WO2022068785A1 - 一种鉴定蜡样芽胞杆菌和苏云金芽胞杆菌的核酸检测方法 - Google Patents
一种鉴定蜡样芽胞杆菌和苏云金芽胞杆菌的核酸检测方法 Download PDFInfo
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Definitions
- the invention belongs to the field of microorganism detection, in particular to a nucleic acid detection method for rapidly identifying Bacillus cereus and Bacillus thuringiensis by high-resolution melting curve.
- Bacillus cereus and Bacillus thuringiensis belong to the same group of Bacillus cereus. Among them, Bacillus cereus is a common food-borne opportunistic pathogen in my country, which can produce diarrhea (enterotoxins) toxins and vomitoxins. During the period from 2008 to 2015, Bacillus cereus ranked third in the prevalence of food-borne pathogens in my country, and was highly harmful. Bacillus thuringiensis is a class of insect pathogenic bacteria. It forms one or more paracellular crystals while forming spores, and its main component is insecticidal crystal protein, so it is widely used as a biological insecticide.
- the physiological and biochemical characteristics of Bacillus cereus and Bacillus thuringiensis are highly similar, and the only difference that can be used to distinguish is the ability to produce paracellular crystals.
- the two have a high degree of genetic similarity, the 16S rRNA sequences are 99%-100% similar, the dDDH (digital DNA-DNA hybridization) value is greater than the species classification threshold of 70%, and the ANI value is greater than the species classification threshold of 95%, all high Due to the standard value of population identification, it is extremely difficult to distinguish. Since there is no molecular rapid detection and identification method that can effectively distinguish Bacillus cereus and Bacillus thuringiensis worldwide, misjudgment may occur during identification, which brings great potential safety hazards to food safety and the industry. Therefore, there is an urgent need to establish a method for the accurate differentiation of Bacillus cereus and Bacillus thuringiensis.
- Bacillus cereus and Bacillus thuringiensis is still the traditional biochemical method, and it is also the national standard method for food microbial safety testing in my country and the US FDA.
- Bacillus thuringiensis needs to be cultured for 4-5 days to produce protein crystals (parascystis), while other Bacillus do not produce protein crystals.
- This process is time-consuming, and if free spore formation is not abundant, the culture needs to be kept at room temperature for 2-3 days for a second check.
- this method is greatly affected by the subjective judgment of observers, which may cause misjudgment, and is not time-sensitive and easy to operate in the actual detection process.
- Korean researchers have discovered a new target XRE, which can be used to identify and differentiate Bacillus thuringiensis from Bacillus closely related. Compared with the existing parasporin gene target cry2, the XRE gene has higher accuracy in the identification of Bacillus thuringiensis.
- the PCR results of XRE showed that neither Bacillus cereus nor non-Bacillus cereus was amplified.
- the real-time PCR method established by XRE could identify Bacillus thuringiensis, and the established standard curve could be used to quantify the number of cells.
- this target failed to detect Bacillus thuringiensis and Bacillus cereus at the same time, and failed to provide a more intuitive and visualized method. Therefore, it is urgent to establish a new molecular method for rapid and accurate identification of the two.
- High-resolution melting curve is an analysis method that has emerged in foreign countries in recent years. Based on PCR technology, it is an analysis technology for gene mutation detection and typing by detecting single-base differences in target fragments. This method is not limited by the site and type of base mutation, does not require specific probes, and runs high-resolution melting directly after the completion of PCR to complete the analysis of sample single nucleotide polymorphism (SNP), Analysis of methylation, matching, etc. Compared with other molecular typing diagnostic methods, HRM has the advantages of simple and rapid operation, low cost, high sensitivity and good specificity, and realizes a true closed-tube operation. The HRM method has received widespread attention and has been applied to various fields, including food safety, environmental monitoring, clinical diagnosis, and biological defense.
- the first object of the present invention is to provide a new molecular target ispD, encoding 2-C-methyl-D-erythritol-4-phosphocysyltransferase (2-C-methyl-D-erythritol- 4-phosphate cytidylyltransferase), used as a target gene in distinguishing Bacillus cereus and Bacillus thuringiensis, this application is for non-disease and therapeutic purposes, and the above two strains can be used in the fields of environment, food and its production chain, clinical and other fields effective distinction.
- the molecular target ispD has a nucleotide sequence shown in SEQ ID NO: 1 (corresponding to Bacillus cereus) or a nucleotide sequence shown in SEQ ID NO: 2 (corresponding to Bacillus thuringiensis).
- the second object of the present invention provides a primer pair for distinguishing Bacillus cereus and Bacillus thuringiensis, which is designed according to the nucleotide sequence shown in SEQ ID NO.1 (corresponding to Bacillus cereus), based on HRM Methods
- SNP site was used to effectively distinguish the two, wherein the amplified product in Bacillus cereus was the nucleotide sequence shown in SEQ ID NO.3, and the amplified product in Bacillus thuringiensis was the nucleotide sequence shown in SEQ ID NO.4. Nucleotide sequence is shown.
- the primer pairs described are as follows:
- the third object of the present invention is to provide a nucleic acid detection method for rapidly identifying Bacillus cereus and Bacillus thuringiensis, comprising the following steps:
- Extract the genomic DNA of the sample to be tested use the genomic DNA as a template, and use the above primer pairs as amplification primers to effectively distinguish between Bacillus cereus and Bacillus thuringiensis through the different characteristic melting curves of Bacillus cereus and Bacillus thuringiensis by high-resolution melting curve method. .
- the PCR reaction system includes: 2x HRM Analysis PreMix, DNA of the sample to be tested, primer pairs, and sterilized double-distilled water.
- the PCR reaction system was as follows: 2x HRM Analysis PreMix reaction buffer 10 ⁇ L, detection sample DNA 50 ng, 10 ⁇ M upstream and downstream primers each 0.6 ⁇ L, sterilized double distilled water to make up the volume to 20 ⁇ L.
- the high-resolution melting curve method is carried out on LightCycler96 using a two-step reaction program.
- the PCR reaction program is: pre-denaturation at 95°C for 2 min, one cycle; denaturation at 95°C for 10s; annealing and extension at 60°C 30min, a total of 40 cycles;
- the high-resolution melting curve reaction program is: denaturation at 95 °C for 1 min, renaturation at 40 °C for 1 min, the initial melting temperature of 65 °C to start the program, the temperature rises at a rate of 0.07 °C/s and melts to 97 °C,
- the fluorescence signal was monitored in real time at 15 Readings/°C during the process.
- the fourth object of the present invention is to provide a method for accurately screening and distinguishing new molecular targets of Bacillus cereus and Bacillus thuringiensis, comprising the following steps:
- the Bacillus cereus strains in the database include Bacillus cereus and Bacillus thuringiensis genome downloading, de-redundancy and genome annotation using Prokka software;
- the invention collects as many public databases as possible and genomes of Bacillus cereus ethnic group strains in previous studies, carries out authenticity confirmation analysis on existing strain classification information, and mines Bacillus cereus (Bacillus cereus) in public databases by comparing genomes and pan-genome methods. ) and Bacillus thuringiensis (Bacillus thuringiensis) can distinguish the target, use this group of experimental strains to verify and screen the characteristic molecular targets of Bacillus cereus and Bacillus thuringiensis, including selecting different SNP sites in the ispD gene sequence of the molecular target to design Primers and primer pairs were screened. HRM experiments were carried out with Bacillus cereus and Bacillus thuringiensis as templates to obtain the characteristic melting curve information of the corresponding strains to achieve accurate identification of Bacillus cereus and Bacillus thuringiensis.
- the present invention performs sequence comparison on the ispD gene to select the SNP site to design primers and perform screening, so as to realize the detection of the SNP site, thereby effectively identifying Bacillus cereus and Bacillus thuringiensis.
- the present invention is based on a high-resolution melting curve method, without sequence-specific probes, and adopts an HRM analysis kit (EvaGreen) (purchased from TIANGEN), which utilizes a novel saturated dye, has an antibody-modified hot-start DNA polymerase, and has High amplification efficiency, high amplification specificity and wide confidence range further increase the stability of the melting curve and improve the specificity of amplification.
- EvaGreen purchased from TIANGEN
- Figure 1 is a phylogenetic tree analysis of Bacillus cereus and Bacillus thuringiensis based on rMLST;
- Figure 2 is a phylogenetic tree analysis of Bacillus cereus and Bacillus thuringiensis based on the full sequence of ispD gene;
- Figure 3 is a phylogenetic tree analysis of Bacillus cereus and Bacillus thuringiensis based on ispD gene amplicon sequences;
- Fig. 4 is the HRM melting curve result of identifying Bacillus cereus ATCC 14579 and Bacillus thuringiensis ATCC 10792 based on ispD gene amplicon, wherein A and B are normalized melting peak map and melting curve respectively;
- Fig. 5 is the HRM identification result of increasing Bacillus cereus and Bacillus thuringiensis, wherein A and B are normalized melting peak map and melting curve respectively;
- Fig. 6 is the specific identification of distinguishing Bacillus cereus and Bacillus thuringiensis based on HRM method, wherein A, B are respectively the normalized melting peak map and melting curve obtained by different standard strains;
- Figures 7 and 8 show the sensitivity detection for the identification of Bacillus cereus and Bacillus thuringiensis based on the HRM method, wherein A and B in Figures 7 and 8 are the normalized melting peak map and melting curve, respectively, and C and D in Figure 7 are respectively Correlation analysis between Bacillus cereus and Bacillus thuringiensis genomic DNA concentration and fluorescence intensity, C and D of Figure 8 are the correlation analysis of the number of Bacillus cereus and Bacillus thuringiensis in unit volume and fluorescence intensity, respectively.
- Bacillus cereus group or related species in GenBank and the existing Bacillus cereus and Bacillus thuringiensis download the genome, remove redundant genome data (contigs ⁇ 200) that do not meet the analysis requirements, and modify the genome Name, use Prokka software for genome annotation; Bacillus cereus group strain genome classification information authenticity confirmation analysis: average nucleotide homology analysis (ANI), based on existing international current Bacillus cereus group or closely related species.
- ANI average nucleotide homology analysis
- 25 reference strains (Table 1) are used as the standard strains of Bacillus cereus group, and the genome similarity between unknown strains and standard strains is compared and analyzed, and the one with the highest similarity with the above-mentioned standard strains is selected, and it is preliminarily believed that the unknown strain is this species;
- PhyloSuite software performed ribosomal protein multi-site sequence typing (rMLST) analysis, extracted 53 ribosomal protein genes (Table 2) and concatenated them for maximum likelihood tree construction.
- the strains with the same evolutionary relationship and ANI analysis results were confirmed to be Bacillus cereus or Bacillus thuringiensis ( Figure 1).
- SEQ ID No: 1 is Bacillus cereus (such as Bacillus cereus ATCC). 14579) ispD nucleotide sequence
- SEQ ID No: 2 is the ispD nucleotide sequence of Bacillus thuringiensis (such as Bacillus thuringiensis ATCC 10792).
- Table 1 25 reference strains of Bacillus cereus group or closely related species in international current
- the specific primers are as follows:
- ispD gene forward primer solution and reverse primer solution with a concentration of 10 ⁇ M in sterile water
- sequence of ispD gene forward primer AACGAAGAAGAACGCCCGTA
- sequence of ispD gene reverse primer TCTTGTCTTTCGGCTCCACC
- LightCycler96 software was used to analyze the high-resolution melting curve results. From the results, it seems that the melting curve based on the gene ispD can achieve a good distinction between Bacillus cereus and Bacillus thuringiensis, as shown in Figure 4. The results were consistent, which theoretically verified the feasibility of using this primer pair to identify Bacillus cereus and Bacillus thuringiensis based on HRM.
- the PCR reaction conditions were pre-denaturation at 95 °C for 2 min, one cycle; denaturation at 95 °C for 10 s, annealing and extension at 60 °C for 30 min, a total of 40 cycles; HRM reaction conditions: denaturation at 95 °C 1min, renaturation at 40°C for 1min, the initial melting temperature was 65°C to start the program, the temperature was increased at a rate of 0.07°C/s and melted to 97°C, and the fluorescence signal was monitored in real time at 15 Readings/°C during the process.
- HRM reaction conditions denaturation at 95 °C 1min, renaturation at 40°C for 1min, the initial melting temperature was 65°C to start the program, the temperature was increased at a rate of 0.07°C/s and melted to 97°C, and the fluorescence signal was monitored in real time at 15 Readings/°C during the process.
- HRM reaction conditions denaturation at 95 °C 1min, renaturation
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Abstract
本发明提供一种快速鉴定蜡样芽胞杆菌和苏云金芽胞杆菌的核酸检测方法，包括选取分子靶标ispD基因序列中不同的SNP位点设计引物，分别以蜡样芽胞杆菌和苏云金芽胞杆菌为模板进行HRM实验，获得对应菌株的特征性熔解曲线信息，实现对蜡样芽胞杆菌和苏云金芽胞杆菌的准确鉴别。
Description
本发明属于微生物检测领域，具体涉及一种通过高分辨率熔解曲线快速鉴定蜡样芽胞杆菌和苏云金芽胞杆菌的核酸检测方法。
蜡样芽胞杆菌(Bacillus cereus)和苏云金芽胞杆菌(Bacillus thuringiensis)同属蜡样芽胞杆菌类群。其中，蜡样芽胞杆菌是我国常见的食源性条件致病菌，可产生腹泻(肠毒素)毒素和呕吐毒素。在2008至2015年期间，蜡样芽胞杆菌在我国食源性致病菌流行率中排名第三，危害性强。苏云金芽胞杆菌是一类昆虫致病菌。其在形成芽胞的同时会形成一个或多个伴胞晶体，其主要成分是杀虫晶体蛋白，因此被广泛地用作生物杀虫剂。蜡样芽胞杆菌和苏云金芽胞杆菌生理生化特征高度相似，可用于辨别的唯一区别是伴胞晶体的产生能力。此外，二者具有高度的遗传相似性，16S rRNA序列具有99％-100％相似性，dDDH(digital DNA-DNA hybridization)值大于物种划分阈值70％，ANI值大于物种划分阈值95％，都高于种群鉴定的标准值，极难区分。由于世界范围内没有对蜡样芽胞杆菌和苏云金芽胞杆菌进行有效区分的分子快速检测鉴定方法，鉴定时可能造成误判，给食品安全和产业带来极大的安全隐患。因此，亟需建立一种对蜡样芽胞杆菌和苏云金芽胞杆菌的准确区分的方法。
目前，能够将蜡样芽胞杆菌和苏云金芽胞杆菌区分的方法仍然是传统生化方法，也是我国和美国FDA食品微生物安全检测的国家标准方法。依照此方法，需要培养苏云金芽胞杆菌4-5天来产生蛋白结晶体(伴胞晶体)，而其他芽胞杆菌不产生蛋白结晶体。该过程耗时，且如果游离芽胞形成不丰富，还需将培养物室温继续放置2-3天进行二次检查。此外，该方法 受观察人员主观判断影响大，可能产生误判，在实际检测过程中不具有时效性与易操作性。近些年来，随着高通量测序技术的不断发展，基于基因组信息的分型与比对可实现蜡样芽胞杆菌和苏云金芽胞杆菌的区分鉴定，但该方法需要进行DNA提取、二代测序、下游信息学分析等，耗时长、成本与操作要求高，不能满足实验室常规使用、高通量快速检测鉴定与基层检测的需要。
已有韩国研究学者发现新型靶标XRE，可用于鉴别和区分苏云金芽胞杆菌与近缘芽胞杆菌。与已有的伴胞晶体蛋白基因靶标cry2相比，XRE基因在苏云金芽胞杆菌的鉴定中具有较高的准确性。XRE的PCR结果显示蜡样芽胞杆菌或非蜡样芽胞杆菌均未扩增，利用XRE建立的实时PCR方法可鉴定苏云金芽胞杆菌，并可利用所建立的标准曲线对细胞数量进行定量。但该靶标未能同时将苏云金芽胞杆菌和蜡样芽胞杆菌进行检测，未能提供一种更直观并具可视化的方法。因此，亟待建立对二者进行快速准确鉴定的新型分子方法。
高分辨率熔解曲线(high-resolution melting，HRM)是近年来在国外兴起的分析方法，以PCR技术为基础，通过检测目标片段单碱基差异从而进行基因变异检测与分型的分析技术。该方法不受碱基突变位点与类型局限，无需特异性探针，在PCR完成后直接运行高分辨率熔解，就可完成对样品单核苷酸多态性(single nucleotide polymorphism，SNP)、甲基化、配型等的分析。相比较于其他分子分型诊断方法，HRM具有操作简便快速、成本低、灵敏度高、特异性好，实现了真正的闭管操作。HRM方法受到普遍关注，并已应用到各个领域，包括食品安全、环境监测、临床诊断和生物防御等领域，对细菌的鉴定及药敏试验、药物的鉴定、疾病诊断等发挥了重要作用。
发明内容：
本发明的第一个目的是提供一种新分子靶标ispD，编码了2-C-甲基-D-赤藓醇-4-磷酸胱氨酰转移酶(2-C-methyl-D-erythritol-4-phosphate cytidylyltransferase)，作为靶标基因在区分蜡样芽胞杆菌和苏云金芽胞杆菌中进行应用，此应用是非疾病的和治疗目的，可在环境、食品及其生产链、临床等领域进行上述两种菌株的有效区分。所述的分子靶标ispD，其核苷酸序列如SEQ ID NO：1所示(对应蜡样芽胞杆菌)或核苷酸序列如SEQ ID NO：2所示(对应苏云金芽胞杆菌)。
本发明的第二个目的提供一种区分蜡样芽胞杆菌和苏云金芽胞杆菌的引物对，其是根据如SEQ ID NO.1所示核苷酸序列(对应蜡样芽胞杆菌)设计得到，基于HRM方法利用SNP位点对两者进行有效区分，其中在蜡样芽胞杆菌中扩增产物为SEQ ID NO.3所示核苷酸序列，在苏云金芽胞杆菌中扩增产物为SEQ ID NO.4所示核苷酸序列。
所述的引物对如下所示：
5’-AACGAAGAAGAACGCCCGTA-3’；
5’-TCTTGTCTTTCGGCTCCACC-3’。
本发明的第三个目的是提供一种快速鉴定蜡样芽胞杆菌和苏云金芽胞杆菌的核酸检测方法，包括以下步骤：
提取待测样品的基因组DNA，以基因组DNA为模板，以上述引物对作为扩增引物用高分辨率熔解曲线方法通过蜡样芽胞杆菌和苏云金芽胞杆菌不同特征性熔解曲线从而对二者进行有效区分。
所述的用高分辨率熔解曲线方法，其PCR反应体系包括：2x HRM Analysis PreMix、待测样品DNA、引物对、灭菌双蒸水。
所述的PCR反应体系为：2x HRM Analysis PreMix反应缓冲液10μL，检测样品DNA 50ng，10μM上、下游引物各0.6μL，灭菌双蒸水补足体积至20μL。
所述的用高分辨率熔解曲线方法，是在LightCycler96上采用两步法反应程序进行反应，其PCR反应程序为：95℃预变性2min，一个循环；95℃变性10s；60℃进行退火及延伸30min，共进行循环40个循环；高分辨率熔解曲线反应程序为：95℃变性1min，40℃复性1min，初始熔解温度65℃开始程序，以0.07℃/s的速率升温熔解至97℃，过程中以15Readings/℃实时监测荧光信号。
本发明的第四个目的是提供一种准确筛选区分蜡样芽胞杆菌和苏云金芽胞杆菌的新分子靶标的方法，包括如下步骤：
(1)数据库中蜡样芽胞杆菌群菌株包括蜡样芽胞杆菌和苏云金芽胞杆菌基因组下载、去冗余和应用Prokka软件进行基因组注释；
(2)蜡样芽胞杆菌群菌株基因组分类信息真实性确证分析：平均核苷酸同源性分析(average nucleotide identity，ANI)，以已有国际现行蜡样芽胞杆菌群或近缘物种的参考菌株作为蜡样芽胞杆菌群的标准菌株，未知菌株与标准菌株全基因组相似度进行比较分析，选用与上述标准菌株相似度最高的，初步认为未知菌株是该种；
(3)蜡样芽胞杆菌群菌株基因组分类信息真实性确证分析：应用PhyloSuite软件进行核糖体蛋白多位点序列分型(rMLST)分析，提取核糖体蛋白基因并将其串联，进行最大似然树构建，以进一步确证相关菌株种属身份。
(4)筛选区分蜡样芽胞杆菌和苏云金芽胞杆菌的新分子靶标：对获得的身份信息真实的蜡样芽胞杆菌和苏云金芽胞杆菌应用Roary软件进行泛基因组分析，设定阈值(Identity)为 95％，选取在95％阈值处可分开的蜡样芽胞杆菌和苏云金芽胞杆菌的核心基因为潜在的分子靶标。
本发明收集尽量多的公共数据库和前期研究中蜡样芽胞杆菌族群菌株基因组，对已有的菌株分类信息进行真实性确证分析，比较基因组和泛基因组方法挖掘公共数据库中蜡样芽胞杆菌(Bacillus cereus)和苏云金芽胞杆菌(Bacillus thuringiensis)可区分靶标，利用本组实验菌株验证和筛选区分蜡样芽胞杆菌和苏云金芽胞杆菌的特征性分子靶标，包括选取分子靶标ispD基因序列中不同的SNP位点设计引物，引物对的筛选，分别以蜡样芽胞杆菌和苏云金芽胞杆菌为模板进行HRM实验，获得对应菌株的特征性熔解曲线信息，实现对蜡样芽胞杆菌和苏云金芽胞杆菌的准确鉴别。
本发明对ispD基因进行序列比对，以选择SNP位点设计引物并进行筛选，以实现对该SNP位点的检测，从而有效鉴定蜡样芽胞杆菌和苏云金芽胞杆菌。本发明基于高分辨率熔解曲线方法，无需序列特异性探针，采用HRM分析试剂盒(EvaGreen)(购自TIANGEN)，该试剂盒利用新型饱和染料，具有抗体修饰的热启动DNA聚合酶，具有高扩增效率、高扩增特异性和宽广的可信范围，进一步增加了熔解曲线的稳定性，提高扩增的特异性，整个流程操作简单，不仅具有灵敏度高、特异性好、成本低、检测速度快等优点，而且分辨率高，可区分单个碱基的变化，同时全部反应在封闭的反应管中完成，有效的避免了交叉污染。
图1为基于rMLST对蜡样芽胞杆菌和苏云金芽胞杆菌的系统发育树分析；
图2为基于ispD基因全序列对蜡样芽胞杆菌和苏云金芽胞杆菌的系统发育树分析；
图3为基于ispD基因扩增子序列对蜡样芽胞杆菌和苏云金芽胞杆菌的系统发育树分析；
图4为基于ispD基因扩增子鉴别蜡样芽胞杆菌ATCC 14579和苏云金芽胞杆菌ATCC 10792的HRM熔解曲线结果，其中A、B分别为归一化熔解峰图和熔解曲线；
图5为增加蜡样芽胞杆菌和苏云金芽胞杆菌的HRM鉴别结果，其中A、B分别为归一化熔解峰图和熔解曲线；
图6为基于HRM方法区分蜡样芽胞杆菌及苏云金芽胞杆菌的特异性鉴定，其中A、B分别为不同标准菌株得归一化熔解峰图和熔解曲线；
图7、8为基于HRM方法鉴定蜡样芽胞杆菌和苏云金芽胞杆菌的灵敏度检测，其中图7和8的A、B分别为归一化熔解峰图和熔解曲线，图7的C、D分别为蜡样芽胞杆菌和苏云金芽胞杆菌基因组DNA浓度与荧光强度的相关性分析，图8的C、D分别为单位体积内蜡样芽胞杆菌数和单位体积内苏云金芽胞杆菌与荧光强度的相关性分析。
下面结合具体实施例及其附图对本发明进行详细描述。
实施例1：
1.以GenBank蜡样芽胞杆菌群或近缘物种基因组和现有蜡样芽胞杆菌和苏云金芽胞杆菌为基础，进行基因组下载、去掉冗余不符合分析要求的基因组数据(contigs≥200)，修改基因组名称，应用Prokka软件进行基因组注释；蜡样芽胞杆菌群菌株基因组分类信息真实性确证分析：平均核苷酸同源性分析(ANI)，以已有国际现行蜡样芽胞杆菌群或近缘物种的25个参考菌株(表1)作为蜡样芽胞杆菌群的标准菌株，未知菌株与标准菌株全基因组相似度进行比较分析，选用与上述标准菌株相似度最高的，初步认为未知菌株是该种；应用PhyloSuite软件进行核糖体蛋白多位点序列分型(rMLST)分析，提取53个核糖体蛋白基因(表2)并 将其串联，进行最大似然树构建。进化关系和ANI分析结果一致的菌株即为确证身份清晰的蜡样芽胞杆菌或苏云金芽胞杆菌(图1)。如图1所示，不同颜色代表不同的芽胞杆菌，基于rMLST方法可以将蜡样芽胞杆菌和苏云金芽胞杆菌菌株进行有效区分。筛选区分蜡样芽胞杆菌和苏云金芽胞杆菌的新分子靶标：对获得的身份信息真实的蜡样芽胞杆菌和苏云金芽胞杆菌应用Roary软件进行泛基因组分析，设定阈值(Identity)为95％，选取在95％阈值处可分开的蜡样芽胞杆菌和苏云金芽胞杆菌的核心基因为潜在的分子靶标ispD，核苷酸序列如下所示，SEQ ID No:1为蜡样芽胞杆菌(如蜡样芽胞杆菌ATCC 14579)的ispD核苷酸序列，SEQ ID No:2为苏云金芽胞杆菌(如苏云金芽胞杆菌ATCC 10792)的ispD核苷酸序列。
表1国际现行蜡样芽胞杆菌群或近缘物种的25个参考菌株
表2 53个核糖体蛋白基因
2.参考GenBank上提供及现有测序完成的蜡样芽胞杆菌和苏云金芽胞杆菌的基因序列，通过比对全基因组序列，挖掘到差异基因ispD，并基于基因序列比对得到的SNP位点设计引物。
具体引物如下：
F：5’-AACGAAGAAGAACGCCCGTA-3’；
R：5’-TCTTGTCTTTCGGCTCCACC-3’。
用此引物对进行扩增，其中在蜡样芽胞杆菌中扩增产物为SEQ ID NO.3所示核苷酸序列，在苏云金芽胞杆菌中扩增产物为SEQ ID NO.4所示核苷酸序列。
3.采用细菌DNA试剂盒(美基生物)按照说明书操作从1mL蜡样芽胞杆菌ATCC 14579培养液及1mL苏云金芽胞杆菌ATCC 10792培养液中分别提取基因组DNA，用Thermo Scientific
TMNanoDrop
TMOne超微量紫外-可见光分光光度计对基因组DNA的浓度和纯度进行检测。
4.为了初步探究所设计引物对蜡样芽胞杆菌及苏云金芽胞杆菌的鉴别效果，基于ispD基因及其扩增子进行系统发育树分析(图2和图3)，根据邻接法进行系统发育树分析，从图2和图3得到差异基因ispD可以将蜡样芽胞杆菌和苏云金芽胞杆菌菌株进行有效区分。利用以 上所设计的引物(步骤2中的)进行菌株PCR扩增，具体为：
以无菌水配制浓度为10μM的ispD基因正向引物溶液及反向引物溶液，ispD基因正向引物的序列：AACGAAGAAGAACGCCCGTA；ispD基因反向引物的序列：TCTTGTCTTTCGGCTCCACC；
在八联管的每个反应孔中依次加入2x HRM Analysis PreMix 10μL和ispD基因正向引物溶液0.6μL、ispD基因反向引物溶液0.6μL，然后在不同的反应孔中分别加入基因组DNA 50ng，以及作为阴性对照的无菌水，将每个反应孔以无菌水补足至20μL；在LightCycler96上采用两步法反应程序进行反应，PCR反应条件为95℃预变性2min，一个循环；95℃变性10s，60℃进行退火及延伸30min，共进行循环40个循环；HRM反应条件：95℃变性1min，40℃复性1min，初始熔解温度65℃开始程序，以0.07℃/s的速率升温熔解至97℃，过程中以15Readings/℃实时监测荧光信号。
应用LightCycler96软件分析高分辨率熔解曲线结果，从结果看来基于基因ispD的熔解曲线可以达到对蜡样芽胞杆菌和苏云金芽胞杆菌很好的区分效果，如图4所示，同时与系统发育树分析的结果一致，从理论上验证了基于HRM利用该引物对鉴别蜡样芽胞杆菌及苏云金芽胞杆菌的可行性。
5.基于ispD基因增加蜡样芽胞杆菌和苏云金芽胞杆菌的菌株数(表3)以进行检测，在八联管的每个反应孔中依次加入2x HRM Analysis PreMix 10μL和ispD基因正向引物溶液0.6μL、ispD基因反向引物溶液0.6μL，然后在不同的反应孔中分别加入待检测样品基因组DNA50ng，以及作为阴性对照的无菌水，将每个反应孔以无菌水补足至20μL；在LightCycler96上采用两步法反应程序进行反应，PCR反应条件为95℃预变性2min，一个循环；95℃变性 10s，60℃进行退火及延伸30min，共进行循环40个循环；HRM反应条件：95℃变性1min，40℃复性1min，初始熔解温度65℃开始程序，以0.07℃/s的速率升温熔解至97℃，过程中以15Readings/℃实时监测荧光信号。应用LightCycler96软件分析HRM结果，如图5所示，蜡样芽胞杆菌和苏云金芽胞杆菌得到不同的特征性熔解曲线可对二者进行有效区分。
表3高分辨率熔解曲线验证实验菌株信息
6.准确鉴定蜡样芽胞杆菌和苏云金芽胞杆菌后，基于ispD基因以其他标准菌株菌按照步骤5的HRM方法进行特异性检测，表4介绍了实验所采用的标准菌株，证明可有效实现对蜡样芽胞杆菌和苏云金芽胞杆菌的鉴定(图6)，特异性好。同时以不同的模板量进行灵敏度检测并建立标准曲线，当基因组DNA浓度范围在10
-7g-10
-12g时可进行有效检测，即该方法的检测限可达到1pg(图7)；以不同单位体积菌落数进行检测时，该方法对蜡样芽胞杆菌的线性检测范围为3.7×10
2cfu/mL-3.7×10
8cfu/mL，对苏云菌芽胞杆菌的线性检测范围为3.3×10
2cfu/mL-3.3×10
8cfu/mL(图8)。
表4高分辨率熔解曲线实验标准菌株信息
Claims (8)
- 分子靶标ispD作为靶标基因在区分蜡样芽胞杆菌和苏云金芽胞杆菌中的应用，此应用是非疾病的治疗目的，所述的分子靶标ispD其核苷酸序列如SEQ ID NO：1所示，对应蜡样芽胞杆菌，或核苷酸序列如SEQ ID NO：2所示，对应苏云金芽胞杆菌。
- 一种区分蜡样芽胞杆菌和苏云金芽胞杆菌的引物对，其特征在于，是根据如SEQ ID NO.1所示的核苷酸序列设计得到，基于HRM方法利用SNP位点对两者进行有效区分。
- 根据权利要求2所述的引物对，其特征在于，所述的引物对如下所示：5’-AACGAAGAAGAACGCCCGTA-3’；5’-TCTTGTCTTTCGGCTCCACC-3’。
- 一种快速鉴定蜡样芽胞杆菌和苏云金芽胞杆菌的核酸检测方法，其特征在于，包括以下步骤：提取待测样品的基因组DNA，以基因组DNA为模板，以权利要求2或3所述的引物对作为扩增引物用高分辨率熔解曲线方法通过蜡样芽胞杆菌和苏云金芽胞杆菌不同特征性熔解曲线从而对二者进行有效区分。
- 根据权利要求4所述的核酸检测方法，其特征在于，所述的用高分辨率熔解曲线方法，其PCR反应体系包括：2x HRMAnalysis PreMix、待测样品DNA、引物对、灭菌双蒸水。
- 根据权利要求5所述的核酸检测方法，其特征在于，所述的PCR反应体系为：2x HRM Analysis PreMix反应缓冲液10μL，检测样品DNA 50 ng，10μM上、下游引物各0.6μL，灭菌双蒸水补足体积至20μL。
- 根据权利要求4所述的核酸检测方法，其特征在于，所述的用高分辨率熔解曲线方法，是在LightCycler96上采用两步法反应程序进行反应，其PCR反应程序为：95℃预变性2 min，一个循环；95℃变性10s；60℃进行退火及延伸30min，共进行循环40个循环；高分辨率熔解曲线反应程序为：95℃变性1min，40℃复性1min，初始熔解温度65℃开始程序，以0.07℃/s的速率升温熔解至97℃，过程中以15 Readings/℃实时监测荧光信号。
- 一种准确筛选区分蜡样芽胞杆菌和苏云金芽胞杆菌的新分子靶标的方法，其特征在于，包括如下步骤：(1)数据库中蜡样芽胞杆菌群菌株包括蜡样芽胞杆菌和苏云金芽胞杆菌基因组下载、去冗余和应用Prokka软件进行基因组注释；(2)蜡样芽胞杆菌群菌株基因组分类信息真实性确证分析：平均核苷酸同源性分析(average nucleotide identity,ANI)，以已有国际现行蜡样芽胞杆菌群或近缘物种的参考菌株作为蜡样芽胞杆菌群的标准菌株，未知菌株与标准菌株全基因组相似度进行比较分析，选用与上述标准菌株相似度最高的，初步认为未知菌株是该种；(3)蜡样芽胞杆菌群菌株基因组分类信息真实性确证分析：应用PhyloSuite软件进行核糖体蛋白多位点序列分型分析，提取核糖体蛋白基因并将其串联，进行最大似然树构建，进行菌株种属身份确认；(4)筛选区分蜡样芽胞杆菌和苏云金芽胞杆菌的新分子靶标：对获得的身份信息真实的蜡样芽胞杆菌和苏云金芽胞杆菌应用Roary软件进行泛基因组分析，设定阈值为95％，选取在95％阈值处可分开的蜡样芽胞杆菌和苏云金芽胞杆菌的核心基因为潜在的分子靶标。
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