryotic Ago protein plays a key role in the RNA interference pathway, and can recognize and even cut complementary RNA under the guidance of small RNA or small DNA. Prokaryotic Ago proteins may be involved in host defense and DNA replication, preferring to cut complementary DNA under the guidance of small DNA. Based on these characteristics, Ago protein can be applied in nucleic acid detection and genetic manipulation.

慢性便秘(CC)是一种常见的多因素疾病,其特征是排便减少、大便硬和大便过度紧张。一些慢性便秘患者可以治愈,然而大约三分之一的慢性转移性功能性便秘患者(FC)对治疗没有反应并反复发作,这种医学上难治性慢运输型便秘被认为是结肠切除术的并发症(STC),而这种病又称为对常规药物无效的顽固性功能性便秘(IFC)。到目前为止,已经确定了与难治性慢运输型便秘相关的几个因素,特别是,最近的全基因组分析和粪便移植表明,肠道微生物志贺菌属(Shigella sp.variant)可能是影响肠道转运和慢性便秘表型的重要因素。Chronic constipation (CC) is a common multifactorial disorder characterized by decreased bowel movements, hard stools, and excessive straining of stools. Some patients with chronic constipation can be cured, however approximately one-third of patients with chronic metastatic functional constipation (FC) do not respond to treatment and have recurrent episodes, the medically refractory slow transit constipation considered the cause of colectomy Complications (STC), which is also known as intractable functional constipation (IFC) unresponsive to conventional medications. So far, several factors associated with refractory slow transit constipation have been identified, in particular, recent genome-wide analyzes and fecal transplants suggest that the gut microbiota Shigella sp.variant may be influencing Important factors in intestinal transit and the chronic constipation phenotype.

人体肠道中存在的数量庞大的微生物,这群微生物依靠人体的肠道生活,同时帮助人体完成多种生理生化功能。肠道微生物不仅在膳食和宿主之间起到了重要的桥梁作用,其本身以及代谢产物也能调节人体健康,因此肠道微生物与人体健康密切相关。因此,基于原核Argonaute蛋白开发一种灵敏度高、特异性好、快速高效的志贺菌属核酸检测方法是非常有必要的。There are a large number of microorganisms in the human intestinal tract. These microorganisms rely on the human intestinal tract to live and help the human body to complete various physiological and biochemical functions. Gut microbes not only serve as an important bridge between diet and host, but also regulate human health by themselves and their metabolites. Therefore, gut microbes are closely related to human health. Therefore, it is very necessary to develop a highly sensitive, specific, fast and efficient detection method for Shigella nucleic acid based on prokaryotic Argonaute protein.

鉴于此,特提出本发明。In view of this, the present invention is proposed.

发明内容Contents of the invention

本发明的目的在于提供一种用于检测志贺菌属核酸分子的方法及试剂盒,以提高志贺菌属核酸分子的检测灵敏度。The object of the present invention is to provide a method and kit for detecting Shigella nucleic acid molecules, so as to improve the detection sensitivity of Shigella nucleic acid molecules.

本发明是这样实现的:The present invention is achieved like this:

本发明提供了一种志贺菌属核酸分子的检测方法,其包括利用Argonaute蛋白的特性,初级向导ssDNAs介导Argonaute蛋白剪切靶标DNA后,断裂产生的次级向导ssDNA再次被Argonaute蛋白利用去剪切与次级向导ssDNA互补的荧光报告核酸;The invention provides a method for detecting nucleic acid molecules of the genus Shigella, which comprises utilizing the characteristics of the Argonaute protein, and after the primary guide ssDNAs mediate the shearing of the target DNA by the Argonaute protein, the secondary guide ssDNA produced by the breakage is again used by the Argonaute protein to remove Cut the fluorescent reporter nucleic acid complementary to the secondary guide ssDNA;

上述靶标DNA的序列如SEQ ID NO.1所示。The sequence of the above target DNA is shown in SEQ ID NO.1.

在一些实施方式中,Argonaute蛋白是以DNA为向导指导剪切靶标DNA的中等温度Argonaute蛋白。In some embodiments, the Argonaute protein is a medium-temperature Argonaute protein that guides the shearing of target DNA by a DNA guide.

在一些实施方式中,中等温度Argonaute蛋白包括BlAgo酶。In some embodiments, the intermediate temperature Argonaute protein comprises BlAgo enzyme.

在一些实施方式中,BlAgo酶的工作温度为36-38℃。In some embodiments, the BlAgo enzyme operates at a temperature of 36-38°C.

在一些实施方式中,靶标DNA在检测体系中的浓度为≥1拷贝/微升。In some embodiments, the concentration of target DNA in the detection system is ≥1 copy/microliter.

在一些实施方式中,靶标DNA在检测体系中的浓度为≥4拷贝/微升。In some embodiments, the concentration of target DNA in the detection system is ≥4 copies/microliter.

在一些实施方式中,靶标DNA在检测体系中的浓度为≥10拷贝/微升。In some embodiments, the concentration of target DNA in the detection system is ≥10 copies/microliter.

在一些实施方式中,初级向导ssDNAs与靶标DNA互补配对,其包括初级向导ssDNAA链和初级向导ssDNA B链。In some embodiments, the primary guide ssDNAs are complementary paired with the target DNA, which includes the primary guide ssDNA A strand and the primary guide ssDNA B strand.

初级向导ssDNA A链的核苷酸序列如SEQ ID NO.2所示;The nucleotide sequence of the primary guide ssDNA A chain is shown in SEQ ID NO.2;

初级向导ssDNA B链的核苷酸序列如SEQ ID NO.3所示。The nucleotide sequence of the primary guide ssDNA B chain is shown in SEQ ID NO.3.

在一些实施方式中,初级向导ssDNAs为5’-磷酸化的单链DNA分子。In some embodiments, primary guide ssDNAs are 5'-phosphorylated single-stranded DNA molecules.

在一些实施方式中,初级向导ssDNAs的长度为18-25nt。In some embodiments, the primary guide ssDNAs are 18-25 nt in length.

在一些实施方式中,靶标DNA和Argonaute蛋白与初级向导ssDNAs在检测体系中的摩尔比为8:60:1-40:600:1。In some embodiments, the molar ratio of target DNA and Argonaute protein to primary guide ssDNAs in the detection system is 8:60:1-40:600:1.

在一些实施方式中,次级向导ssDNA的长度为10-25nt。In some embodiments, the secondary guide ssDNA is 10-25 nt in length.

在一些实施方式中,荧光报告核酸的长度为25-50nt。In some embodiments, the fluorescent reporter nucleic acid is 25-50 nt in length.

在一些实施方式中,荧光报告核酸在检测体系中的浓度为200-1000nM。In some embodiments, the concentration of the fluorescent reporter nucleic acid in the detection system is 200-1000 nM.

在一些实施方式中,靶标DNA与荧光报告核酸的摩尔比为:102:1至2×105:1;优选地,靶标DNA与荧光报告核酸的摩尔比102:1至2×103:1。In some embodiments, the molar ratio of target DNA to fluorescent reporter nucleic acid is: 10 2 : 1 to 2×10 5 : 1; preferably, the molar ratio of target DNA to fluorescent reporter nucleic acid is 10 2 : 1 to 2×10 3 :1.

本发明还提供了一种用于检测志贺菌属核酸分子的试剂盒,其包括上述方法中采用的Argonaute蛋白、初级向导ssDNAs和荧光报告核酸。The present invention also provides a kit for detecting nucleic acid molecules of the genus Shigella, which comprises the Argonaute protein used in the above method, primary guide ssDNAs and fluorescent reporter nucleic acid.

本发明具有以下有益效果:The present invention has the following beneficial effects:

本发明利用以DNA为向导指导剪切靶标DNA的Argonaute蛋白的特性:即在首次由初级向导ssDNA(guide ssDNA)介导剪切后,在合适的反应温度下,断裂的5’核酸片段可再次被Argonaute蛋白利用去剪切与之互补的荧光报告核酸链,通过荧光报告核酸断裂产生的荧光信号检测样本中志贺菌属核酸含量。检测结果表明,该检测方法在现有技术的基础上有更好的灵敏性和重复性。The present invention utilizes the characteristics of the Argonaute protein that uses DNA as a guide to guide the shearing of target DNA: that is, after the shearing is mediated by the primary guide ssDNA (guide ssDNA) for the first time, at a suitable reaction temperature, the fragmented 5' nucleic acid fragments can be regenerated. It is used by the Argonaute protein to cut the complementary fluorescent reporter nucleic acid chain, and the content of Shigella nucleic acid in the sample is detected by the fluorescent signal generated by the fragmentation of the fluorescent reporter nucleic acid. The detection results show that the detection method has better sensitivity and repeatability on the basis of the existing technology.

附图说明Description of drawings

为了更清楚地说明本发明实施例的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,应当理解,以下附图仅示出了本发明的某些实施例,因此不应被看作是对范围的限定,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他相关的附图。In order to illustrate the technical solutions of the embodiments of the present invention more clearly, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention, and thus It should be regarded as a limitation on the scope, and those skilled in the art can also obtain other related drawings based on these drawings without creative work.

图1为实验例中两种方法的检测结果对比:A为RT-qPCR试剂盒分别对反应体系中0拷贝至1000拷贝靶基因的检测结果,数据来自九次重复实验;B为BlAgo介导的核酸检测方法分别对反应体系中0拷贝至1000拷贝靶基因的检测结果,数据来自九次重复实验。Figure 1 is a comparison of the detection results of the two methods in the experimental example: A is the detection results of 0 to 1000 copies of the target gene in the reaction system by the RT-qPCR kit, and the data comes from nine repeated experiments; B is BlAgo-mediated Nucleic acid detection methods are the detection results of 0 copy to 1000 copies of the target gene in the reaction system, and the data come from nine repeated experiments.

具体实施方式Detailed ways

为使本发明实施例的目的、技术方案和优点更加清楚,下面将对本发明实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased from the market.

Argonautes是一种核酸酶,其普遍存在于细菌、植物、真菌、哺乳细胞内。其除了参与RNA干扰机制,某些物种的Argonaute还可作为DNA核酸内切酶。Argonautes are nucleases commonly found in bacteria, plants, fungi, and mammalian cells. In addition to participating in the RNA interference mechanism, some species of Argonaute can also act as DNA endonucleases.

中等温度Argonaute蛋白(Ago酶)包括Brevibacillus laterosporus(BlAgo)、Clostridiumbutyricum(CbAgo)、Intestinibacter bartlettii(IbAgo)及其突变体,通过CbAgo的系统进化树分析,我们从嗜温细菌中发现了Brevibacillus laterosporus(BlAgo),并研究了它们在广泛条件下切割ssDNA和dsDNA的生化特性。其中BlAgo酶切特性为:该酶能够在中等温度条件下利用5’磷酸化或羟基化的寡聚核苷酸作为向导ssDNA指导该酶对靶标DNA序列的精确剪切;剪切位点位于与向导ssDNA的第10与11位核苷酸对应的靶标DNA(ssDNA)之间的磷酸二酯键。Moderate temperature Argonaute proteins (Ago enzymes) include Brevibacillus laterosporus (BlAgo), Clostridium butyricum (CbAgo), Intestinibacter bartlettii (IbAgo) and their mutants. Through the phylogenetic tree analysis of CbAgo, we found Brevibacillus laterosporus (BlAgo) from mesophilic bacteria ), and studied their biochemical properties for cleaving ssDNA and dsDNA under a wide range of conditions. Among them, the enzyme cutting characteristics of BlAgo are: the enzyme can use 5' phosphorylated or hydroxylated oligonucleotides as guide ssDNA to guide the enzyme to accurately cut the target DNA sequence under moderate temperature conditions; The phosphodiester bond between the target DNA (ssDNA) corresponding to the 10th and 11th nucleotides of the guide ssDNA.

目前已知的BlAao会产生一种独特的独木舟状板层体附于孢子一侧,该物种的全基因测序揭示其能产生多酮物质,以此BlAao成为生物防治虫害的热门菌种。还未有相关报道证明BlAao具有切割ssDNA和dsDNA的生化特性。The currently known BlAao can produce a unique canoe-shaped lamellar body attached to one side of the spore. Whole-gene sequencing of this species revealed that it can produce polyketide substances, so BlAao has become a popular strain for biological control of pests. There is no relevant report to prove that BlAao has the biochemical properties of cutting ssDNA and dsDNA.

基于此,本发明首次开发了一种针对Shigella sp.variant 16sRNA的操作简便、兼容性好的核酸检测方法。本发明方法利用以DNA为向导指导剪切靶标DNA的BlAgo酶的特性,即在首次由初级向导ssDNA(guide ssDNA)介导剪切后,在合适的反应温度下,断裂的5’核酸片段可再次被BlAgo酶利用去剪切与之互补的荧光报告核酸链。Based on this, the present invention firstly develops a nucleic acid detection method for Shigella sp.variant 16sRNA which is easy to operate and has good compatibility. The method of the present invention utilizes the characteristics of the BlAgo enzyme that uses DNA as a guide to guide the shearing of target DNA, that is, after the shearing is mediated by the primary guide ssDNA (guide ssDNA) for the first time, at a suitable reaction temperature, the cleaved 5' nucleic acid fragment can be It is used again by the BlAgo enzyme to cut the complementary fluorescent reporter nucleic acid chain.

在本发明中,靶标DNA来源于Shigella sp.variant特异性16s rRNA,位于山梨糖醇脱氢酶基因上的SNP,其核苷酸序列如SEQ ID NO.1所示。In the present invention, the target DNA is derived from Shigella sp.variant-specific 16s rRNA, the SNP located on the sorbitol dehydrogenase gene, and its nucleotide sequence is shown in SEQ ID NO.1.

SEQ ID NO.1:SEQ ID NO.1:

5’-GCAAATATAACATCTGCCCGAACGTTGATTTTATGGCGACACAACCTAACTACCGCGGCGCATTAACGCACTATCTGTGTCATCCGGAGAGCTTTACTTACAAACTGCCCGACAATATGGACACGATGGAAGGGGCGCTGGTGGAGCCTGCCGCAGTCGGGATGCATGCCGCGATGCTGGCAGATGTTAAACCGGGTAAGAAGATAATTATTCTGGGAGCGGGTTGTATTGGTTTGATGACGTTGCAAGC-3’。5’-GCAAATATAACATCTGCCCGAACGTTGATTTTATGGCGACACAACCTAACTACCGCGGCGCATTAACGCACTATCTGTGTCATCCGGAGAGCTTTACTTACAAACTGCCCGACAATATGGACACGATGGAAGGGGCGCTGGTGGAGCCTGCCGCAGTCGGGATGCATGCCGCGATGCTGGCAGATGTTAAACCGGGTAAGAAGATAATTATTCTGGGAGCGGGTTGTATTGGTTTGATGACGTTGCAAGC-3’。

在一些实施例中,向导ssDNA对包括初级向导ssDNA和次级向导ssDNA。In some embodiments, the guide ssDNA pair includes a primary guide ssDNA and a secondary guide ssDNA.

初级向导ssDNAs与靶标DNA互补配对,由初级向导ssDNAs引导BlAgo酶的特异性切割,产生次级向导ssDNA及另外两段单链DNA。The primary guide ssDNAs are complementary to the target DNA, and the primary guide ssDNAs guide the specific cleavage of the BlAgo enzyme to generate the secondary guide ssDNA and the other two single-stranded DNAs.

在一些实施例中,初级向导ssDNA可以一对或多对。In some embodiments, there can be one or more pairs of primary guide ssDNA.

在一些实施例中,初级向导ssDNAs为5’-磷酸化的单链DNA分子。In some embodiments, primary guide ssDNAs are 5'-phosphorylated single-stranded DNA molecules.

在一些实施例中,所述初级向导ssDNAs的长度为18-30nt。In some embodiments, the primary guide ssDNAs are 18-30 nt in length.

在一些实施例中,初级向导ssDNA包括初级向导ssDNA A链和初级向导ssDNAB链;初级向导ssDNA A链的核苷酸序列如SEQ ID NO.2所示;初级向导ssDNA B链的核苷酸序列如SEQ ID NO.3所示。In some embodiments, the primary guide ssDNA includes a primary guide ssDNA A strand and a primary guide ssDNA B strand; the nucleotide sequence of the primary guide ssDNA A strand is shown in SEQ ID NO.2; the nucleotide sequence of the primary guide ssDNA B strand As shown in SEQ ID NO.3.

SEQ ID NO.2:SEQ ID NO.2:

5’P-AGGCTCCACCATCGCCCCTTCCATCG 3’;5'P-AGGCTCCACCATCGCCCCTTCCATCG 3';

SEQ ID NO.3:SEQ ID NO.3:

5’P-ACTGCGGCAGGCTCCACCAGCGCC 3’。5'P-ACTGCGGCAGGCTCCACCAGCGCC 3'.

由上述初级向导ssDNAs引导BlAgo酶的特异性切割,产生次级向导ssDNA,其长度为10-20nt。次级向导ssDNA的序列如SEQ ID NO.4所示。The specific cleavage of the BlAgo enzyme is guided by the above-mentioned primary guide ssDNAs to generate the secondary guide ssDNA, which is 10-20nt in length. The sequence of the secondary guide ssDNA is shown in SEQ ID NO.4.

SEQ ID NO.4:SEQ ID NO.4:

5’-GCGCTGGTGGAGCCT-3’。5'-GCGCTGGTGGAGCCT-3'.

次级向导ssDNA与荧光报告核酸的序列互补结合后,引导BlAgo酶对荧光报告核酸进行切割,从而产生可检测的信号。After the secondary guide ssDNA is complementary to the sequence of the fluorescent reporter nucleic acid, it guides the BlAgo enzyme to cut the fluorescent reporter nucleic acid, thereby generating a detectable signal.

在一些实施例中,与次级向导ssDNA互补的荧光报告核酸的长度为20-30nt。In some embodiments, the fluorescent reporter nucleic acid complementary to the secondary guide ssDNA is 20-30 nt in length.

在一些实施例中,报告分子是荧光分子或荧光基团。报告核酸分子是分别携带荧光基团和淬灭基团的核酸分子。其中,在5’端标记荧光基团(F),3’端标记淬灭基团(Q)。In some embodiments, the reporter molecule is a fluorescent molecule or fluorophore. The reporter nucleic acid molecule is a nucleic acid molecule carrying a fluorescent group and a quencher group respectively. Among them, the fluorescent group (F) is labeled at the 5' end, and the quencher group (Q) is labeled at the 3' end.

在一些实施例中,荧光报告核酸的序列如SEQ ID NO.5所示。In some embodiments, the sequence of the fluorescent reporter nucleic acid is shown in SEQ ID NO.5.

SEQ ID NO.5:SEQ ID NO.5:

3’F-AGCGACTTGACGCGACCACCTCGGAGTGC-Q 5’。3'F-AGCGACTTGACGCGACCACCTCGGAGTGC-Q 5'.

在一些实施例中,靶标DNA在检测体系中的浓度为≥1拷贝/微升。In some embodiments, the concentration of target DNA in the detection system is ≥1 copy/microliter.

在一些实施例中,靶标DNA在检测体系中的浓度为≥4拷贝/微升。In some embodiments, the concentration of the target DNA in the detection system is ≥4 copies/microliter.

在一些实施例中,靶标DNA、Argonaute蛋白、初级向导ssDNAs在检测体系中的摩尔比为8:60:1-40:600:1。In some embodiments, the molar ratio of target DNA, Argonaute protein, and primary guide ssDNAs in the detection system is 8:60:1-40:600:1.

在一些实施例中,荧光报告核酸在检测体系中的浓度为:200-1000nM。In some embodiments, the concentration of the fluorescent reporter nucleic acid in the detection system is: 200-1000 nM.

在一些实施例中,靶标DNA与荧光报告核酸的摩尔比为:102:1至105:1;优选地,靶标DNA与荧光报告核酸的摩尔比102:1至103:1。In some embodiments, the molar ratio of target DNA to fluorescent reporter nucleic acid is 10 2 :1 to 10 5 :1; preferably, the molar ratio of target DNA to fluorescent reporter nucleic acid is 10 2 :1 to 10 3 :1.

在一些实施例中,上述检测方法是体外方法。In some embodiments, the above detection methods are in vitro methods.

在一些实施例中,上述检测方法是非诊断性和非治疗性的。In some embodiments, the detection methods described above are non-diagnostic and non-therapeutic.

本发明还提供了一种用于检测志贺菌属核酸分子的试剂盒,其包括上述方法中采用的Argonaute蛋白、初级向导ssDNAs和荧光报告核酸。The present invention also provides a kit for detecting nucleic acid molecules of the genus Shigella, which comprises the Argonaute protein used in the above method, primary guide ssDNAs and fluorescent reporter nucleic acid.

上述试剂盒的使用方法如下:The usage method of above-mentioned kit is as follows:

1、获取的待检样本作为模板加入PCR体系中对其进行扩增,获得靶标DNA;1. The obtained sample to be tested is used as a template and added to the PCR system to amplify it to obtain the target DNA;

2、加入特异性的寡核苷酸向导ssDNAs、与之相应的荧光报告核酸及BlAgo酶在36-38℃持续保温的条件下进行特异性剪切,并对荧光信号进行采集;2. Add specific oligonucleotide guide ssDNAs, corresponding fluorescent reporter nucleic acid and BlAgo enzyme to perform specific shearing under the condition of continuous incubation at 36-38°C, and collect the fluorescent signal;

3、分析荧光信号值,调节Baseline的Start值、End值和阈值线,判定结果。3. Analyze the fluorescence signal value, adjust the Start value, End value and threshold line of Baseline, and judge the result.

以下结合实施例对本发明的特征和性能作进一步的详细描述。The characteristics and performance of the present invention will be described in further detail below in conjunction with the examples.

实施例1Example 1

本实施例提供了一种用于检测志贺菌属核酸分子的方法。This example provides a method for detecting Shigella nucleic acid molecules.

其中,检测试剂包括:Among them, the detection reagents include:

(1)初级向导ssDNA,包括初级向导ssDNA A链和初级向导ssDNAB链;初级向导ssDNA A链的核苷酸序列如下:(1) The primary guide ssDNA, including the primary guide ssDNA A strand and the primary guide ssDNA B strand; the nucleotide sequence of the primary guide ssDNA A strand is as follows:

5’P-AGGCTCCACCATCGCCCCTTCCATCG 3’;5'P-AGGCTCCACCATCGCCCCTTCCATCG 3';

初级向导ssDNA B链的核苷酸序列如下:The nucleotide sequence of the primary guide ssDNA B strand is as follows:

5’P-ACTGCGGCAGGCTCCACCAGCGCC 3’。5'P-ACTGCGGCAGGCTCCACCAGCGCC 3'.

(2)荧光报告核酸,其序列如下:(2) fluorescent reporter nucleic acid, its sequence is as follows:

3’F-AGCGACTTGACGCGACCACCTCGGAGTGC-Q 5’。3'F-AGCGACTTGACGCGACCACCTCGGAGTGC-Q 5'.

(3)BlAgo酶。(3) B1Ago enzyme.

(4)反转录引物为:Oligo(dT)(4) The reverse transcription primer is: Oligo(dT)

(5)反转录反应酶及缓冲液:2×ES Reaction mix。(5) Reverse transcription reaction enzyme and buffer: 2×ES Reaction mix.

(6)dNTP。(6) dNTPs.

(7)小鼠RNase抑制剂。(7) Mouse RNase inhibitor.

(8)MgCl2溶液。(8) MgCl 2 solution.

检测方法如下:The detection method is as follows:

(1)将初级向导ssDNA、荧光报告核酸以及BlAgo酶用超纯水配制成浓度为10μM的溶液;反转录引物R-primer干粉用超纯水溶解制成2μM的溶液。(1) Prepare primary guide ssDNA, fluorescent reporter nucleic acid and BlAgo enzyme with ultrapure water to make a solution with a concentration of 10 μM; reverse transcription primer R-primer dry powder is dissolved with ultrapure water to make a 2 μM solution.

(2)获取的待检样本作为模板加入PCR体系中对其进行扩增,获得靶标DNA;扩增体系为:5μL RNA、10μL 2×ES Reaction mix和1μL Oligo(dT)最后定容到20μL体积。(2) Add the sample to be tested as a template to the PCR system to amplify it to obtain the target DNA; the amplification system is: 5 μL RNA, 10 μL 2×ES Reaction mix and 1 μL Oligo (dT), and finally make it to 20 μL volume .

(3)加入特异性的寡核苷酸向导ssDNAs、与之相应的荧光报告核酸及BlAgo酶在37℃持续保温的条件下反应2h,进行特异性剪切,并对荧光信号进行采集,每分钟检测一次荧光信号。其中向导ssDNAs、荧光报告核酸及BlAgo酶的使用量分别为0.27mM、0.625nM、40.5nM;(3) Add specific oligonucleotide guide ssDNAs, corresponding fluorescent reporter nucleic acid and BlAgo enzyme to react for 2 hours under the condition of continuous incubation at 37°C, perform specific shearing, and collect the fluorescent signal, every minute Fluorescence signal is detected once. The usage amounts of guide ssDNAs, fluorescent reporter nucleic acid and BlAgo enzyme were 0.27mM, 0.625nM and 40.5nM respectively;

具体地,将靶标DNA与反转录引物R-primer、dNTP、ProtoScript II RT、5×ProtoScript II Buffer、DTT、小鼠RNase抑制剂、BlAgo酶、MgCl2、初级向导ssDNA A链B链、荧光报告核酸、RNase-free双蒸水混合,配置成30μL体系。该体系为:R-primer终浓度为400nM,dNTP终浓度为1mM,ProtoScriptII RT 200U,小鼠RNase抑制剂8U,DTT终浓度为100mM,MgCl2终浓度为0.83mM,荧光报告核酸终浓度为0.27mM,靶标核酸分子终浓度分别为5nM、12.5nM、25nM、50nM、125nM、250nM、500nM,靶标核酸分子与BlAgo与向导ssDNAs的终浓度摩尔比40:324:5。Specifically, combine target DNA with reverse transcription primer R-primer, dNTP, ProtoScript II RT, 5×ProtoScript II Buffer, DTT, mouse RNase inhibitor, BlAgo enzyme, MgCl 2 , primary guide ssDNA A chain B chain, fluorescent Mix the reporter nucleic acid and RNase-free double distilled water to make a 30 μL system. The system is: the final concentration of R-primer is 400nM, the final concentration of dNTP is 1mM, ProtoScriptII RT 200U, mouse RNase inhibitor 8U, the final concentration of DTT is 100mM, the final concentration of MgCl 2 is 0.83mM, and the final concentration of fluorescent reporter nucleic acid is 0.27 mM, the final concentrations of target nucleic acid molecules were 5nM, 12.5nM, 25nM, 50nM, 125nM, 250nM, and 500nM, and the molar ratio of the final concentration of target nucleic acid molecules to BlAgo to guide ssDNAs was 40:324:5.

(4)分析荧光信号值,调节Baseline的Start值、End值和阈值线,判定结果。(4) Analyze the fluorescent signal value, adjust the Start value, End value and threshold line of the Baseline, and judge the result.

对比例comparative example

本对比例是采用RT-qPCR检测方法志贺菌属核酸分子,具体操作步骤如下:In this comparative example, RT-qPCR is used to detect Shigella nucleic acid molecules, and the specific operation steps are as follows:

其中,检测试剂包括:Among them, the detection reagents include:

(1)2 × HSYBR qPCR Mix 5 μl(1) 2 × HSYBR qPCR Mix 5 μl

(2)DNA模板(1-10 ng cDNA)2 μl(2) DNA template (1-10 ng cDNA) 2 μl

(3)primer-F、primer-R(10 μM)各0.4 μl(3) 0.4 μl each of primer-F and primer-R (10 μM)

(4)RNase-free water 2.2μl(4) RNase-free water 2.2μl

PCR程序为:The PCR program is:

(1)95 ℃ 10 min(1) 95°C for 10 minutes

(2)95 ℃ 15 s,55 ℃ 30s 72℃ for 30s,42个循环(2) 95°C for 15 s, 55°C for 30s, 72°C for 30s, 42 cycles

(3)4℃ 10min(3) 10min at 4°C

实验例Experimental example

实施例与对比例的检测结果如图1所示。其中A为RT-qPCR试剂盒分别对反应体系中0拷贝至1000拷贝靶基因的检测结果,数据来自九次重复实验;B为BlAgo介导的核酸检测方法分别对反应体系中0拷贝至1000拷贝靶基因的检测结果,数据来自九次重复实验。The detection results of Examples and Comparative Examples are shown in Figure 1. Among them, A is the detection result of 0 copy to 1000 copy target gene in the reaction system by RT-qPCR kit, and the data comes from nine repeated experiments; The detection results of target genes, the data come from nine repeated experiments.

从图1中可以看出,当反应体系中核酸浓度大于等于100拷贝时两种方法均能有效地检测出目标核酸。It can be seen from Figure 1 that both methods can effectively detect the target nucleic acid when the nucleic acid concentration in the reaction system is greater than or equal to 100 copies.

当反应体系中核酸浓度为10拷贝时,RT-qPCR试剂盒的九次重复中只有三次的实验结果的Ct值小于37,当反应体系中核酸浓度为8拷贝或者4拷贝时,相应地Ct值或者为0或者大于38,表明此时已无法准确检测该浓度的核酸样本。When the concentration of nucleic acid in the reaction system is 10 copies, the Ct value of the experimental results in only three of the nine repetitions of the RT-qPCR kit is less than 37. When the concentration of nucleic acid in the reaction system is 8 copies or 4 copies, the corresponding Ct value Or it is 0 or greater than 38, indicating that the nucleic acid sample of this concentration cannot be accurately detected at this time.

而BlAgo介导的核酸检测方法可以准确检测出反应体系中含有8拷贝或者4拷贝的核酸样品,荧光信号的强度均高于阴性对照,在反应体系中只含有1拷贝的核酸样品时,九次重复中有四次实验检测到高于阴性对照的荧光信号,表明BlAgo介导的核酸检测方法的检测下限可以达到1拷贝每反应。The BlAgo-mediated nucleic acid detection method can accurately detect nucleic acid samples containing 8 copies or 4 copies in the reaction system, and the intensity of the fluorescent signal is higher than that of the negative control. When only 1 copy of the nucleic acid sample is contained in the reaction system, nine times Fluorescent signals higher than those of the negative control were detected in four replicates, indicating that the detection limit of the BlAgo-mediated nucleic acid detection method can reach 1 copy per reaction.

因此相比于RT-qPCR,BlAgo介导的核酸检测方法在检测极低浓度的核酸时具有更高的灵敏度和稳定性。Therefore, compared with RT-qPCR, the BlAgo-mediated nucleic acid detection method has higher sensitivity and stability when detecting extremely low concentrations of nucleic acids.

以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims (10)

1.一种志贺菌属核酸分子的检测方法,其特征在于,包括利用Argonaute蛋白的特性,初级向导ssDNAs介导Argonaute蛋白剪切靶标DNA后,断裂产生的次级向导ssDNA再次被Argonaute蛋白利用去剪切与次级向导ssDNA互补的荧光报告核酸;1. A detection method for Shigella nucleic acid molecules, characterized in that, comprising utilizing the characteristics of the Argonaute protein, after the primary guide ssDNAs mediates the shearing target DNA of the Argonaute protein, the secondary guide ssDNA produced by the breakage is utilized by the Argonaute protein again To cut the fluorescent reporter nucleic acid complementary to the secondary guide ssDNA; 所述靶标DNA的序列如SEQ ID NO.1所示。The sequence of the target DNA is shown in SEQ ID NO.1. 2.根据权利要求1所述的检测方法,其特征在于,所述Argonaute蛋白是以DNA为向导指导剪切靶标DNA的中等温度Argonaute蛋白;2. The detection method according to claim 1, wherein the Argonaute protein is a medium-temperature Argonaute protein that guides shearing target DNA with DNA as a guide; 优选地,所述中等温度Argonaute蛋白包括BlAgo酶;Preferably, said intermediate temperature Argonaute protein comprises a BlAgo enzyme; 优选地,所述BlAgo酶的工作温度为36-38℃。Preferably, the working temperature of the BlAgo enzyme is 36-38°C. 3.根据权利要求1所述的检测方法,其特征在于,所述靶标DNA在检测体系中的浓度为≥1拷贝/微升;3. The detection method according to claim 1, wherein the concentration of the target DNA in the detection system is ≥ 1 copy/microliter; 优选地,所述靶标DNA在检测体系中的浓度为≥4拷贝/微升。Preferably, the concentration of the target DNA in the detection system is ≥4 copies/microliter. 4.根据权利要求1所述的检测方法,其特征在于,所述初级向导ssDNAs与靶标DNA互补配对,其包括初级向导ssDNA A链和初级向导ssDNAB链;4. detection method according to claim 1, is characterized in that, described primary guide ssDNAs and target DNA complementary pairing, it comprises primary guide ssDNA A chain and primary guide ssDNA B chain; 所述初级向导ssDNAA链的核苷酸序列如SEQ ID NO.2所示;The nucleotide sequence of the primary guide ssDNAA chain is shown in SEQ ID NO.2; 所述初级向导ssDNA B链的核苷酸序列如SEQ ID NO.3所示。The nucleotide sequence of the primary guide ssDNA B chain is shown in SEQ ID NO.3. 5.根据权利要求4所述的检测方法,其特征在于,所述初级向导ssDNAs为5’-磷酸化的单链DNA分子;5. detection method according to claim 4, is characterized in that, described primary guide ssDNAs is the single-stranded DNA molecule of 5 '-phosphorylation; 优选地,所述初级向导ssDNAs的长度为16-40nt。Preferably, the length of the primary guide ssDNAs is 16-40nt. 6.根据权利要求1-5任一项所述的检测方法,其特征在于,所述靶标DNA和Argonaute蛋白与所述初级向导ssDNAs在检测体系中的摩尔比为8:60:1-40:600:1。6. according to the detection method described in any one of claim 1-5, it is characterized in that, the mol ratio of described target DNA and Argonaute protein and described primary guide ssDNAs in detection system is 8:60:1-40: 600:1. 7.根据权利要求1所述的检测方法,其特征在于,所述次级向导ssDNA的长度为10-25nt。7. The detection method according to claim 1, characterized in that, the length of the secondary guide ssDNA is 10-25nt. 8.根据权利要求1所述的检测方法,其特征在于,所述荧光报告核酸的长度为25-50nt;8. detection method according to claim 1, is characterized in that, the length of described fluorescent reporter nucleic acid is 25-50nt; 优选地,所述荧光报告核酸在检测体系中的浓度为200-1000nM。Preferably, the concentration of the fluorescent reporter nucleic acid in the detection system is 200-1000 nM. 9.根据权利要求8所述的检测方法,其特征在于,所述靶标DNA与荧光报告核酸的摩尔比为:1*102:1至2*105:1,优选地1*102:1至2*103:1。9. The detection method according to claim 8, wherein the molar ratio of the target DNA to the fluorescent reporter nucleic acid is: 1*10 2 : 1 to 2*10 5 : 1, preferably 1*10 2 : 1 to 2*10 3 :1. 10.一种用于检测志贺菌属核酸分子的试剂盒,其特征在于,所述试剂盒包括1-7任一项所述的方法中采用的Argonaute蛋白、初级向导ssDNAs和荧光报告核酸。10. A kit for detecting nucleic acid molecules of the genus Shigella, characterized in that the kit includes the Argonaute protein, primary guide ssDNAs and fluorescent reporter nucleic acid used in the method described in any one of 1-7.
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