WO2021082590A1 - 一种对副溶血弧菌o9血清型o抗原分子分型的lamp检测方法 - Google Patents

一种对副溶血弧菌o9血清型o抗原分子分型的lamp检测方法 Download PDF

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WO2021082590A1
WO2021082590A1 PCT/CN2020/107355 CN2020107355W WO2021082590A1 WO 2021082590 A1 WO2021082590 A1 WO 2021082590A1 CN 2020107355 W CN2020107355 W CN 2020107355W WO 2021082590 A1 WO2021082590 A1 WO 2021082590A1
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vibrio parahaemolyticus
antigen
lamp
serotype
typing
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王磊
宁可馨
王晓晨
曹勃阳
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南开大学
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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
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • the invention relates to a LAMP technology for typing the O9 serotype strain O antigen of Vibrio parahaemolyticus in a sample and a preparation method thereof.
  • the present invention also designs a detection method using the LAMP primer.
  • Vibrio parahaemolyticus is a gram-negative bacterium with arc, rod, filament and other shapes without spores. It is a kind of halophilic bacteria. It belongs to the Vibrio genus of the Vibrio family. Vibrio parahaemolyticus is found in water, fish, salted foods and human intestines. It can cause abdominal pain, vomiting, and diarrhea. It is an important intestinal pathogen. At present, the classification and identification of Vibrio parahaemolyticus are mainly based on: morphological characteristics of bacteria, physiological and biochemical characteristics of bacteria, serological reactions and other methods. The O antigen typing of Vibrio parahaemolyticus is a kind of serological reaction. Due to the diversity of the environment and antibodies, the diversity of O antigens is formed. According to the diversity of O antigens, different strains of Vibrio parahaemolyticus can be typed and identified.
  • Loop-mediated Isothermal Amplification technology can perform nucleic acid amplification in a short time (usually within one hour) under isothermal (60-65°C) conditions. It is a "simple, fast, and accurate , Low-cost" gene amplification method. Compared with conventional PCR, it does not require thermal denaturation, temperature cycling, electrophoresis, and ultraviolet observation of the template.
  • the loop-mediated isothermal amplification method is a new nucleic acid amplification method, which is simple, rapid and specific. This technology is comparable to or even better than PCR technology in indicators such as sensitivity, specificity and detection range. It does not rely on any special equipment to achieve high-throughput and rapid detection on site, and the detection cost is much lower than fluorescent quantitative PCR.
  • 60-65°C is the intermediate temperature for double-stranded DNA renaturation and extension, and DNA is in a dynamic equilibrium state at about 65°C. Therefore, DNA synthesis is possible at this temperature. Utilize 4 kinds of specific primers and rely on a kind of high activity strand displacement DNA polymerase. This makes strand-displacement DNA synthesis continue to circulate on its own.
  • Amplification is divided into two stages: the first stage is the initial stage.
  • the first stage is the initial stage.
  • the F2 sequence of the upstream internal primer FIP first binds to the template F2c and extends forward under the action of strand displacement DNA polymerase to initiate strand displacement synthesis.
  • the external primer F3 binds to the template F3c and extends, replacing the complete FIP-linked complementary single strand.
  • F1c on FIP and F1 on this single strand are complementary structures. Self-base pairing forms a ring structure.
  • the downstream primers BIP and B3 successively initiate the synthesis similar to FIP and F3, forming a single strand with a dumbbell-like structure.
  • the F1 section at the 3'end Use itself as a template to synthesize and extend DNA to form a stem loop structure.
  • This structure is the starting structure of the LAMP gene amplification cycle.
  • the second stage is the amplification cycle stage.
  • FIP binds to the F2c region of the stem-loop. Start strand displacement synthesis, and a ring structure will also be formed on the dissociated single-stranded nucleic acid.
  • B1 on the BIP primer hybridizes with it to start a new One round of amplification, and the product DNA length doubled.
  • Two loop primers LF and LB were added to the reaction system, and they were also combined with the stem loop structure to initiate strand replacement synthesis. Repeatedly, the final product of the amplification is a mixture of DNA with different numbers of stem-loop structures and different lengths, and the product DNA is an alternating inverted repeat sequence of the amplified target sequence.
  • the inventor has applied for: nucleotides specific to Vibrio parahaemolyticus K36, K37, K68 and their applications; nucleotides specific to Vibrio parahaemolyticus K36, K37, K68 and their applications.
  • the difference between this application and the published patent application is that the detection method no longer uses gene chips (due to separate amplification and hybridization, which takes a little longer), but uses LAMP technology to detect (one-step reaction amplification detection, simple and fast, no Rely on any special equipment to achieve high-throughput and rapid on-site testing, with low testing costs).
  • the present invention discloses a LAMP primer for typing the Vibrio parahaemolyticus O9 serotype O antigen in a sample.
  • the primer contains FIP primer, F3 primer, BIP primer and B3 primer.
  • LAMP primers are mainly aimed at six different regions of the target gene.
  • Four primers are designed based on 6 different sites including the F3c, F2c and Flc regions at the 3'end of the target gene and the Bl, B2 and B3 regions at the 5'end.
  • FIP Forward Inner Primer
  • upstream internal primer composed of F2 region and F1C region.
  • F2 region is complementary to the F2c region at the 3'end of the target gene, and the F1C region has the same sequence as the Flc region at the 5'end of the target gene.
  • F3 primer the upstream outer primer (Forward Outer Primer), which consists of the F3 region and is complementary to the F3c region of the target gene.
  • BIP primer Backward Inner Primer, composed of B1C and B2 regions.
  • the B2 region is complementary to the B2c region at the 3'end of the target gene, and the B1C domain has the same sequence as the Blc region at the 5'end of the target gene.
  • B3 primer Backward Outer Primer, composed of the B3 region, and complementary to the B3c region of the target gene.
  • Vibrio parahaemolyticus O9 serotype O antigen typing refers to the specific gene sequence of the O antigen gene cluster from the Vibrio parahaemolyticus O9 serotype, with the sequence from SEQ ID NO:1-SEQ ID NO : A DNA sequence in the DNA sequence shown in 4.
  • the LAMP primer for typing the Vibrio parahaemolyticus O9 serotype O antigen in the sample of the present invention is characterized in that the primer sequence has the primer shown in SEQ ID NO: 1-SEQ ID NO: 4.
  • the present invention further discloses the application of LAMP primers for typing the O antigen molecule of Vibrio parahaemolyticus O9 serotype O in the sample, and the application in the detection of O antigen molecule typing of Vibrio parahaemolyticus O9.
  • the said Vibrio parahaemolyticus refers to the crude extraction of the pure culture of the sample obtained by separating it from any environment suitable for the living of Vibrio parahaemolyticus.
  • the experimental results show that the present invention can type the O antigen of Vibrio parahaemolyticus at a lower DNA concentration.
  • the present invention provides a LAMP system for detecting a serotype in an environment.
  • the system includes: WarmStart Colorimetric LAMP 2X Master Mix (NEB), 10 ⁇ M FIP, F3, BIP and B3 primers, 1 ⁇ L DNA and ddH 2 O.
  • the described primers are designed based on the specific gene sequence of the O antigen gene cluster of Vibrio parahaemolyticus O9 serotype: use http://primerexplorer.jp/ from the O antigen gene cluster specific gene sequence of Vibrio parahaemolyticus O9 serotype e/
  • the length of the designed B3 and F3 primers is about 20 nt, and the Tm is between 55-60°C; the length of the BIP and FIP primers is about 50 nt; the "-" in the primer sequence represents "TTTT".
  • the present invention is the practical application of the LAMP loop-mediated isothermal amplification technology, and is used for the typing and identification of Vibrio parahaemolyticus O9 serotype.
  • the present invention introduces LAMP technology into the field of Vibrio parahaemolyticus O antigen typing for the first time, and establishes a rapid, sensitive, highly accurate, and reproducible sample of Vibrio parahaemolyticus O9 serotype LAMP
  • the detection method, the use of the LAMP probe of the present invention can achieve the purpose of identifying common serotype Vibrio parahaemolyticus O strains in the sample. Because of its simple operation, high accuracy and strong reproducibility, this invention is useful for real-time and rapid detection of medical departments at all levels.
  • the O antigen typing of Vibrio parahaemolyticus in samples has important application value.
  • Figure 1 VP O9 LAMP reaction positive and specific detection: add Vibrio parahaemolyticus O1, O2, O3, O4, O5, O6, O7, O8, O9, O10, respectively to the LAMP system of the Vibrio parahaemolyticus genome sample O9
  • the genomes of O11, O12 and O13 have no crossover with genomes other than O9, indicating that the LAMP primers of Vibrio parahaemolyticus O9 have good specificity;
  • FIG. 1 PS O9 LAMP reaction foreign strain detection: the LAMP system of Vibrio parahaemolyticus genome sample O9 was added with the genomes of P. shigella O45 and Legionella pneumophila O5, and the LAMP primers of Vibrio parahaemolyticus O9 Good specificity.
  • Vibrio parahaemolyticus used in the present invention are shown in Table 1 below:
  • O-unit processing genes wzx and wzy
  • glycosyltransferase genes are generally highly specific for each O-antigen, and are commonly used as targets in PCR-based O-serotyping methods.
  • specific primers were mainly designed based on the specific glycosyltransferase gene located in the variable region of Vibrio parahaemolyticus OGD.
  • Vibrio parahaemolyticus O9 serotype uses wvdH gene.
  • the all_vs_all_blast method is performed on all genes in the gene cluster to perform comparison, and the number of matches of specific genes will inevitably be much smaller than that of conservative genes. Synthesize the above methods to find specific genes and design primers for them.
  • a specific gene of Vibrio parahaemolyticus was selected as a template to design LAMP primers.
  • FIP Forward Inner Primer
  • upstream internal primer composed of F2 region and F1C region, F2 region is complementary to the F2c region at the 3'end of the target gene, and the F1C region has the same sequence as the Flc region at the 5'end of the target gene
  • F3 primer upstream external primer (Forward Outer Primer), consisting of the F3 region and complementary to the F3c region of the target gene
  • BIP primer Backward Inner Primer, consisting of the B1C and B2 regions, and the B2 region is complementary to the B2c region at the 3'end of the target gene , B1C domain is the same sequence as the Blc region at the 5'end of the target gene
  • B3 primer Backward Outer Primer, composed of the B3 region, and complementary to the B3c region of the target gene.
  • Table 2 The quantity and sequence information of each primer are listed in Table 2.
  • the length of B3 and F3 primers is about 20 nt, and the Tm is between 55-60°C; the length of BIP and FIP primers is about 50 nt.
  • the primers were synthesized by Invitrogen (Shanghai, China).
  • sample nucleic acid isolated in any environment suitable for the life of Vibrio parahaemolyticus, crude extract of pure culture of the sample
  • Sample processing Take 1 mL of the overnight cultured bacteria solution into a 1.5 mL centrifuge tube, centrifuge at 8000 rpm at room temperature for 1 min, discard the supernatant, and collect the bacteria. Add 400 ⁇ L Buffer Digestion, shake and mix, and bath at 65°C for 1h until the cells are completely lysed.
  • the obtained DNA is dissolved in 50-100 ⁇ L ddH 2 O, and stored in the refrigerator at -20°C for later use;
  • the LAMP system of Vibrio parahaemolyticus O9, O1, O2, O3, O4, O5, O6, O7, O8, O9, O10, O11, O12, and O13 were added to the LAMP system of the Vibrio parahaemolyticus genome sample O9.
  • Genome the LAMP primer of Vibrio parahaemolyticus O9 has good specificity.
  • the genomes of Vibrio parahaemolyticus O9, P. shiga-like O45, and Legionella pneumophila O10 were added to the LAMP system of Vibrio parahaemolyticus O9.
  • the LAMP of Vibrio parahaemolyticus O9 The primer has good specificity.

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Abstract

本发明公开了一种对副溶血弧菌O9血清型O抗原分子分型的LAMP检测方法。本发明以副溶血弧菌O9的O抗原基因簇内的特异基因，即wvdH为靶基因，建立了对副溶血弧菌O9的O抗原分型的四条引物，为肠道及水环境中副溶血弧菌的O抗原分型提供一条途径。利用本发明的LAMP引物检测肠道及水环境中的副溶血弧菌，并且对其进行O抗原分型。

Description

一种对副溶血弧菌O9血清型O抗原分子分型的LAMP检测方法
本申请要求于2019年10月30日提交中国专利局、申请号为201911043452.7、发明名称为“一种对副溶血弧菌O9血清型O抗原分子分型的LAMP检测方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及对样品中副溶血弧菌O9血清型菌株O抗原分型的LAMP技术及其制备方法。本发明还设计利用所述的LAMP引物进行检测的方法。
背景技术
副溶血弧菌，是一种革兰氏阴性菌，呈弧状、杆状、丝状等多种形状，无芽孢。是一种嗜盐性细菌。隶属于弧菌科弧菌属。副溶血弧菌在水、鱼类、含盐分较高的腌制食品及人类肠道中发现，能引起人的腹痛、呕吐、腹泻，是一种重要的肠道致病菌。目前对副溶血弧菌的分型鉴定主要根据有：细菌的形态学特征、细菌的生理生化特征、血清学反应等方法，副溶血弧菌的O抗原分型属于血清学反应的一种。由于环境和抗体的多样性，形成了O抗原的多样性，而根据O抗原的多样性可以对副溶血弧菌的不同菌株进行分型鉴定。
环介导等温扩增技术(Loop-mediated Isothermal Amplification)能在等温(60-65℃)条件下，短时间(通常是一小时内)内进行核酸扩增，是一种“简便、快速、精确、低价”的基因扩增方法。与常规PCR相比，不需要模板的热变性、温度循环、电泳及紫外观察等过程。环介导等温扩增法是一种全新的核酸扩增方法，具有简单、快速、特异性强的特点。该技术在灵敏度、特异性和检测范围等指标上能媲美甚至优于PCR技术，不依赖任何专门的仪器设备实现现场高通量快速检测，检测成本远低于荧光定量PCR。
其技术原理为：60-65℃是双链DNA复性及延伸的中间温度，DNA 在65℃左右处于动态平衡状态。因此，DNA在此温度下合成是可能的。利用4种特异引物依靠一种高活性链置换DNA聚合酶。使得链置换DNA合成在不停地自我循环。
扩增分两个阶段：第1阶段为起始阶段，任何一个引物向双链DNA的互补部位进行碱基配对延伸时，另一条链就会解离，变成单链。上游内部引物FIP的F2序列首先与模板F2c结合，在链置换型DNA聚合酶的作用下向前延伸启动链置换合成。外部引物F3与模板F3c结合并延伸，置换出完整的FIP连接的互补单链。FIP上的F1c与此单链上的F1为互补结构。自我碱基配对形成环状结构。以此链为模板，下游引物BIP与B3先后启动类似于FIP和F3的合成，形成哑铃状结构的单链。迅速以3’末端的F1区段为起点。以自身为模板，进行DNA合成延伸形成茎环状结构。该结构是LAMP基因扩增循环的起始结构。
第2阶段是扩增循环阶段。以茎环状结构为模板，FIP与茎环的F2c区结合。开始链置换合成，解离出的单链核酸上也会形成环状结构。迅速以3’末端的B1区段为起点，以自身为模板，形成DNA合成延伸及链置换，形成长短不一的2条新茎环状结构的DNA，BIP引物上的B2与其杂交，启动新一轮的扩增，且产物DNA长度增加一倍。在反应体系中添加2条环状引物LF和LB，它们也分别与茎环状结构结合启动链置换合成。周而复始，扩增的最后产物是具有不同个数茎环结构、不同长度DNA的混合物，且产物DNA为扩增靶序列的交替反向重复序列。
发明人曾申请过：对副溶血弧菌K36，K37，K68特异的核苷酸及其应用；对副溶血弧菌K36，K37，K68特异的核苷酸及其应用。本申请和已经公开的专利申请的区别在于：检测手段不再利用基因芯片(因扩增和杂交分开进行，时间稍长)，而是利用LAMP技术检测(一步反应扩增检测，简单快速，不依赖任何专门的仪器设备实现现场高通量快速检测，检测成本低)。
发明内容
为实现上述目的，本发明公开了一种对样品中副溶血弧菌O9血清型 O抗原分型的LAMP引物，该引物含有FIP引物、F3引物、BIP引物以及B3引物。
LAMP引物的设计主要是针对靶基因的六个不同的区域，基于靶基因3'端的F3c、F2c和Flc区以及5'端的Bl、B2和B3区等6个不同的位点设计4种引物。
FIP(Forward Inner Primer)：上游内部引物，由F2区和F1C区域组成，F2区与靶基因3’端的F2c区域互补，F1C区与靶基因5'端的Flc区域序列相同。
F3引物：上游外部引物(Forward Outer Primer)，由F3区组成，并与靶基因的F3c区域互补。
BIP引物：下游内部引物(Backward Inner Primer)，由B1C和B2区域组成，B2区与靶基因3'端的B2c区域互补，B1C域与靶基因5'端的Blc区域序列相同.
B3引物：下游外部引物(Backward Outer Primer)，由B3区域组成，和靶基因的B3c区域互补。
其主要特征在于所述的副溶血弧菌O9血清型O抗原分型指的是：从副溶血弧菌O9血清型的O抗原基因簇特异基因序列，具有从SEQ ID NO:1-SEQ ID NO:4所示的DNA序列中的一种DNA序列。
本发明所述对样品中副溶血弧菌O9血清型O抗原分型的LAMP引物，其特征在于所述的引物序列：具有SEQ ID NO:1-SEQ ID NO:4所示的引物。
本发明进一步公开了对样品中副溶血弧菌O9血清型O抗原分子分型的LAMP引物，在用于副溶血弧菌O9的O抗原分子分型检测方面的应用。所述的副溶血弧菌指的是分离于任何适合副溶血弧菌生活的环境中所得到的，样品的纯培养物的粗提。实验结果显示：本发明可以在较低的DNA浓度下对副溶血弧菌O抗原进行分型。
本发明提供检测环境中一种血清型的LAMP体系，该体系包括：WarmStart Colorimetric LAMP 2X Master Mix(NEB)，10μM FIP、F3、BIP以及B3引物，1μL DNA和ddH 2O。所述的根据从副溶血弧菌O9血清 型的O抗原基因簇特异基因序列设计的引物：从副溶血弧菌O9血清型的O抗原基因簇特异基因序列中用http://primerexplorer.jp/e/设计的B3、F3引物长度约为20nt，Tm在55-60℃之间；BIP、FIP引物长度约为50nt；其中引物序列中“-”代表“TTTT”。
本发明是LAMP环介导等温扩增技术的实际应用，用于副溶血弧菌O9血清型的副溶血弧菌分型鉴定。
由上述的技术方案可见，本发明首次将LAMP技术引入副溶血弧菌O抗原分型领域，建立了一种快速、灵敏、准确性高、重复性强的样品中副溶血弧菌O9血清型LAMP检测方法，利用本发明的LAMP探针可以达到鉴定样品中常见的副溶血弧菌O血清型菌株的目的，由于操作简便，准确性高，重复性强，该发明对于各级医疗部门实时快速检测样品中副溶血弧菌O抗原分型具有重要的应用价值。
附图说明
图1 VP O9 LAMP反应阳性及特异性检测：在副溶血弧菌基因组样品O9的LAMP体系中分别加入副溶血弧菌O1、O2、O3、O4、O5、O6、O7、O8、O9、O10、O11、O12以及O13的基因组，与除O9外的基因组无交叉，说明副溶血弧菌O9的LAMP引物特异性良好；
图2 PS O9 LAMP反应外源菌株检测：在副溶血弧菌基因组样品O9的LAMP体系中分别加入类志贺邻单胞菌O45以及嗜肺军团菌O5的基因组，副溶血弧菌O9的LAMP引物特异性良好。
具体实施方式
下面通过具体的实施方案叙述本发明。除非特别说明，本发明中所用的技术手段均为本领域技术人员所公知的方法。另外，实施方案应理解为说明性的，而非限制本发明的范围，本发明的实质和范围仅由权利要求书所限定。对于本领域技术人员而言，在不背离本发明实质和范围的前提下，对这些实施方案中的物料成分和用量进行的各种改变或改动也属于本发明的保护范围。本发明所用原料及试剂均有市售。
本发明所用到的副溶血弧菌菌种来源如下表1所示：
表1本实验所用到的菌种
Figure PCTCN2020107355-appb-000001
a，BCRC:台湾生物资源保存及研究中心
b，CDC：中国疾病预防控制中心
c，CNCTC：Czech National Collection of Type Cultures,the Czech Republic
实施例1
引物的设计
1、特异基因的筛选
据调查显示，27％的临床副溶血性弧菌对tdh和trh呈阴性。另一方面，毒素基因之间显著的核苷酸序列相似性经常出现在不同的细菌物种中，特别是在同一属内，这使得这些毒素基因无法用于细菌病原体的物种特异性鉴定。因此，在血清型鉴别检测中我们通常选择其O抗原簇中的特异基因(wzx或wzy)；
O-单元加工基因(wzx和wzy)和糖基转移酶基因通常对每一个O-抗原高度特异，并且通常用作基于PCR的O-血清群分型方法中的靶标。然而，由于副溶血弧菌中不存在O-侧链，副溶血弧菌不含O-单元加工基因。因此，在该研究中，主要基于位于副溶血性弧菌OGD的可变区中的特定糖基转移酶基因设计特异性引物。副溶血性弧菌O9血清型选用wvdH基因。本发明通过对基因簇中所有的基因进行all_vs_all_blast的方法，进行比对，特异基因的匹配数必然会远小于保守基因。综合上述方法找到特异基因并对其设计引物。
2、引物的设计
以挑选出来的1种副溶血弧菌的特异基因为模板设计LAMP的引物。
使用http://primerexplorer.jp/e/根据加工wzx基因的序列设计LAMP引物。FIP(Forward Inner Primer)：上游内部引物，由F2区和F1C区域组成，F2区与靶基因3’端的F2c区域互补，F1C区与靶基因5'端的Flc区域序列相同；F3引物：上游外部引物(Forward Outer Primer)，由F3区组成，并与靶基因的F3c区域互补；BIP引物：下游内部引物(Backward Inner Primer)，由B1C和B2区域组成，B2区与靶基因3'端的B2c区域互补，B1C域与靶基因5'端的Blc区域序列相同；B3引物：下游外部引物(Backward Outer Primer)，由B3区域组成，和靶基因的B3c区域互补。每种引物的数量和序列信息列于表2中。
B3、F3引物长度约为20nt，Tm在55-60℃之间；BIP、FIP引物长度约为50nt。引物由Invitrogen(中国上海)合成。
表2 LAMP所用到的引物
Figure PCTCN2020107355-appb-000002
Figure PCTCN2020107355-appb-000003
实施例2
样本核酸的提取(分离于任何适合副溶血弧菌生活的环境中所得到的，样品的纯培养物的粗提液)
1、样品处理：取1mL过夜培养的细菌菌液加入1.5mL离心管中，室温8000rpm，离心1min，弃上清，收集菌体。加入400μL Buffer Digestion，震荡混匀，65℃水浴1h至细胞完全裂解。
水浴过程中，每10min颠倒混匀一次，可促进样品裂解，混合液变澄清透明为裂解完全；
2、加入200μL Buffer PB，充分颠倒混匀，-20℃冰箱放置5min；
3、室温10000rpm离心5min，将上清液(500-550μL)转移到新的1.5mL离心管中；
4、加入等体积的异丙醇，颠倒5-8次使之充分混匀，室温放置2-3min，室温10000rpm离心5min，弃上清；
5、加入1mL 75％乙醇，颠倒漂洗1-3min，10000rpm离心2min，弃上清；
6、重复步骤5一次；
7、开盖室温倒置5-10min至残留的乙醇完全挥发；
8、得到的DNA用50-100μL ddH 2O溶解，并于-20℃冰箱备用；
9、定浓度至300ng/μL。
实施例3
阳性及特异性检测
以提取的核酸溶液作为LAMP反应的模板，LAMP反应体系及反应条件：
1、LAMP反应体系(25μL)：
表3
Figure PCTCN2020107355-appb-000004
表4
Figure PCTCN2020107355-appb-000005
表5
Figure PCTCN2020107355-appb-000006
表6
Figure PCTCN2020107355-appb-000007
表7
Figure PCTCN2020107355-appb-000008
表8
Figure PCTCN2020107355-appb-000009
表9
Figure PCTCN2020107355-appb-000010
表10
Figure PCTCN2020107355-appb-000011
表11
Figure PCTCN2020107355-appb-000012
表12
Figure PCTCN2020107355-appb-000013
表13
Figure PCTCN2020107355-appb-000014
表14
Figure PCTCN2020107355-appb-000015
表15
Figure PCTCN2020107355-appb-000016
2、LAMP反应条件：
Figure PCTCN2020107355-appb-000017
3、检测结果：
见图1所示，在副溶血弧菌基因组样品O9的LAMP体系中分别加入副溶血弧菌O1、O2、O3、O4、O5、O6、O7、O8、O9、O10、O11、O12以及O13的基因组，副溶血弧菌O9的LAMP引物特异性良好。
实例4
外源菌株检测
1、LAMP反应体系(25μL)：
表16
Figure PCTCN2020107355-appb-000018
表17
Figure PCTCN2020107355-appb-000019
2、LAMP反应条件：
Figure PCTCN2020107355-appb-000020
3、检测结果：
见图2所示，在副溶血弧菌基因组样品O9的LAMP体系中分别加入副溶血弧菌O9、类志贺邻单胞菌O45以及嗜肺军团菌O10的基因组，副溶血弧菌O9的LAMP引物特异性良好。
以上对本发明所提供的一种对副溶血弧菌O9血清型O抗原分子分型的LAMP检测方法进行了详细介绍。本文应用了具体个例对本发明的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。

Claims (7)

  1. 一种对副溶血弧菌O9血清型O抗原分子分型的LAMP引物组，其特征在于，所述引物组的引物序列如SEQ ID NO:1-SEQ ID NO:4所示；所述的副溶血弧菌O抗原分型指的是：从副溶血弧菌O9的O抗原基因簇特异基因序列。
  2. 如权利要求1所述的LAMP引物在制备副溶血弧菌O9血清型中O抗原分型检测的试剂和/或试剂盒中的应用。
  3. 如权利要求2所述的应用，其特征在于，所述副溶血弧菌指的是分离于任何适合副溶血弧菌生活的环境中所得到的，样品的纯培养物的粗提。
  4. 对副溶血弧菌O9血清型O抗原分子分型的试剂，其特征在于，包括如权利要求1所述的LAMP引物。
  5. 对副溶血弧菌O9血清型O抗原分子分型的试剂盒，其特征在于，包括如权利要求4所述的试剂。
  6. 对副溶血弧菌O9血清型O抗原分子分型的LAMP检测方法，其特征在于，取待测样本与如权利要求4所述的试剂混合，扩增。
  7. 如权利要求6所述的LAMP检测方法，其特征在于，LAMP反应条件为：
    Figure PCTCN2020107355-appb-100001
PCT/CN2020/107355 2019-10-30 2020-08-06 一种对副溶血弧菌o9血清型o抗原分子分型的lamp检测方法 WO2021082590A1 (zh)

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