CN112375833A - Loop-mediated isothermal amplification detection method for O antigen serotype typing of Enterobacter sakazakii - Google Patents
Loop-mediated isothermal amplification detection method for O antigen serotype typing of Enterobacter sakazakii Download PDFInfo
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Abstract
The invention relates to a loop-mediated isothermal amplification (LAMP) detection method for typing of serotype O antigen of Enterobacter sakazakii O1-O7. The invention designs and screens four respective primers for O antigen typing of Enterobacter sakazakii O1-O7 by taking specific genes, namely wzx and wzy, in an O antigen gene cluster of the Enterobacter sakazakii O1-O7 as target genes, and establishes a loop-mediated isothermal amplification (LAMP) reaction system. Provides a corresponding technology for detecting and serotyping Enterobacter sakazakii in food, especially infant formula. The LAMP system is used for detecting the Enterobacter sakazakii and serotyping the Enterobacter sakazakii, and has the advantages of simple operation, rapidness and high efficiency.
Description
Technical Field
The invention relates to a LAMP technology for typing an O antigen of an enterobacter sakazakii O1-O7 serotype strain in a sample and a preparation method thereof. The invention also designs a method for detecting by using the LAMP primer.
Background
Infection with enterobacter sakazakii can cause very serious diseases, especially in premature infants. In particular to meningitis, sepsis, necrotizing enterocolitis and septicemia. The centers for disease and prevention control estimate the mortality rate of cases infected with enterobacter sakazakii as high as 40%. The fatality rate of infected newborns and infants is reported to be 50-80%, with 20% of survivors developing severe neurological disorders. In older children, enterobacter sakazakii can colonize the gastrointestinal lumen, and reports have shown that the fatality rate caused thereby is 10-55%. The bacteria can be isolated from many food products including milk powder, dried vegetables and cereal flour, but are most commonly found in infant formulas.
In 27.6.2017, a food safety national standard (GB 4789.40-2016) issued by the national food and drug administration of the State Committee for health and family planning performs biochemical identification on Enterobacter sakazakii by adopting a detection method of food microbiology, and the method is economical and practical, but has the defects of complex experimental process and long time (about 5-6 days); in the business standard (SN/T2754.15-2011) of entry and exit inspection and quarantine implemented by the national quality supervision, inspection and quarantine headquarters of the people's republic of China in 7/1/2011, the enterobacter sakazakii in the export food is detected by a Loop-mediated Isothermal Amplification (LAMP) detection method, but the standard only detects the enterobacter sakazakii to the species level, does not detect serotypes, and is difficult to meet the requirement for tracing food-borne disease pathogens.
The principle of the loop-mediated isothermal amplification (LAMP) technology 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 relies on a highly active strand-displacing DNA polymerase. So that strand displacement DNA synthesis is continuously self-circulating.
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. Starting immediately with the F1 segment at the 3' end. DNA synthesis and extension are carried out by taking the self as a template to form a stem-loop structure. 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 started, and a loop structure is also formed in 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 method has the advantages of rapidness, simplicity and convenience, and can amplify nucleic acid in a short time (usually within one hour) under the condition of isothermal temperature (60-65 ℃). Compared with the conventional PCR, the method does not need thermal denaturation and temperature circulation of a template, does not depend on any special instrument and equipment to realize on-site high-flux rapid detection, and has lower detection cost.
Disclosure of Invention
In order to achieve the aim, the invention discloses an LAMP primer for typing an O antigen of serotype O of Enterobacter sakazakii O1-O7 in a sample, which comprises an FIP primer, an 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.
Primer) Inner FIP (Forward: the upstream inner primer consists of an F2 region and an F1C region, wherein the F2 region is complementary with an 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 LAMP primer is mainly characterized in that the LAMP primer is 4 DNA fragments respectively selected from wzy genes of Enterobacter sakazakii O1-O2 type and O4-O6 type and wzx genes of O3 type and O7 type, and the nucleotide sequence of the LAMP primer is shown as SEQ ID NO. 1-SEQ ID NO. 28.
The invention also provides a loop-mediated isothermal amplification (LAMP) reaction system which comprises the following components:
the invention further discloses application of the loop-mediated isothermal amplification (LAMP) reaction system in detecting O antigen serotype of the Enterobacter sakazakii strain, wherein the Enterobacter sakazakii strain refers to a pure culture or a crude nucleic acid DNA extract of the bacterium isolated from the infant formula where the bacterium is located. Experimental results showed that the reaction system can realize detection of at least one of enterobacter sakazakii of all 7O antigens serous type (fig. 1 to fig. 7).
The invention mainly provides a technical means for detecting all O antigen serotypes of common pathogenic bacteria, namely enterobacter sakazakii, in infant formula milk powder by using a loop-mediated isothermal amplification (LAMP) method. The positive effects are as follows:
(1) the technical means for detecting all O antigen serotypes of the enterobacter sakazakii by using a loop-mediated isothermal amplification (LAMP) method is disclosed for the first time, and an effective method is provided for clinical detection and epidemiological monitoring of the enterobacter sakazakii.
(2) The operation is simple and convenient, expensive reaction equipment is not needed, and the reaction can be finished in the environment of a common water area.
(3) The detection time is short: by using the technical means, the detection can be completed within about 1.5 hours after the genomic DNA crude extract is obtained.
Drawings
FIG. 1 LAMP reaction specificity detection of Enterobacter sakazakii O1: the genomes of the Enterobacter sakazakii O1, O2, O3, O4, O5, O6 and O7 are respectively added into the LAMP system of the Enterobacter sakazakii genome sample O1, and no amplification band appears in electrophoresis detection except the O1 genome, so that the LAMP primer specificity of the Enterobacter sakazakii O1 is good;
FIG. 2 LAMP reaction specificity detection of Enterobacter sakazakii O2: the genomes of the Enterobacter sakazakii O1, O2, O3, O4, O5, O6 and O7 are respectively added into the LAMP system of the Enterobacter sakazakii genome sample O2, and no amplification band appears in electrophoresis detection except the O2 genome, so that the LAMP primer specificity of the Enterobacter sakazakii O2 is good;
FIG. 3 LAMP reaction specificity detection of Enterobacter sakazakii O3: the genomes of the Enterobacter sakazakii O1, O2, O3, O4, O5, O6 and O7 are respectively added into the LAMP system of the Enterobacter sakazakii genome sample O3, and no amplification band appears in electrophoresis detection except the O3 genome, so that the LAMP primer specificity of the Enterobacter sakazakii O3 is good;
FIG. 4 LAMP reaction specificity detection of Enterobacter sakazakii O4: the genomes of the Enterobacter sakazakii O1, O2, O3, O4, O5, O6 and O7 are respectively added into the LAMP system of the Enterobacter sakazakii genome sample O4, and no amplification band appears in electrophoresis detection except the O4 genome, so that the LAMP primer specificity of the Enterobacter sakazakii O4 is good;
FIG. 5 LAMP reaction specificity detection of Enterobacter sakazakii O5: the genomes of the Enterobacter sakazakii O1, O2, O3, O4, O5, O6 and O7 are respectively added into the LAMP system of the Enterobacter sakazakii genome sample O5, and no amplification band appears in electrophoresis detection except the O5 genome, so that the LAMP primer specificity of the Enterobacter sakazakii O5 is good;
FIG. 6 LAMP reaction specificity detection of Enterobacter sakazakii O6: the genomes of the Enterobacter sakazakii O1, O2, O3, O4, O5, O6 and O7 are respectively added into the LAMP system of the Enterobacter sakazakii genome sample O6, and no amplification band appears in electrophoresis detection except the O6 genome, so that the LAMP primer specificity of the Enterobacter sakazakii O6 is good;
FIG. 7 LAMP reaction specificity detection of Enterobacter sakazakii O7: the genomes of the Enterobacter sakazakii O1, O2, O3, O4, O5, O6 and O7 are respectively added into the LAMP system of the Enterobacter sakazakii genome sample O7, and no amplification band appears in electrophoresis detection except the O7 genome, which shows that the LAMP primer specificity of the Enterobacter sakazakii O7 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.
Example 1
Design of primers
1. Screening for specific genes
The O antigen processing genes wzy, wzx, wzm and wzt are highly serotype determinative and have therefore been widely used as target genes for the serotyping of many gram-negative bacterial molecules. The invention takes wzy genes of O1-O2 type and O4-O6 type of Enterobacter sakazakii and wzx genes of O3 type and O7 type as target genes.
According to the invention, all genes in the gene cluster are compared by the all _ vs _ all _ blast method, and the matching number of specific genes is inevitably far smaller than that of conserved genes. The above method is combined to find a specific gene and design a primer for the specific gene.
2. Design of primers
And (3) designing LAMP primers by taking the selected specific genes of 1 Enterobacter sakazakii as templates.
The Primer c Inner c (Backward, consisting of B1c and B c regions, the B1c region being complementary to the B2c region at the 3' end of the target gene, the B1c region being identical to the Blc region at the 5' end of the target gene, the B c region being complementary to the B2c region at the 3' end of the target gene, the B c region being identical to the sequence of the Blc region at the 72 ' end of the target gene, the B c Primer: the downstream Inner Primer) c Primer c (Backward, consisting of B1c and B c regions, the B c region being complementary to the B2c region at the 3' end of the target gene c, the B c Primer: the downstream Outer Primer) c/c filter c (Backward, consisting of B c region, B3 region, the B3 region of the target gene, the sequence of the Primer and the sequence of the Primer: the Primer/Primer: the sequence of the target gene/Primer: the sequence of the Forward gene/Primer: the sequence: the Primer: the sequence/Primer: the sequence of the Forward gene/Primer: the following table c/Primer)
The length of the B3 and F3 primers is about 20 nt, and the Tm is between 55 and 60 ℃; the length of BIP and FIP primers is about 50 nt. The primers were synthesized by GENEWIZ (tianjin, china).
Primers for LAMP
Example 2
Extraction of sample nucleic acid
1. The obtained pure cultured bacteria were treated in the following manner:
(1) and picking a single bacterial colony of the bacteria into 10 muL of deionized water or 10 muL of bacteria liquid cultured overnight, and treating for 15 min in a boiling water bath.
(2) After being placed on ice for 1 min, the mixture was centrifuged at 8000 rpm for 1 min.
(3) And taking 3 mu L of supernatant as a template for the next LAMP reaction.
2. Taking 10g of infant formula milk powder, and treating the infant formula milk powder in the following way:
(1) the sample is placed in 20 mL LB culture medium and shake cultured for 3 h at 37 ℃.
(2) 1 mL of the culture of (1) was centrifuged at 8000 rpm for 5min, and the supernatant was discarded.
(3) Adding 500 mu L of deionized water, re-suspending and uniformly mixing, centrifuging at 8000 rpm for 5min, and discarding the supernatant.
(4) And adding 100 mu L of deionized water, and treating for 15 min in a boiling water bath.
(5) After being placed on ice for 1 min, the mixture was centrifuged at 8000 rpm for 1 min.
(6) And taking 3 mu L of supernatant as a template for the next LAMP reaction.
Example 3
LAMP reaction
Using the nucleic acid solution extracted in example 2 as a template for the reaction, O1-O7 were added to each LAMP reaction system, and the reaction was carried out in a water bath under the following conditions.
Example 4
Agarose gel electrophoresis detection
From each reaction system of O1-O7 of example 3, 2. mu.L of the reaction product was aspirated, and subjected to agarose (2%) gel electrophoresis at a voltage of 120v for an electrophoresis time of 20 min. And then observing under an ultraviolet lamp.
Experimental results showed that the reaction system can realize detection of at least one of enterobacter sakazakii of all 7O antigens serous type (fig. 1 to fig. 7).
SEQUENCE LISTING
<110> university of southern kayak
<120> a loop-mediated isothermal amplification detection method for serotype typing of Enterobacter sakazakii O antigen
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Claims (3)
1. The LAMP primers are respectively specific to different O antigen serological Enterobacter sakazakii, and are characterized in that 4 DNA fragments are respectively selected from wzy genes of Enterobacter sakazakii O1-O2 and O4-O6 and wzx genes of O3 and O7, and the nucleotide sequences of the DNA fragments are shown as SEQ ID NO. 1-SEQ ID NO. 28.
2. A loop-mediated isothermal amplification (LAMP) reaction system, which contains the primers for molecular typing of the O antigen of the serotype O1-O7 of Enterobacter sakazakii according to claim 1, and comprises the following components:
。
3. use of the loop-mediated isothermal amplification (LAMP) reaction system according to claim 2 for detecting O antigen serotype of Enterobacter sakazakii strain; the Enterobacter sakazakii strain refers to a pure culture or a crude extract of nucleic acid DNA of the bacterium isolated from infant formula.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154451A (en) * | 2010-12-30 | 2011-08-17 | 广东省微生物研究所 | Loop-mediated isothermal amplification detection primer group, detection method and detection kit for enterobacter sakazakii |
| CN102154270A (en) * | 2011-01-11 | 2011-08-17 | 天津生物芯片技术有限责任公司 | Cronobacter sakazakii O antigen specific nucleotides and use thereof |
| CN108866217A (en) * | 2018-07-20 | 2018-11-23 | 暨南大学 | For detecting 7 nest type qPCR primers, kit and the detection method of 7 kinds of pathogenic bacteria in milk powder |
| CN109182469A (en) * | 2018-08-24 | 2019-01-11 | 暨南大学 | Primer and kit and method based on digital LAMP technology detection Enterobacter sakazakii |
| CN111676304A (en) * | 2020-07-08 | 2020-09-18 | 南开大学 | Method for real-time fluorescence PCR detection of Enterobacter sakazakii O antigen and application |
-
2020
- 2020-11-23 CN CN202011316948.XA patent/CN112375833A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154451A (en) * | 2010-12-30 | 2011-08-17 | 广东省微生物研究所 | Loop-mediated isothermal amplification detection primer group, detection method and detection kit for enterobacter sakazakii |
| CN102154270A (en) * | 2011-01-11 | 2011-08-17 | 天津生物芯片技术有限责任公司 | Cronobacter sakazakii O antigen specific nucleotides and use thereof |
| CN108866217A (en) * | 2018-07-20 | 2018-11-23 | 暨南大学 | For detecting 7 nest type qPCR primers, kit and the detection method of 7 kinds of pathogenic bacteria in milk powder |
| CN109182469A (en) * | 2018-08-24 | 2019-01-11 | 暨南大学 | Primer and kit and method based on digital LAMP technology detection Enterobacter sakazakii |
| CN111676304A (en) * | 2020-07-08 | 2020-09-18 | 南开大学 | Method for real-time fluorescence PCR detection of Enterobacter sakazakii O antigen and application |
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Application publication date: 20210219 |