CN115725608A - Pathogen specific nucleic acid gene and acquisition and detection method - Google Patents
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Abstract
本发明公开了一种病原体特异性核酸基因及获取和检测方法,获取全基因组序列:对获取的全基因组序列建立数据库并进行多序列比对分析,得到的重叠序列即为该种细菌的种内共同序列;获取种间特异序列:将得到的每种细菌的种内共同序列依次与所有细菌的种内共同序列、所有细菌的基因组序列、人类的基因组序列进行三次BLAST比对,然后将没有重叠的序列作为种间特异序列,种间特异序列即为病原体特异性核酸基因;去除重复序列:将病原体特异性核酸基因中的重复序列覆盖起来。本发明可用于对病原体进行快速鉴定,在临床上应用时阳性检出率高,检测周期短。
The invention discloses a pathogen-specific nucleic acid gene and its acquisition and detection method. Obtaining the whole genome sequence: establishing a database for the acquired whole genome sequence and performing multiple sequence comparison and analysis, and the obtained overlapping sequence is the intraspecies of the bacterium Common sequence; obtain interspecies-specific sequence: perform three BLAST comparisons of the obtained intraspecies common sequence of each bacterium with the intraspecies common sequence of all bacteria, the genome sequence of all bacteria, and the human genome sequence in turn, and then there will be no overlap The sequence is used as the inter-species specific sequence, and the inter-species specific sequence is the pathogen-specific nucleic acid gene; removing repetitive sequences: covering the repetitive sequences in the pathogen-specific nucleic acid genes. The invention can be used for rapid identification of pathogens, has high positive detection rate and short detection period when applied clinically.
Description
技术领域technical field
本发明涉及一种病原体特异性核酸基因及获取和检测方法,属于病原菌检测技术领域。The invention relates to a pathogen-specific nucleic acid gene and an acquisition and detection method, belonging to the technical field of pathogen detection.
背景技术Background technique
基于菌落培养从患者体液样本中鉴定病原菌属于传统病原体检测方法,也是目前临床微生物检验的金标准。该方法需要通过无菌操作收集患者的体液样本,然后将其转移至需氧或厌氧的无菌培养瓶中首先进行病原菌富集培养,随后接种至平板培养皿,最终挑取单克隆进行染色镜检观察、进行选择性培养基上的差异生长实验或进行各种生化测试,根据病原菌的表型特征及生理特征实现对患者样本中病原菌的鉴定。传统培养法不仅耗时(通常需2-5天才能获取病原学信息),同时因为某些厌氧致病菌所需要的严苛生长条件,传统培养法无法培养出目标病原菌,导致检验结果出现假阴性。此外,在细菌培养和分离过程中可能由于操作问题引入污染,产生假阳性结果,从而导致误诊和抗菌药物的误用。The identification of pathogenic bacteria from patient body fluid samples based on colony culture is a traditional pathogen detection method and is currently the gold standard for clinical microbiological testing. This method requires aseptically collecting the patient's body fluid samples, and then transferring them to an aerobic or anaerobic sterile culture bottle, first enriching the pathogenic bacteria, then inoculating it into a flat petri dish, and finally picking a single clone for staining Microscopic observation, differential growth experiments on selective media, or various biochemical tests are performed to identify pathogenic bacteria in patient samples based on their phenotypic and physiological characteristics. The traditional culture method is not only time-consuming (it usually takes 2-5 days to obtain the etiological information), but also because of the harsh growth conditions required by some anaerobic pathogenic bacteria, the traditional culture method cannot cultivate the target pathogenic bacteria, which leads to the failure of the test results. false negative. In addition, contamination may be introduced due to operational problems during the bacterial culture and isolation process, resulting in false-positive results, leading to misdiagnosis and misuse of antimicrobials.
在主要的致病肠杆菌科中,粘质沙雷菌是迄今为止研究最少的病原菌之一。长久以来,研究人员始终认为粘滞沙雷氏菌属于非致病菌或机会致病菌,主要影响免疫功能不成熟的新生儿、儿童及老年人。目前已知粘质沙雷菌可引起呼吸系统、泌尿系统、血液以及眼部感染,其中气管、支气管与泌尿道是最易贮存该菌的部位。同时,儿童的消化道也常检测出该菌。在机体免疫功能低下时也可以引起肺部感染、颅内感染及脓毒症等各类感染。过去20年中,粘质沙雷菌在发达国家的新生儿病房,以及ICU的成年患者人群中发生过连续的感染暴发。意大利学者研究发现,肺炎与脓毒症是患儿感染该细菌最严重的并发症,感染后的病死率高达7%,并且血液与痰液是该病原菌的主要临床分离来源,暗示临床医生需要重点关注该病原菌感染的并发症。对于ICU,神经科等科室,急需快速准确获取病原学信息从而及时合理地开展抗菌治疗,以改善患者的临床表征,增加救治率。然而,目前的临床病原菌检测技术-传统培养法还远远不能满足临床诊断和治疗的需要。Among the major pathogenic Enterobacteriaceae, Serratia marcescens is one of the least studied pathogens to date. For a long time, researchers have always believed that Serratia viscosus is a non-pathogenic or opportunistic pathogen, mainly affecting newborns, children and the elderly with immature immune function. It is currently known that Serratia marcescens can cause respiratory system, urinary system, blood and eye infections, among which the trachea, bronchi and urinary tract are the most likely to store the bacteria. At the same time, the bacterium is often detected in the digestive tract of children. When the body's immune function is low, it can also cause various infections such as pulmonary infection, intracranial infection and sepsis. Over the past 20 years, there have been successive outbreaks of Serratia marcescens infection in neonatal wards and adult ICU populations in developed countries. Italian scholars have found that pneumonia and sepsis are the most serious complications of children infected with this bacterium, and the fatality rate after infection is as high as 7%, and blood and sputum are the main sources of clinical isolation of this pathogen, suggesting that clinicians need to focus on Pay attention to the complications of this pathogen infection. For ICU, neurology and other departments, there is an urgent need to quickly and accurately obtain pathogenic information so as to carry out antibacterial treatment in a timely and reasonable manner, so as to improve the clinical manifestations of patients and increase the treatment rate. However, the current clinical pathogen detection technology - the traditional culture method is far from meeting the needs of clinical diagnosis and treatment.
发明内容Contents of the invention
发明目的:为了克服现有技术中存在的不足,本发明提供一种病原体特异性核酸基因及获取和检测方法。Purpose of the invention: In order to overcome the deficiencies in the prior art, the present invention provides a pathogen-specific nucleic acid gene and its acquisition and detection method.
技术方案:为实现上述目的,本发明采用的技术方案为:Technical scheme: in order to achieve the above object, the technical scheme adopted in the present invention is:
一种病原体特异性核酸基因获取方法,包括以下步骤:A method for obtaining pathogen-specific nucleic acid genes, comprising the following steps:
步骤1,获取全基因组序列:获取常见微生物的全基因组序列。Step 1, obtain the whole genome sequence: obtain the whole genome sequence of common microorganisms.
步骤2,获取同种细菌的种内共同序列:对获取的全基因组序列建立数据库并进行多序列比对分析,首先使用一条某细菌全基因组序列中测序质量高的序列作为目标序列,分别与属于同种细菌的其他菌株全基因组序列进行双序列比对,得到两两的共同序列,再对两两的共同序列进行多序列比对综合分析,得到的重叠序列即为该种细菌的种内共同序列。Step 2. Obtain the common sequence within the same species of bacteria: establish a database for the obtained whole genome sequence and perform multiple sequence comparison analysis. First, use a sequence with high sequencing quality in the whole genome sequence of a certain bacterium as the target sequence, respectively, and the sequences belonging to The whole genome sequences of other strains of the same bacterium are compared in pairs to obtain the common sequences of the two pairs, and then the common sequences of the two pairs are analyzed by multiple sequence alignment, and the overlapping sequences obtained are the intraspecific common sequences of the bacteria. sequence.
步骤3,获取种间特异序列:将得到的每种细菌的种内共同序列依次与所有细菌的种内共同序列、所有细菌的基因组序列、人类的基因组序列进行三次BLAST比对,然后将没有重叠的序列作为种间特异序列,种间特异序列即为病原体特异性核酸基因。
步骤4,去除重复序列:将病原体特异性核酸基因中的重复序列覆盖起来。Step 4, removing repetitive sequences: covering the repetitive sequences in the pathogen-specific nucleic acid gene.
优选的:包括去除重复序列:将病原体特异性核酸基因中的重复序列覆盖起来。Preferably: including removing repetitive sequences: covering the repetitive sequences in the pathogen-specific nucleic acid gene.
一种粘滞沙雷氏菌病原体特异性基因,粘滞沙雷氏菌病原体特异性基因通过病原体特异性核酸基因获取方法获取,粘滞沙雷氏菌病原体特异性基因的核苷酸序列分别如序列表SEQ ID NO:4-40。A Serratia stickies pathogen-specific gene, the Serratia sticks pathogen-specific gene is obtained by a method for acquiring a pathogen-specific nucleic acid gene, and the nucleotide sequences of the Serratia sticks pathogen-specific gene are as follows: SEQUENCE LISTING SEQ ID NO: 4-40.
一种重组表达载体,包括粘滞沙雷氏菌病原体特异性基因。A recombinant expression vector comprising Serratia sticks pathogen-specific genes.
一种转化子,包括重组表达载体的宿主细胞。A transformant, including the host cell of the recombinant expression vector.
一种粘滞沙雷氏菌病原体特异性基因的应用,用于粘滞沙雷氏菌病原体的检测。Application of a Serratia stickies pathogen-specific gene for the detection of the Serratia sticks pathogen.
一种粘滞沙雷氏菌病原体特异性基因的鉴定方法,包括以下步骤:A kind of identification method of Serratia stickies pathogen-specific gene, comprises the following steps:
S101,获取带有粘滞沙雷氏菌病原体的样品。S101, obtaining a sample with the Serratia stickies pathogen.
S102,检测所述样品中是否存在粘滞沙雷氏菌病原体特异性基因或者检测所述样品中粘滞沙雷氏菌病原体特异性基因的含量。对应的粘滞沙雷氏菌病原体特异性基因的核苷酸序列分别如序列表SEQ ID NO:4-40中任一序列的至少一部分,或其互补序列,其中所述粘滞沙雷氏菌病原体特异性基因是否存在或其含量分别反映与所述粘滞沙雷氏菌病原体特异性基因对应的病原体在所述样品中的是否存在或其含量。所述核酸分子的长度不小于50个核苷酸。S102. Detecting whether there is a Serratia stickiesa pathogen-specific gene in the sample or detecting the content of the Serratia stickiesa pathogen-specific gene in the sample. The nucleotide sequences of the corresponding Serratia stickies pathogen-specific genes are respectively at least a part of any sequence in the sequence table SEQ ID NO: 4-40, or its complementary sequence, wherein the Serratia stickies Whether the pathogen-specific gene exists or its content respectively reflects whether the pathogen corresponding to the Serratia stickies pathogen-specific gene exists or its content in the sample. The length of the nucleic acid molecule is not less than 50 nucleotides.
优选的:检测所述样品中是否存在粘滞沙雷氏菌病原体特异性基因或者检测所述样品中粘滞沙雷氏菌病原体特异性基因的含量的方法为:对粘滞沙雷氏菌病原体特异性基因进行扩增反应,通过检测扩增产物是否存在或数量来确定所述粘滞沙雷氏菌病原体特异性基因是否存在或含量。所述扩增反应为PCR扩增反应。Preferably: the method for detecting whether there is a Serratia stickies pathogen-specific gene in the sample or detecting the content of the Serratia sticks pathogen-specific gene in the sample is: for the Serratia sticks pathogen The specific gene is amplified, and the presence or content of the Serratia stickies pathogen-specific gene is determined by detecting the presence or quantity of the amplified product. The amplification reaction is a PCR amplification reaction.
优选的:扩增反应中,粘滞沙雷氏菌病原体特异性基因进行扩增的引物如序列表SEQ ID NO:1和SEQ ID NO:2所示的序列。Preferably: in the amplification reaction, the primers for amplifying the Serratia stickies pathogen-specific gene are the sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence table.
优选的:扩增产物检测利用Crispr/Cas家族核酸酶的反式切割活性进行,与所述Crispr/Cas家族核酸酶联合使用的crRNA的靶序列如序列表SEQ ID NO:3所示的序列。所述Crispr/Cas家族核酸酶为Cas12或者所述Crispr/Cas家族核酸酶为LbCas12。Preferably: detection of the amplification product is carried out using the trans-cleavage activity of the Crispr/Cas family nuclease, and the target sequence of the crRNA used in combination with the Crispr/Cas family nuclease is the sequence shown in SEQ ID NO:3 in the sequence table. The Crispr/Cas family nuclease is Cas12 or the Crispr/Cas family nuclease is LbCas12.
一种用于在样品中检测病原体的试剂盒,其包括A test kit for detecting pathogens in a sample, comprising
1)用于对所述样品中的病原体特异性核酸片段进行扩增以产生扩增产物的引物;以及1) primers for amplifying the pathogen-specific nucleic acid fragments in the sample to generate amplification products; and
2)具有反式切割活性的Crispr/Cas家族核酸酶,以所述扩增产物的至少部分序列作为靶序列的crRNA,和在5’和3’末端分别带有荧光基团和淬灭基团的单链DNA报告分子,其中2) A Crispr/Cas family nuclease with trans-cleavage activity, a crRNA with at least a partial sequence of the amplified product as a target sequence, and a fluorescent group and a quencher group at the 5' and 3' ends respectively The single-stranded DNA reporter molecule, in which
与粘滞沙雷氏菌对应的病原体特异性核酸片段选自SEQ ID NO:4-40中任一序列的至少一部分,或其互补序列The pathogen-specific nucleic acid fragment corresponding to Serratia stickies is selected from at least a part of any sequence in SEQ ID NO: 4-40, or its complementary sequence
在一些实施方案中,所述试剂盒还包括用作阳性标准品的上述核酸片段。In some embodiments, the kit further includes the nucleic acid fragments described above for use as positive standards.
在一些实施方案中,所述Crispr/Cas家族蛋白为LbCas12。In some embodiments, the Crispr/Cas family protein is LbCas12.
在一些实施方案中,用于对粘质沙雷菌菌的病原体特异性核酸片段进行扩增的引物包括SEQ ID NO:1和SEQ ID NO:2所示的序列In some embodiments, the primers used to amplify the pathogen-specific nucleic acid fragments of Serratia marcescens include the sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2
在一些实施方案中,用于粘滞沙雷氏菌的病原体特异性核酸片段进行扩增的引物包括SEQ ID NO:1和SEQ ID NO:2所示的序列,所述crRNA的靶序列包括SEQ ID NO:3所示的序列。In some embodiments, the primers used to amplify the pathogen-specific nucleic acid fragment of Serratia stickies include the sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2, and the target sequence of the crRNA includes SEQ ID NO: 1 and SEQ ID NO: 2. The sequence shown in ID NO:3.
在一些实施方案中,所述样品为来自重症肺炎患者的痰液或肺泡灌洗液。In some embodiments, the sample is sputum or alveolar lavage fluid from a patient with severe pneumonia.
在一些实施方案中,所述试剂盒用于对所述样品中的粘质沙雷菌菌。In some embodiments, the kit is for Serratia marcescens in the sample.
本发明相比现有技术,具有以下有益效果:Compared with the prior art, the present invention has the following beneficial effects:
本发明可用于对病原体进行快速鉴定,在临床上应用时阳性检出率高,检测周期短(例如少于3个小时)。The present invention can be used for rapid identification of pathogens, and has high positive detection rate and short detection period (for example, less than 3 hours) in clinical application.
附图说明Description of drawings
图1为本文描述的获取病原体特异性核酸片段的方法的流程图。Figure 1 is a flowchart of the method for obtaining pathogen-specific nucleic acid fragments described herein.
图2为显示Cas12a的反式切割活性的示意图。Figure 2 is a schematic diagram showing the trans-cleavage activity of Cas12a.
图3为以针对粘滞沙雷氏菌的引物对SEQ ID NO:1和SEQ ID NO:2测试临床常见各种病原体的DNA模板进行常规PCR扩增后扩增产物的电泳结果图。Fig. 3 is a graph showing the electrophoresis results of the amplified products after routine PCR amplification using the primer pair SEQ ID NO: 1 and SEQ ID NO: 2 for Serratia stickies to test the DNA templates of various common clinical pathogens.
图4显示了利用LbCas12a和crRNA对不同扩增产物进行检测的荧光信号结果。Figure 4 shows the fluorescent signal results of detecting different amplification products by using LbCas12a and crRNA.
具体实施方式Detailed ways
下面结合附图和具体实施例,进一步阐明本发明,应理解这些实例仅用于说明本发明而不用于限制本发明的范围,在阅读了本发明之后,本领域技术人员对本发明的各种等价形式的修改均落于本申请所附权利要求所限定的范围。Below in conjunction with accompanying drawing and specific embodiment, further illustrate the present invention, should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention, after having read the present invention, those skilled in the art will understand various aspects of the present invention All modifications of the valence form fall within the scope defined by the appended claims of the present application.
“病原体特异性核酸片段”,也可称为病原菌或病原体特有核酸片段(通常指DNA序列,也可以包括RNA序列),指:生物分类学上属于同一种病原菌的菌株的基因组中均广泛存在的DNA序列,但是该序列在其他种类的病原菌基因组中并不存在,即种内共有种间特异的DNA序列。可以依据病原菌特有DNA序列来准确地检测鉴定细菌的种类。"Pathogen-specific nucleic acid fragments" may also be referred to as pathogenic bacteria or pathogen-specific nucleic acid fragments (usually referring to DNA sequences, but may also include RNA sequences), and refer to: the genomes of strains belonging to the same pathogenic bacteria in biological taxonomy are widely present DNA sequence, but this sequence does not exist in the genomes of other types of pathogenic bacteria, that is, there are inter-species specific DNA sequences shared within the species. The type of bacteria can be accurately detected and identified based on the unique DNA sequence of the pathogenic bacteria.
在一些实施方案中,本文通过如下步骤获取病原菌特有DNA序列(参见图1):In some embodiments, the specific DNA sequence of pathogenic bacteria is obtained by the following steps (see Figure 1):
1)获取全基因组序列:从NCBI等公共数据库获取数百种常见微生物的全基因组序列;1) Obtain the whole genome sequence: obtain the whole genome sequence of hundreds of common microorganisms from public databases such as NCBI;
2)获取同种细菌的种内共同序列:对获取的全基因组序列建立数据库并进行多序列比对分析,首先使用一条某细菌全基因组序列中准确度较高(测序质量高)的序列作为目标序列,分别与属于同种细菌的其他菌株全基因组序列进行双序列比对,得到两两的共同序列,再对两两的共同序列进行多序列比对综合分析,得到的重叠(overlap)序列即为该种细菌的种内共同序列;2) Obtain the common sequence within the same species of bacteria: establish a database for the obtained whole genome sequence and perform multiple sequence comparison analysis, first use a sequence with high accuracy (high sequencing quality) in the whole genome sequence of a certain bacterium as the target Sequences are compared with the whole genome sequences of other strains belonging to the same bacterium respectively to obtain the common sequences of the two pairs, and then the multiple sequence comparison and comprehensive analysis of the common sequences of the two pairs are carried out, and the overlap sequence obtained is is the common sequence within the species of the bacteria;
3)获取种间特异序列:将得到的每种细菌的种内共同序列依次与所有细菌的种内共同序列、所有细菌的基因组序列(比对的时候排除自身比对)、人类的基因组(hg19)序列进行三次BLAST比对,然后将没有重叠的序列作为种间特异序列,种间特异序列即为病原菌特有DNA序列;3) Obtain inter-species specific sequences: the obtained intra-species common sequence of each bacterium is sequenced with the intra-species common sequence of all bacteria, the genome sequence of all bacteria (excluding self-alignment when comparing), human genome (hg19 ) sequences were compared three times by BLAST, and then the non-overlapping sequences were used as inter-species specific sequences, which were the specific DNA sequences of pathogenic bacteria;
4)去除重复序列:使用RepeatMasker等工具将重复序列覆盖起来。4) Remove repetitive sequences: Use tools such as RepeatMasker to cover the repetitive sequences.
采用这种方式,我们获得了多种细菌的多个特异性核酸片段,其中粘滞沙雷氏菌的核苷酸序列分别显示在SEQ ID NO:4-40中。In this way, we obtained multiple specific nucleic acid fragments of various bacteria, wherein the nucleotide sequences of Serratia stickies are shown in SEQ ID NO: 4-40, respectively.
在获得这些病原体特异性核酸片段的序列后,可方便地用于对患者或患者样品中的各种病原体进行检测。例如,特定病原体特异性核酸片段的存在可用于指示患者或患者样品中包括该特定病原体;特定病原体特异性核酸片段的含量可用于指示患者或患者样品中该特定病原体的含量。这些病原体特异性核酸片段的序列本身也可包括在一些检测试剂盒中,例如可作为阳性对照品。可理解的是,在检测过程中,可以检测本文提供的针对粘滞沙雷氏菌的特异性核酸片段(SEQ ID NO:4-40)的全长;或者,仅检测其部分片段,例如长度为50、60、80、100个核苷酸或更多核苷酸的片段。类似地,试剂盒包括的病原体特异性核酸片段也可以为全长片段或部分片段(例如长度为50、60、80、100个核苷酸或更多核苷酸的片段)。在一些优选的实施方案中,病原体特异性核酸片段的长度至少为60个核苷酸。After obtaining the sequences of these pathogen-specific nucleic acid fragments, they can be conveniently used to detect various pathogens in patients or patient samples. For example, the presence of a particular pathogen-specific nucleic acid fragment can be used to indicate that a patient or patient sample contains that particular pathogen; the amount of a particular pathogen-specific nucleic acid fragment can be used to indicate the amount of that particular pathogen in a patient or patient sample. The sequences of these pathogen-specific nucleic acid fragments themselves can also be included in some detection kits, for example, as positive controls. It can be understood that, in the detection process, the full length of the specific nucleic acid fragment (SEQ ID NO: 4-40) provided herein for Serratia stickies can be detected; or, only a partial fragment thereof, such as the length are fragments of 50, 60, 80, 100 nucleotides or more. Similarly, the pathogen-specific nucleic acid fragments included in the kit can also be full-length fragments or partial fragments (eg, fragments with a length of 50, 60, 80, 100 nucleotides or more). In some preferred embodiments, the pathogen-specific nucleic acid fragment is at least 60 nucleotides in length.
核酸扩增nucleic acid amplification
核酸扩增技术的应用改变了微生物病原体的诊断方式,也是目前病原微生物检测鉴定手段中常用的分子生物学技术。在20世纪80年代,先后出现了多种用于DNA扩增的技术,主要包括聚合酶链式反应(PCR)技术、连接酶链式反应(LCR)技术以及等温扩增技术。The application of nucleic acid amplification technology has changed the way of diagnosing microbial pathogens, and it is also a molecular biology technique commonly used in the detection and identification of pathogenic microorganisms. In the 1980s, a variety of technologies for DNA amplification have emerged, mainly including polymerase chain reaction (PCR) technology, ligase chain reaction (LCR) technology and isothermal amplification technology.
具有耐高温特性的DNA聚合酶的发现与应用,使PCR技术成为最常用的DNA扩增技术。PCR技术检测病原微生物的原理是利用特异性的寡核苷酸链作为引物,以含待扩增序列的靶核酸作为模板,通过不断转换温度以实现双链DNA的指数型扩增,从而得到大量目的DNA片段(即扩增产物)用于后续鉴定。该技术的优势在于高灵敏性和易操作性,能够对极微量的病原菌完成检测,对于生长周期较长、培养条件苛刻或生化反应特点不典型的病原微生物检测至关重要。The discovery and application of DNA polymerase with high temperature resistance make PCR technology the most commonly used DNA amplification technology. The principle of PCR technology to detect pathogenic microorganisms is to use specific oligonucleotide chains as primers, use the target nucleic acid containing the sequence to be amplified as a template, and achieve exponential amplification of double-stranded DNA by continuously changing the temperature, thereby obtaining a large number of The target DNA fragment (i.e. the amplified product) is used for subsequent identification. The advantage of this technology lies in its high sensitivity and ease of operation, and it can complete the detection of very small amounts of pathogenic bacteria, which is very important for the detection of pathogenic microorganisms with long growth cycles, harsh culture conditions or atypical biochemical reaction characteristics.
LCR是PCR技术问世后开发的另一种体外扩增技术。该技术的原理是:使用耐高温的DNA连接酶和四条引物,两条相邻的正向引物及两条与其反向互补配对的反向引物,在两条相邻引物之间通常存在1个缺口,其充当DNA连接酶连接的模板。DNA连接酶特异性较高,不耐受碱基错配,所以该项技术经常被用于SNP的检测。LCR is another in vitro amplification technique developed after the advent of PCR technology. The principle of this technology is: use high temperature resistant DNA ligase and four primers, two adjacent forward primers and two reverse primers that are complementary to their reverse counterparts, usually there is one primer between the two adjacent primers. nick, which serves as a template for DNA ligase ligation. DNA ligase has high specificity and is not tolerant to base mismatches, so this technology is often used for SNP detection.
核酸的等温扩增是一项简便的技术,该技术可以在恒定温度下快速且有效地积累核酸序列,自20世纪90年代初以来,已经开发了多种等温扩增技术作为PCR的替代方法。与PCR技术相比,等温扩增技术无需复杂的热循环过程(也因此可降低扩增成本),仅在一个特定温度下实现扩增反应。本领域常用的等温扩增技术包括:基于核酸序列的扩增(NuclearAcid Sequence-Based Amplification,NASBA)、链置换扩增(Strand DisplacementAmplification,SDA)、重组酶聚合酶扩增(Recombinase Polymerase Amplification,RPA)、解旋酶依赖性恒温DNA扩增(Helicase-dependent Isothermal DNA Amplification,HDA)、环介导恒温扩增(Loop-mediated Isothermal Amplification,LAMP)、滚环扩增(Rolling Circle Amplification,RCA)、等等。这些等温扩增技术为本领域技术人员所熟知,在此不另加赘述。Isothermal amplification of nucleic acids is a facile technique that can rapidly and efficiently accumulate nucleic acid sequences at a constant temperature, and since the early 1990s, various isothermal amplification techniques have been developed as alternatives to PCR. Compared with PCR technology, isothermal amplification technology does not require complex thermal cycle process (and therefore can reduce the cost of amplification), and only realizes the amplification reaction at a specific temperature. Isothermal amplification techniques commonly used in the art include: Nucleic Acid Sequence-Based Amplification (NASBA), Strand Displacement Amplification (SDA), Recombinase Polymerase Amplification (RPA) , Helicase-dependent Isothermal DNA Amplification (HDA), Loop-mediated Isothermal Amplification (LAMP), Rolling Circle Amplification (RCA), etc. wait. These isothermal amplification techniques are well known to those skilled in the art and will not be described in detail here.
在本文的一些实施方案中,可以在检测病原体特异性核酸片段之前,对样品中的核酸进行扩增,以增加检测的灵敏度。In some embodiments herein, the nucleic acid in the sample can be amplified before detecting the pathogen-specific nucleic acid fragment, so as to increase the detection sensitivity.
CRISPR/Cas基因编辑系统和Cas12a的反式切割活性CRISPR/Cas gene editing system and the trans-cleavage activity of Cas12a
CRISPR/Cas系统具有靶向切割核酸分子的潜力,基于此系统的基因编辑工具逐步被大量开发和利用。目前,基于CRISPR/Cas9和CRISPR/Cas12a系统的基因编辑工具应用最为普遍。它们的工作原理简述如下:首先,借助于RNA分子的靶向功能,引导Cas蛋白定向切割目标基因的双链DNA,造成DNA链的完整性被破坏;然后,细胞内相应的DNA修复系统被激活,主要包括NHEJ修复机制与HDR修复机制,从而完成对目标基因的破坏或定向改造。The CRISPR/Cas system has the potential to target nucleic acid molecules, and gene editing tools based on this system have been developed and utilized in large numbers. Currently, gene editing tools based on the CRISPR/Cas9 and CRISPR/Cas12a systems are most commonly used. Their working principles are briefly described as follows: First, with the help of the targeting function of RNA molecules, the Cas protein is guided to cut the double-stranded DNA of the target gene, causing the integrity of the DNA chain to be destroyed; then, the corresponding DNA repair system in the cell is activated. Activation mainly includes NHEJ repair mechanism and HDR repair mechanism, so as to complete the destruction or directional transformation of the target gene.
与以往的基因编辑工具相比,基于CRISPR/Cas的基因编辑技术存在相当大的优势:组成简单,仅由一个Cas蛋白及sgRNA(对于Cas12a,仅需crRNA)组成,因而针对不同位点的编辑只需要更换不同的sgRNA(或者仅更换其中与靶核酸结合的种子区(seed region)序列)即可。可以通过设计多对sgRNA以实现多个基因位点的同时编辑,为多拷贝基因的功能研究提供了可能。Compared with previous gene editing tools, CRISPR/Cas-based gene editing technology has considerable advantages: the composition is simple, consisting of only one Cas protein and sgRNA (for Cas12a, only crRNA is required), so it can edit different sites It is only necessary to replace different sgRNAs (or only replace the sequence of the seed region (seed region) that binds to the target nucleic acid). Multiple pairs of sgRNAs can be designed to simultaneously edit multiple gene loci, providing the possibility for functional studies of multiple copies of genes.
近两年研究发现,Cas12a不仅具有靶向切割DNA的顺式(cis)切割活性,也具有非特异性切割任意单链DNA的反式(trans)切割活性(参见图2)。Cas12a-crRNA复合体与crRNA反向互补配对的目标DNA结合后,会激发Cas12a的反式切割活性,导致Cas12a无差别的切割附近的任意单链DNA分子(即无序列特异性)。所以,在单链报告DNA分子存在时,可以利用Cas12a的反式切割活性检测目标DNA分子。检测过程例如包括:首先利用PCR或RPA等技术对待测样本中的目标DNA进行指数型扩增,以提高检测的灵敏度;然后利用Cas12a、crRNA以及单链报告DNA对扩增产物进行检测,如果扩增产物中存在与crRNA的种子区对应的DNA序列(5’上游还存在PAM序列,如TTTA),则会激发Cas12a的反式切割活性从而对报告DNA进行切割,而产生荧光信号。Studies in the past two years have found that Cas12a not only has the cis (cis) cleavage activity of targeting DNA, but also has the trans (trans) cleavage activity of non-specifically cutting any single-stranded DNA (see Figure 2). After the Cas12a-crRNA complex binds to the target DNA of the reverse complementary pairing of crRNA, it will stimulate the trans-cleavage activity of Cas12a, causing Cas12a to indiscriminately cut any single-stranded DNA molecule nearby (that is, without sequence specificity). Therefore, in the presence of single-stranded reporter DNA molecules, the trans-cleavage activity of Cas12a can be used to detect target DNA molecules. The detection process includes, for example: firstly, using techniques such as PCR or RPA to perform exponential amplification on the target DNA in the sample to be tested to improve the sensitivity of detection; then using Cas12a, crRNA and single-stranded reporter DNA to detect the amplification product, if the amplification The presence of a DNA sequence corresponding to the seed region of crRNA in the amplification product (there is also a PAM sequence such as TTTA upstream of the 5' will stimulate the trans-cleavage activity of Cas12a to cut the reporter DNA and generate a fluorescent signal.
在一些实施方案中,与Cas12crRNA配合使用的crRNA由21nt的骨架区(backbone)以及20~24nt用于识别目标靶序列的种子区(seed region/spacer)组成。在更具体的实施方案中,体外转录合成的crRNA的序列为:5’-AAUUUCUACUAAGUGUAGAUCAGGUAAGGCGCCUCGGGUG-3’(SEQ ID NO:41)。在一些实施方案中,可通过以DNA为模板,利用T7 RNA聚合酶在体外转录得到对应的crRNA,并且通过纯化得到纯净的crRNA。In some embodiments, the crRNA used in conjunction with Cas12crRNA consists of a 21nt backbone region (backbone) and a 20-24nt seed region (seed region/spacer) for identifying the target sequence. In a more specific embodiment, the sequence of the crRNA synthesized by in vitro transcription is: 5'-AAUUUUCUACUAAGUGUAGAUCAGGUAAGGCGCCUCGGGUG-3' (SEQ ID NO: 41). In some embodiments, the corresponding crRNA can be obtained by in vitro transcription using DNA as a template and using T7 RNA polymerase, and purified crRNA can be obtained by purification.
单链报告DNA分子的一端具有荧光基团修饰,一端具有淬灭基团修饰。由于淬灭基团的存在,完整单链报告DNA分子不产生荧光信号,当Cas12a的反式切割活性被激发后,切割该单链报告DNA分子,使得淬灭基团与荧光基团分离,产生荧光信号。荧光信号的是否存在或强度提示是否存在扩增产物或扩增产物的量。The single-stranded reporter DNA molecule is modified with a fluorescent group on one end and a quencher group on the other. Due to the existence of the quencher group, the complete single-stranded reporter DNA molecule does not produce a fluorescent signal. When the trans-cleavage activity of Cas12a is activated, the single-stranded reporter DNA molecule is cut, so that the quencher group is separated from the fluorescent group, resulting in fluorescent signal. The presence or intensity of the fluorescent signal indicates the presence or amount of amplification product.
在本研究中,我们首先利用生物信息学方法获得了针对粘滞沙雷氏菌的种内特异性DNA序列,并且根据这些序列设计并合成了用于靶向识别该病原菌的特异性crRNA。接着,我们利用大肠杆菌原核表达并纯化了来源于毛螺杆菌(Lachnospiraceae bacterium)的LbCas12a蛋白,验证了该蛋白具备顺式与反式切割活性。随后利用单链报告DNA,LbCas12a蛋白与特异的crRNA,辅助于PCR扩增技术,开发了基于CRISPR/Cas12a的病原菌检测方法,并且验证了其准确性及特异性。最终,利用基于CRISPR/Cas12a的病原菌检测工具在3个小时以内实现了重症肺炎病人临床样本的快速检测。本文提供的基于CRISPR/Cas12a的病原菌检测方法有望成为一种新型快速的临床病原菌检测手段,为提高重症肺炎的诊治水平提供重要的技术支持。In this study, we first obtained the intraspecies-specific DNA sequences for Serratia stickies by using bioinformatics methods, and based on these sequences, we designed and synthesized specific crRNAs for targeted recognition of the pathogen. Next, we expressed and purified the LbCas12a protein derived from Lachnospiraceae bacterium using Escherichia coli prokaryotic, and verified that the protein had cis- and trans-cleavage activities. Subsequently, using single-stranded reporter DNA, LbCas12a protein and specific crRNA, assisted by PCR amplification technology, a pathogen detection method based on CRISPR/Cas12a was developed, and its accuracy and specificity were verified. Finally, the rapid detection of clinical samples from patients with severe pneumonia was achieved within 3 hours using the CRISPR/Cas12a-based pathogen detection tool. The CRISPR/Cas12a-based pathogen detection method provided in this paper is expected to become a new and rapid clinical pathogen detection method, and provide important technical support for improving the diagnosis and treatment of severe pneumonia.
在本文的一些实施方案中,利用Cas12a的反式切割活性来对病原体特异性核酸片段或其扩增产物进行检测,由于需要crRNA与目标DNA互补配对结合,该检测方法进一步增加了检测的特异性。In some embodiments herein, the trans-cleavage activity of Cas12a is used to detect pathogen-specific nucleic acid fragments or their amplified products. Since crRNA is required to be complementary to the target DNA, this detection method further increases the specificity of detection. .
以下通过具体实施例进一步说明本发明。The present invention is further illustrated below by specific examples.
实施例1基于CRISPR/Cas的病原菌检测方法的特异性验证Example 1 Specificity verification of the pathogenic bacteria detection method based on CRISPR/Cas
用于检测的病原菌均来源于鼓楼医院重症监护室,由重症肺炎患者的临床样本中分离培养而来,并通过鼓楼医院微生物检验科确定菌株种类(每种病原菌具有两组,分别来源于不同病人)。The pathogenic bacteria used for detection are all from the intensive care unit of Gulou Hospital, isolated and cultured from clinical samples of patients with severe pneumonia, and the types of strains are determined by the Department of Microbiology of Gulou Hospital (each pathogenic bacteria has two groups, each from different patients) ).
1.特异性检验1. Specificity test
为了证明基于CRISPR/Cas系统的病原菌检测方法的特异性与可靠性,我们对扩增引物及相应的crRNA分别进行了交叉检测实验。In order to prove the specificity and reliability of the pathogen detection method based on the CRISPR/Cas system, we conducted cross-detection experiments on the amplification primers and the corresponding crRNA respectively.
1.1引物特异性检验1.1 Primer specificity test
为了明确扩增引物的特异性,针对粘滞沙雷氏菌的扩增引物分别以粘滞沙雷氏菌,以及临床常见的其它11种病原菌所提取的基因组DNA为模板,进行交叉式PCR反应。In order to clarify the specificity of the amplification primers, the amplification primers for Serratia stickies were used as templates for the amplification primers of Serratia stickies and other 11 common clinical pathogenic bacteria to carry out cross-type PCR reaction .
所使用的扩增引物中正向和反向引物分别为:The forward and reverse primers in the amplification primers used are:
SEQ ID NO:1和2,用于粘滞沙雷氏菌的基因组DNA进行扩增;SEQ ID NO: 1 and 2, used for the amplification of the genomic DNA of Serratia sticks;
与以上引物对配合使用的crRNA的靶序列(或称为种子区(seed region))如SEQID NO:3所示。The target sequence (or called the seed region) of the crRNA used in conjunction with the above primer pair is shown in SEQ ID NO:3.
如下显示了利用粘滞沙雷氏菌病原体特异性核酸片段来设计扩增引物和crRNA序列的实例,其中与上游引物和下游引物(SEQ ID NO:1和2)对应的序列加有下划线,crRNA的靶序列(SEQ ID NO:3)加有下划线,PAM序列加有方框底色。The example of using the Serratia stickies pathogen-specific nucleic acid fragment to design amplification primers and crRNA sequences is shown below, wherein the sequences corresponding to the upstream primer and the downstream primer (SEQ ID NO: 1 and 2) are underlined, crRNA The target sequence (SEQ ID NO: 3) is underlined and the PAM sequence is boxed.
使用I-5TM Master Mix(TsingKe)进行PCRPCR using I-5 TM Master Mix (TsingKe)
TIANSeq HiFi Amplification Mix PCR反应体系:TIANSeq HiFi Amplification Mix PCR reaction system:
PCR反应条件:PCR reaction conditions:
PCR扩增都设置相应的阳性对照和阴性对照,分别以目标病原菌的基因组DNA和水作为扩增模板。Corresponding positive controls and negative controls were set for PCR amplification, and the genomic DNA and water of the target pathogen were respectively used as amplification templates.
PCR反应结束后,加入相应体积的6×Gel Loading Dye(NEB),混合均匀后通过琼脂糖电泳检测。根据胶图结果有无目的DNA大小的明显亮带判断引物特异性。After the PCR reaction, a corresponding volume of 6×Gel Loading Dye (NEB) was added, mixed evenly, and detected by agarose electrophoresis. Judge the specificity of the primers according to the presence or absence of obvious bright bands of the size of the target DNA in the results of the gel map.
1.2 crRNA特异性检验1.2 crRNA specificity test
利用LbCas12a及针对病原菌特异DNA序列设计的上述crRNA分别检测对应引物扩增的PCR产物,每个样品设置3个重复。LbCas12a and the above-mentioned crRNA designed for the specific DNA sequence of the pathogenic bacteria were used to detect the PCR products amplified by the corresponding primers, and three replicates were set for each sample.
反应体系:reaction system:
其中ssDNA-reporter的结构和序列为:5’-FAM-TTATT-BHQ1-3’。The structure and sequence of ssDNA-reporter are: 5'-FAM-TTATT-BHQ1-3'.
将上述反应液加入到384孔板,37℃反应30~45min。反应结束后,利用酶标仪(Infinite M200 Pro多功能酶标仪,Austria Tecan)检测每孔的荧光值,检测参数设置如下:The above reaction solution was added to a 384-well plate, and reacted at 37°C for 30-45min. After the reaction, use a microplate reader (Infinite M200 Pro multi-functional microplate reader, Austria Tecan) to detect the fluorescence value of each well, and the detection parameters are set as follows:
2.实验结果2. Experimental results
2.1引物特异性检验结果2.1 Primer specificity test results
如图3所示,仅个别的非目标病原菌存在少量的非特异性杂带,但是在以对应病原菌基因组DNA为模板时可以扩增出大量目的DNA产物,说明针对粘滞沙雷氏菌扩增的引物特异性良好。As shown in Figure 3, only a small amount of non-specific bands exist in individual non-target pathogenic bacteria, but a large number of target DNA products can be amplified when the corresponding pathogenic bacteria genomic DNA is used as a template, indicating that the amplification of Serratia sticks Primer specificity is good.
2.2 crRNA特异性检验结果2.2 crRNA specificity test results
利用LbCas12a以及针对粘滞沙雷氏菌的crRNA对上述对应引物扩增后的PCR反应液进行检测。图4的荧光结果显示:粘滞沙雷氏菌病原菌的检测体系仅在存在粘滞沙雷氏菌病原菌的基因组DNA作为模板进行PCR扩增后的扩增产物时才能出现明显的荧光信号,而其余非目标病原菌的荧光信号强度和阴性对照一致,说明与crRNA结合使得这种检测方法的特异性极好。LbCas12a and crRNA against Serratia stickies were used to detect the PCR reaction solution amplified by the above corresponding primers. The fluorescence results in Figure 4 show that the detection system of the Serratia stickies pathogenic bacterium can only produce obvious fluorescent signals when there is the genomic DNA of the Serratia stickies pathogenic bacterium as a template for the amplification product after PCR amplification, while The fluorescence signal intensity of the remaining non-target pathogenic bacteria was consistent with that of the negative control, indicating that the combination with crRNA made the specificity of this detection method excellent.
实施例2利用CRISPR/Cas系统检测重症肺炎病患临床样本Example 2 Using the CRISPR/Cas system to detect clinical samples from patients with severe pneumonia
1.实验操作1. Experimental operation
12个临床样本(痰液或肺泡灌洗液)均来源于鼓楼医院重症监护室,并与鼓楼医院微生物检验科利用传统的培养分离检测法确定的病原菌类型进行对比。The 12 clinical samples (sputum or alveolar lavage fluid) all came from the intensive care unit of Gulou Hospital, and were compared with the types of pathogenic bacteria determined by traditional culture isolation and detection methods in the Department of Microbiology of Gulou Hospital.
1.1试剂盒法提取临床样本DNA1.1 Kit method to extract DNA from clinical samples
临床样本DNA的提取参照Quick-DNA/RNATM Pathogen Miniprep Kit(ZYMORESEARCH)产品说明书进行,操作步骤简述如下:(离心转速均为16,000×g)The extraction of DNA from clinical samples was carried out according to the Quick-DNA/RNA TM Pathogen Miniprep Kit (ZYMORESEARCH) product manual. The operation steps are briefly described as follows: (centrifugation speed is 16,000×g)
a)吸取50-200μL样本,加入800μL DNA/RNA Shield试剂,涡旋60s。a) Pipette 50-200 μL sample, add 800 μL DNA/RNA Shield reagent, and vortex for 60 seconds.
b)16,000×g离心1min,吸取200μL上清液。b) Centrifuge at 16,000×g for 1 min, and absorb 200 μL of supernatant.
c)加入2μl Proteinase K试剂至200μl上清液中,混匀。c) Add 2 μl Proteinase K reagent to 200 μl supernatant and mix well.
d)加入1ml Pathogen DNA/RNA缓冲液试剂,混匀后室温静止5min。d) Add 1ml of Pathogen DNA/RNA buffer reagent, mix well and let stand at room temperature for 5min.
e)吸取上述溶液转移至DNA结合柱(已置于回收管),离心30s,弃去过柱液。e) Transfer the above solution to a DNA binding column (placed in a recovery tube), centrifuge for 30 seconds, and discard the column liquid.
f)加入500μl Pathogen DNA/RNA Wash缓冲液至柱子中,离心30s,弃去过柱液。该步骤重复一次。f) Add 500 μl of Pathogen DNA/RNA Wash buffer to the column, centrifuge for 30 seconds, and discard the column liquid. This step is repeated once.
g)加入500μl乙醇(95-100%)至柱子中,16,000×g离心1min,确保乙醇清除干净,丢弃回收管,将DNA结合柱放置于无DNA酶的1.5ml离心管。g) Add 500 μl of ethanol (95-100%) to the column, centrifuge at 16,000×g for 1 min to ensure that the ethanol is removed, discard the recovery tube, and place the DNA-binding column in a DNase-free 1.5 ml centrifuge tube.
h)吸取50μl 65℃的双蒸水至柱子的基质中,静止2~5min,16,000×g离心1min,收集洗脱液。h) Pipette 50 μl of double-distilled water at 65° C. into the matrix of the column, let it rest for 2 to 5 minutes, centrifuge at 16,000×g for 1 minute, and collect the eluate.
i)测定DNA浓度,并储存于-20℃冰箱。i) Measure DNA concentration and store in -20°C refrigerator.
1.2PCR反应扩增靶序列1.2 PCR reaction to amplify the target sequence
以提取的临床样本总DNA为模板,分别使用实施例1中描述的1对引物进行PCR扩增反应。阳性对照组所用模板为粘滞沙雷氏菌的基因组DNA,阴性对照组的模板为水。Using the total DNA extracted from clinical samples as a template, a PCR amplification reaction was performed using a pair of primers described in Example 1, respectively. The template used in the positive control group was the genomic DNA of Serratia stickies, and the template in the negative control group was water.
PCR反应体系与反应条件同实施例1。The PCR reaction system and reaction conditions are the same as in Example 1.
1.3 Cas12a检测1.3 Cas12a detection
利用LbCas12a、crRNA及ssDNA-reporter检测未纯化的PCR反应原液。对于同一个待测样品,设置3个重复;Use LbCas12a, crRNA and ssDNA-reporter to detect the unpurified PCR reaction stock solution. For the same sample to be tested, set 3 repetitions;
检测反应体系及反应条件同实施例1。Detection reaction system and reaction conditions are the same as in Example 1.
2.实验结果2. Experimental results
利用1对扩增引物分别对12份重症肺炎病患临床样本的总DNA进行PCR扩增,随后利用单链报告DNA、LbCas12a及粘滞沙雷氏菌的特异crRNA检测相应的PCR反应液。基于荧光信号判定的检测结果显示在表1中,并且与传统培养法进行比较。A pair of amplification primers was used to amplify the total DNA of 12 clinical samples from patients with severe pneumonia, and then single-stranded reporter DNA, LbCas12a and specific crRNA of Serratia stickies were used to detect the corresponding PCR reaction solution. The detection results based on the fluorescence signal judgment are shown in Table 1 and compared with the traditional culture method.
表1 12个肺泡灌洗液临床样本中粘滞沙雷氏菌检测结果的比较Table 1 Comparison of the detection results of Serratia viscosus in 12 clinical samples of alveolar lavage fluid
从表中可知,基于CRISPR/Cas12a检测1号,5号,8号和10号临床样本的中检出粘滞沙雷氏菌,剩余样本显示阴性。临床培养结果显示1号,5号和8号样本中存在粘滞沙雷氏菌。12个样本中有5个样本送检了二代测序技术,其中5号,8号和10号临床样本的中检出粘滞沙雷氏菌,2号和4号样本显示阴性。值得注意的是,10号样本中二代测序技术显示存在少量的粘滞沙雷氏菌病原体,而临床培养方法未检出,基于我们开发的PCR-CRISPR/Cas12a联合检测技术显示阳性。It can be seen from the table that based on the CRISPR/Cas12a detection of clinical samples No. 1, No. 5, No. 8 and No. 10, Serratia viscosus was detected, and the remaining samples were negative. The results of clinical culture showed the presence of Serratia viscosus in samples No. 1, No. 5 and No. 8. Five of the 12 samples were submitted to next-generation sequencing technology, among which Serratia viscosus was detected in clinical samples Nos. 5, 8 and 10, and samples Nos. 2 and 4 were negative. It is worth noting that the next-generation sequencing technology in sample No. 10 showed a small amount of Serratia stickies pathogen, but it was not detected by the clinical culture method, and it was positive based on the PCR-CRISPR/Cas12a combined detection technology developed by us.
总之,相比于依赖于病原菌分离和培养的传统检测方法,本研究开发的基于CRISPR/Cas12a的病原菌检测工具展现出良好的检出效果,尤其是针对于低荷菌量的样本,比如10号样本,并且将传统的检测周期由数天缩短为4个小时以内,初步证实该检测方法可以作为一种快速诊断临床样本中粘滞沙雷氏菌存在情况的潜在检测工具。除了粘滞沙雷氏菌,基于本文提供的众多病原体的特异性核酸片段,可以采用类似方法进行多种其他感染性疾病的病原微生物鉴定。In conclusion, compared with the traditional detection methods that rely on the isolation and cultivation of pathogenic bacteria, the pathogen detection tool based on CRISPR/Cas12a developed in this study shows good detection effect, especially for samples with low bacterial load, such as No. 10 samples, and the traditional detection cycle was shortened from several days to less than 4 hours. It was preliminarily confirmed that this detection method can be used as a potential detection tool for rapid diagnosis of the presence of Serratia sticks in clinical samples. In addition to Serratia stickies, based on the specific nucleic acid fragments of many pathogens provided herein, similar methods can be used to identify pathogenic microorganisms of various other infectious diseases.
以上所述仅是本发明的优选实施方式,应当指出:对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。The above is only a preferred embodiment of the present invention, it should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications are also possible. It should be regarded as the protection scope of the present invention.
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