阅读说明：本技术 一种对斯氏普罗威登斯菌o33血清型o抗原分子分型的检测方法 (Detection method for typing of providencia stuartii O33 serotype O antigen molecules ) 是由 王磊 鲁阁阁 夏香红 郭玺 刘斌 于 2020-07-08 设计创作，主要内容包括：本发明涉及一种对斯氏普罗威登斯菌O33血清型O抗原分子分型的检测方法。本发明以斯氏普罗威登斯菌O33的O抗原基因簇内的特异基因为靶基因,设计并筛选用于斯氏普罗威登斯菌O33的O抗原分型的四条引物,并建立了相应的LAMP检测方法,为斯氏普罗威登斯菌的O33抗原分型提供一条可信的途径。利用本发明的LAMP引物检测腹泻大便、尿道感染、伤口、烧伤和菌血症标本的斯氏普罗威登斯菌,并且对其进行O抗原分型,具有操作简单、快速高效、高灵敏度等诸多优点。(The invention relates to a detection method for typing of providencia stuartii O33 serotype O antigen molecules. According to the invention, a specific gene in an O antigen gene cluster of providencia stuartii O33 is used as a target gene, four primers for O antigen typing of providencia stuartii O33 are designed and screened, and a corresponding LAMP detection method is established, so that a credible way is provided for O33 antigen typing of providencia stuartii. The LAMP primer provided by the invention is used for detecting providencia stuartii of diarrhea stool, urinary tract infection, wound, burn and bacteremia specimens, and performing O antigen typing on the providencia stuartii, and has the advantages of simplicity in operation, rapidness, high efficiency, high sensitivity and the like.)

1. An LAMP detection method for typing providencia stuartii O33 serotype O antigen molecules is mainly characterized by having a nucleotide sequence shown by SEQ ID NO 1-SEQ ID NO 4; the providencia stuartii O antigen typing refers to the following steps: o antigen typing is carried out on providencia stuartii O33 from the O antigen gene cluster specific gene sequence.

2. The use of the LAMP detection method for typing O antigen molecules of providencia stuartii O33 serotype as claimed in claim 1 for typing detection of O antigen in providencia stuartii O33 serotype.

3. The application of the LAMP primer as claimed in claim 2, characterized in that the LAMP primer is used for O antigen typing detection in providencia stuartii O33 serotype.

4. Use according to claim 3, wherein the providencia stuartii refers to a crude extract of a pure culture of a sample isolated in any environment suitable for the life of providencia stuartii.

Technical Field

The invention relates to a LAMP technology for typing providencia stuartii O33 serotype strain O antigen in a sample and a preparation method thereof. The invention also designs a method for detecting by using the LAMP primer.

Background

Providencia stuartii (a)Providencia stuartii) Belongs to providencia, gram-negative corynebacterium, facultative anaerobe and milk white colony. The movement with the periphytic flagella did not result in colonization. Separating from diarrhea, stool, urinary tract infection, wound, burn and bacteremia specimen. At present, the typing and identification of providencia stuartii are mainly based on the following steps: morphological characteristics of bacteria, physiological and biochemical characteristics of bacteria, serological reaction and the like, and O antigen typing of providencia stuartii belongs to one of serological reactions. Due to the diversity of the environment and the antibody, the diversity of the O antigen is formed, and different strains of providencia stuartii can be subjected to typing identification according to the diversity of the O antigen.

Loop-mediated Isothermal Amplification (LAMP) is a brand-new nucleic acid Amplification method, can amplify nucleic acid in a short time (usually within one hour) under the condition of Isothermal temperature (60-65 ℃), and is a simple, convenient, rapid, accurate and low-price gene Amplification method. The technology can be comparable to or even superior to the PCR technology in the indexes such as sensitivity, specificity, detection range and the like, does not need the processes of thermal denaturation, temperature circulation, electrophoresis, ultraviolet observation and the like of a template, does not depend on any special instrument and equipment to realize on-site high-flux rapid detection, and has detection cost far lower than that of fluorescent quantitative PCR.

The technical principle is as follows: the temperature of 60-65 ℃ is the intermediate temperature of renaturation and extension of double-stranded DNA, and the DNA is in a dynamic equilibrium state at about 65 ℃. Thus, DNA synthesis at this temperature is possible. The use of 4 specific primers allows strand-displaced DNA synthesis to be continuously self-cycling by means of a highly active strand-displaced DNA polymerase.

Amplification is in two stages: stage 1 is the initial stage, in which either primer undergoes base-pairing extension to the complementary portion of the double-stranded DNA, the other strand dissociates and becomes single-stranded. The F2 sequence of the upstream inner primer FIP is firstly combined with the template F2c, and is extended forward under the action of strand displacement type DNA polymerase to start strand displacement synthesis. The outer primer F3 binds to and extends from template F3c, displacing the entire FIP-ligated complementary single strand. F1c on FIP and F1 on this single strand are complementary structures. Self base pairing forms a ring structure. Using the strand as a template, the downstream primers BIP and B3 sequentially initiate synthesis similar to FIP and F3 to form a single strand with a dumbbell-shaped structure. The stem loop structure is formed by rapidly synthesizing and extending DNA using the F1 segment at the 3' end as the starting point and itself as a template. This structure is the initial structure of the LAMP gene amplification cycle.

Stage 2 is the amplification cycle stage. FIP binds to the F2c region of the stem loop using the stem loop structure as a template, strand displacement synthesis is initiated, and a loop structure is also formed on the dissociated single-stranded nucleic acid. The B1 segment at the 3' end is taken as a starting point, the self is taken as a template, DNA synthesis extension and strand displacement are formed, 2 pieces of DNA with different lengths and new stem loop structures are formed, B2 on the BIP primer is hybridized with the DNA, a new round of amplification is started, and the length of the product DNA is doubled. 2 circular primers LF and LB are added into the reaction system, and are respectively combined with the stem-loop structure to start strand displacement synthesis. In cycles, the final product of amplification is a mixture of DNAs with different stem-loop structures and different lengths, and the product DNA is an alternating inverted repeat sequence of the amplified target sequence.

The differences between this application and the already published patent applications are: the detection means does not use a gene chip (the amplification and hybridization are carried out separately, the time is long), but uses LAMP technology to detect (one-step reaction amplification detection, is simple and quick, does not depend on any special instrument and equipment to realize on-site high-flux quick detection, and has low detection cost).

Disclosure of Invention

In order to achieve the purpose, the invention discloses a LAMP primer for typing O33 serotype O antigen of providencia stuartii in a sample, which comprises a FIP primer, a F3 primer, a BIP primer and a B3 primer.

The LAMP primers are mainly designed aiming at six different regions of a target gene, and 4 primers are designed based on 6 different sites such as F3c, F2c and Flc regions at the 3 'end of the target gene 3 and Bl, B2 and B3 regions at the 5' end.

FIP primer: upstream Inner Primer) Inner (Forward, consisting of F2 region and F1C region, wherein the 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 5.

F3 primer: upstream Outer Primer) Outer (Forward, consisting of the F3 region, and is complementary to the F3c region of the target gene.

The BIP primer is as follows: downstream Inner Primer) Inner (Backward, consisting of B1C and B2 regions, B2 region is complementary to B2c region at 3 'end of target gene 3, B1C region has the same sequence as Blc region at 5' end of target gene 5.

B3 primer: downstream Outer Primer) Outer (Backward, consisting of the B3 region, complementary to the B3c region of the target gene.

The method is mainly characterized in that the O33 serotype O antigen typing of providencia stuartii refers to the following steps: the O antigen gene cluster specific gene sequence of providencia stuartii O33 serotype has DNA sequence shown in SEQ ID No. 1-SEQ ID No. 4.

The LAMP primer for typing the serotype O antigen of providencia stuartii O33 in the sample is characterized in that the primer sequence is as follows: has primers shown in SEQ ID NO. 1-SEQ ID NO. 4.

The invention further discloses a LAMP primer for typing O33 serotype O antigen molecules of providencia stuartii in a sample and application thereof in O antigen molecule typing detection of providencia stuartii O33. The providencia stuartii refers to a crude extract of a pure culture of a sample isolated in any environment suitable for the life of providencia stuartii. The experimental results show that: the invention can be used for typing providencia stuartii O antigen under lower DNA concentration.

The invention provides a LAMP system for detecting a serotype in an environment, which comprises: WarmStartColorimetric LAMP 2X Master Mix (NEB), 10. mu.M FIP, F3, BIP and B3 primers, 1. mu.L DNA and ddH₂And O. The primer is designed according to the specific gene sequence of O antigen gene cluster of providencia stuartii O33 serotype: jp/e/designed B3 and F from the specific gene sequence of O antigen gene cluster of providencia stuartii O33 serotype using http:// primeredor3 the primer length is about 20 nt, and the Tm is between 55 and 60 ℃; the length of the BIP primer and the FIP primer is about 50 nt; wherein "-" in the primer sequence represents "TTTT".

The invention is the practical application of LAMP loop-mediated isothermal amplification technology, and is used for identifying the providencia stuartii serotype O33.

According to the technical scheme, the LAMP technology is introduced into the field of providencia stuartii O antigen typing for the first time, the LAMP detection method for the providencia stuartii O33 serotype in the sample with rapidness, sensitivity, high accuracy and strong repeatability is established, the LAMP probe can be used for achieving the purpose of identifying the common providencia stuartii O serotype strain in the sample, and the LAMP probe has important application value for rapidly detecting the providencia stuartii O antigen typing in the sample in real time by various medical departments due to simplicity and convenience in operation, high accuracy and strong repeatability.

Drawings

FIG. 1O 33 serotype LAMP reaction positive and specificity detection: the LAMP system of providencia stuartii genome sample O33 is added with genomes of providencia stuartii O3, O9, O20, O29, O33, O40, O44, O45, O46 and O48, and no amplification band appears in electrophoresis detection of the genomes except O33, which shows that the LAMP primer specificity of providencia stuartii O33 is good.

Detailed Description

The invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention. The raw materials and reagents used in the present invention are commercially available.

The sources of providencia stuartii strains used in the present invention are shown in table 1 below:

TABLE 1 bacterial species used in this experiment

a，Department of Immunobiology of Bacteria Institute of Microbiology,Biotechnology and Immunology University of Lodz←Hungarian NationalCollection Medical Bacteria (National Insititute of Hygiene, Budapest ,Hungary)

b，Department of Immunobiology of Bacteria Institute of Microbiology andImmunology University of Lodz←Hungarian National Collection Medical Bacteria(National Insititute of Hygiene,Budapest,Hungary)