CN116287344A - Primer pair, kit and method for detecting Cronobacter sakazakii - Google Patents
Primer pair, kit and method for detecting Cronobacter sakazakii Download PDFInfo
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Abstract
The invention belongs to the technical field of molecular biology application, and particularly relates to a primer pair, a kit and a method for detecting Cronobacter sakazakii. The nucleotide sequence of the target gene fragment is shown as SEQ ID NO. 1; the primer pair is designed according to the target gene fragment, and comprises an upstream primer mngB-F and a downstream primer mngB-R, and the sequences of the primer pair are respectively shown as SEQ ID NO.2 and SEQ ID NO. 3. The primer pair and the RPA detection method based on the target gene fragment of the new target solve the problems of few target gene types and weak specificity of the existing cronobacter sakazakii nucleic acid detection, have the advantages of strong broad spectrum, good specificity, high sensitivity, high accuracy, simple operation, low cost and the like, and have good application prospects in the field of food detection.
Description
Technical Field
The invention belongs to the technical field of molecular biology application, and particularly relates to a primer pair, a kit and a method for detecting Cronobacter sakazakii.
Background
Cronobacter sakazakii (Cronobacter sakazakii), also known as enterobacter sakazakii, is a facultative anaerobic gram-negative bacillus, and is found in various foods such as infant milk powder, infant cereal foods, meats, water, vegetables, and the like. Infants are a high risk group for Cronobacter sakazakii infection. Once infants encounter such bacterial infections, bacteremia, meningitis, necrotizing enterocolitis, etc. can be caused thereby, with mortality rates of up to 40% -80% for the relevant diseases. Cronobacter sakazakii has strong proliferation capability, strong stress resistance to environmental factors, high heat resistance, acid and alkali resistance and extremely strong drying resistance. Therefore, the establishment of a rapid and specific detection method of Cronobacter sakazakii is a key point for effectively preventing and controlling the bacterial contamination in food.
The traditional detection method of the Cronobacter is to separate a chromogenic plate after enrichment and enrichment culture and then carry out biochemical identification. However, this detection method has problems of complicated steps, long process, and the like. Therefore, an accurate, sensitive, rapid and specific detection method is established, and the method can play an important role in food safety detection. Currently, various rapid detection methods have been developed, mainly Polymerase Chain Reaction (PCR) technology, loop-mediated isothermal method (LAMP) technology, and Recombinase Polymerase Amplification (RPA) technology. Recombinase polymerase amplification (Recombinase polymerase amplification, RPA) is an isothermal nucleic acid amplification technology developed by twist dx corporation of england in 2006.
Existing detection techniques of cronobacter sakazakii are generally directed to the α -glucosidase (gluA) gene, the outer membrane protein a (ompA) gene, the 16S rRNA gene, and the like. The basic research of cronobacter sakazakii specific genes is insufficient compared with other food-borne pathogens such as salmonella. Different popular strains of Cronobacter sakazakii are all over, the genetic variation is quick, the specificity of the commonly used detection target genes is insufficient, and a new specific target gene needs to be mined, so that the specificity and the broad spectrum of the detection technology are improved.
Disclosure of Invention
The existing RPA detection technology is generally aimed at the cronobacter sakazakii gluA gene, ompA gene, 16S rRNA gene and the like, but the cronobacter sakazakii has more types and more genetic variations, and a new specific target gene is required to be mined, so that the specificity and the broad spectrum of the detection technology are improved. The first object of the present invention is to provide a novel target for detecting cronobacter sakazakii with high specificity.
The invention systematically analyzes the specific gene of Cronobacter sakazakii based on comparative genomics, digs out a new target glycosylhydrolase gene mngB, and provides a section of nucleotide sequence (target gene fragment) in the gene to be applied to the detection of Cronobacter sakazakii, wherein the nucleotide sequence of the target gene fragment is shown as SEQ ID NO. 1.
The inventor finds that all secondary and tertiary whole genome sequencing strains mn gB of Cronobacter sakazakii in NCBI database have higher homology. The similarity of the ompA gene sequence of different strains of Cronobacter sakazakii is about 96 percent at the minimum, the similarity of the gluA gene sequence is about 97 percent at the minimum, and the similarity of the mngB gene sequence of different strains of Cronobacter sakazakii is about 98 percent at the minimum. Compared with ompA and gluA genes, the method has higher homology of different strains mngB of Cronobacter sakazakii, so the method has excellent broad spectrum in identifying and distinguishing the Cronobacter sakazakii.
In addition, the mngB gene of Cronobacter sakazakii has very low homology with other closely related bacteria such as Cronobacter zurich (C.turicensis), cronobacter malonate (C.malonaticus). The ompA and gluA genes of Cronobacter sakazakii have higher homology with other near bacteria. The method based on the target gene fragment has excellent specificity in identifying and distinguishing the Cronobacter sakazakii and the kindred species thereof.
Based on the target gene fragment of the novel target, the second object of the invention is to provide an RPA primer pair for detecting Cronobacter sakazakii, wherein the primer pair is designed according to the target gene fragment and comprises an upstream primer mngB-F and a downstream primer mngB-R, the sequence of the upstream primer mngB-F is shown as SEQ ID NO.2, and the sequence of the downstream primer mngB-R is shown as SEQ ID NO. 3.
The primer pair can be applied to preparation of an identification reagent and a kit of Cronobacter sakazakii, and based on the primer pair, the three purposes of the invention are to provide an RPA detection kit for providing the Cronobacter sakazakii with accuracy, rapidness, strong specificity and strong broad spectrum, comprising the primer pair; the kit also comprises a buffer solution and sterile double distilled water; gene extraction reagents and/or positive genomic DNA may also be included.
Based on the primer pair, a fourth object of the present invention is to provide a method for detecting cronobacter sakazakii for the purpose of diagnosis or treatment of non-disease, comprising the steps of: and carrying out RPA reaction on the DNA of the sample to be detected by adopting the primer pair, and detecting whether a positive band exists after the reaction is finished.
Preferably, the reaction system of the RPA reaction comprises: reaction buffer A buffer 29.4. Mu.L, reaction buffer B buffer 29.4. Mu.L, 10. Mu.M of the upstream primer 2. Mu.L, 10. Mu.M of the downstream primer 2. Mu.L, sterile double distilled water 13.1. Mu.L, and 1. Mu.L of sample DNA to be detected. Preferred RPA reaction conditions are: 37℃to 42℃for 15min to 40min (more preferably 37℃for 30 min). Can be implemented by using a domestic Twit Amp Basic kit. The reaction system and the reaction conditions are the optimal conditions finally determined by the inventor through continuous software analysis and experiments, and the effect can be better realized.
In detection, whether a positive band exists or not can be detected by agarose gel electrophoresis; or purifying the product, and detecting whether positive bands exist by agarose gel electrophoresis.
The method can be applied to detection of Cronobacter sakazakii in foods such as infant milk powder, infant rice powder, vegetables and meats, and has wide application range.
The beneficial effects of the invention are as follows: the invention provides a new target, provides a primer pair and an RPA detection method based on target gene fragments, solves the problems of few target gene types and weak specificity of the existing cronobacter sakazakii nucleic acid detection, has the advantages of strong broad spectrum, good specificity, high sensitivity, high accuracy, simple operation, low cost and the like, and has good application prospect in the field of food detection.
Drawings
FIG. 1 shows the sequence homology comparison of the NCBI database of Cronobacter sakazakii mngB gene;
FIG. 2 shows the results of sequence homology comparison of the NCBI database of the ompA gene of Cronobacter sakazakii;
FIG. 3 shows the sequence homology comparison of the NCBI database of the GluA gene of Cronobacter sakazakii;
FIG. 4 is a graph showing the experimental results of the detection sensitivity of the mngB primer;
FIG. 5 is a graph showing the results of the experiment for detecting the specificity of the mngB primer.
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described below with reference to the embodiments and the drawings to fully understand the objects, aspects and effects of the present invention.
Example 1: screening of novel target of Cronobacter sakazakii and design of RPA primer pair
(1) Screening of novel targets of Cronobacter sakazakii
A Cronobacter sakazakii JXES-28 (NCBI accession number: CP 098777.1) is selected as a model strain, the whole genome sequence of the model strain is subjected to Blastn comparison with a salmonella strain A7 (NCBI accession number: CP 084001), a non-homologous section is intercepted through a Graphics result of the model strain, the intercepted non-homologous section is compared with a RAST annotation result, a repetitive sequence and a fictitious protein gene are removed, and functional genes positioned in the non-homologous section are screened out for sorting and induction. And then sequentially inputting the functional gene sequences into an NCBI database for Blastn comparison, and reserving a conserved sequence (the matching length with non-Cronobacter sakazakii is <30 bp) with higher homology with Cronobacter sakazakii (namely E < 10-200). These functional genes were initially selected as specific detection targets for cronobacter sakazakii.
The method screens out Cronobacter sakazakii to detect a new target mngB gene. The mngB gene and the common targets ompA and gluA of Cronobacter sakazakii are respectively input into NCBI data for blast comparison, the sequence similarity of the mngB gene in different Cronobacter sakazakii strains is as low as about 98 percent (shown in figure 1), the sequence similarity of the ompA gene is as low as about 96 percent (shown in figure 2), and the sequence similarity of the gluA gene is as low as 97 percent (shown in figure 3).
In addition to Cronobacter sakazakii, other closely related bacteria have very low mngB gene homology (e.g., cronobacter zurich (C. Turicensis), cronobacter malonate (C. Malonaicus), as shown in FIG. 1). The omp A gene of Cronobacter sakazakii has 96% sequence similarity with the closely related bacterium Cronobacter dublinensis. The gluA gene of Cronobacter sakazakii has up to 95% sequence similarity with Cronobacter malonate Cronobacter malonaticus (as shown in FIGS. 2 and 3). Compared with ompA and gluA, the mngB gene has better broad spectrum and specificity as a new target for detecting Cronobacter sakazakii.
(2) Design of RPA primer pair
Downloading a plurality of Cronobacter sakazakii mngB genes from the NCBI database, performing multi-sequence alignment, and designing RPA primers in a sequence conservation region. The primers were then input to NCBI for blast alignment and analyzed for primer specificity. Finally, the primer pair disclosed by the invention is preferred. The primers were synthesized by Shanghai Biotechnology Inc., specifically as follows:
upstream primer mngB-F:5'-GCTGGATCTACTGGAAAAGGATGCGGATTT-3' (SEQ ID NO. 2).
Downstream primer mngB-R:5'-TAGTTGGTGGCGATGGATTTCTGGTTTGGT-3' (SEQ ID NO. 3).
Example 2: RPA method for detecting Cronobacter sakazakii
An RPA method for detecting cronobacter sakazakii, comprising the steps of:
s1, preparing a DNA sample of a sample to be detected;
s2, performing RPA reaction on the DNA sample by using the primer pair mngB-F/mngB-R obtained in the example 1;
and S3, directly performing agarose gel electrophoresis to detect whether positive bands exist after the RPA reaction is finished.
Wherein, the RPA reaction system is: 29.4. Mu.L of A buffer; b buffer 2.5 μl; 2. Mu.L of each of the 10. Mu.M upstream and downstream primers was added; 13.1 mu L of sterile double distilled water; 1 μl of DNA sample. The reaction conditions are as follows: 37℃for 30min. Is implemented by using a domestic twist amp Basic kit (Anpu future biotechnology Co., ltd.).
After the reaction, 5. Mu.L of the reaction solution was taken and subjected to 1.5% agarose gel electrophoresis, goldview was added to the gel for staining, and after the electrophoresis was completed, the gel was observed and photographed under an ultraviolet projector. In other embodiments, the PCR product kit may also be used for product purification, followed by agarose gel electrophoresis and photographic observation.
The effect evaluation of the various strains is carried out by adopting the method, and the method is specifically as follows:
(1) Experimental strains
The 11 reference strains of Cronobacter sakazakii JXES-28 (NCBI accession number: CP 098777.1), cronobacter sakazakii JXES-5, cronobacter sakazakii JXES-14, cronobacter sakazakii JXES-17, cronobacter sakazakii JXES-19, cronobacter sakazakii JXES-30, E.coli ATCC 25922, aeromonas 429, listeria monocytogenes ATCC19114, proteus mirabilis 012 (NCBI accession number: CP 062146), salmonella ATCC 14028, etc. were stored by the university of light industry food safety team laboratory.
(2) Preparation of bacterial DNA templates
Extracting genome DNA of the strain to be detected by a boiling method: culturing the strain in LB liquid medium overnight, taking 1mL of bacterial liquid, centrifuging at 12000rpm for 5min, and discarding the supernatant; the cell pellet was washed with 1mL of sterile water, centrifuged at 12000rpm for 5min, and the supernatant was discarded. Adding 100 mu L of sterile deionized water into the precipitate to form a suspension, boiling for 10min, ice-bathing for 5min, and centrifuging at 12000rpm for 5min, wherein the supernatant is the genomic DNA and is used as a template for RPA amplification.
In other embodiments, strain DNA may also be extracted using commercial kits or organic reagent methods.
(3) Sensitivity detection experiment
DNA of Cronobacter sakazakii JXES-28 was extracted and used as a detection template by ten-fold dilution with sterile water at a concentration of 146 ng/. Mu.L. 1 μl of each concentration gradient was added to the RPA reaction system for sensitivity detection.
The results are shown in FIG. 4, lanes 1-8 show the results of electrophoresis of the JXES-28RPA amplification products at different concentrations. Lanes 1-2, among others: 14.6 ng/. Mu.L; lanes 3-4:1.46 ng/. Mu.L; lanes 5-6:0.146 ng/. Mu.L; lanes 7-8:0.0146 ng/. Mu.L; lane 9: a blank control without DNA template was used.
As shown by the sensitivity verification result of FIG. 4, the RPA technology of the present invention can detect Cronobacter sakazakii having a DNA concentration as low as 0.146 ng/. Mu.L.
(4) Specificity detection experiment
10 bacteria were selected for the RPA-specific experiment. As a result, as shown in FIG. 5, lanes 1-10 are, in order, cronobacter sakazakii JXES-5, cronobacter sakazakii JXES-14, cronobacter sakazakii JXES-17, cronobacter sakazakii JXES-19, cronobacter sakazakii JXES-30, E.coli ATCC 25922, aeromonas 429, listeria monocytogenes ATCC19114, proteus mirabilis 012 (NCBI accession number: CP 062146), salmonella ATCC 14028; lanes 11-12 are blank controls without DNA template.
From the specificity verification result of fig. 5, it was revealed that only cronobacter sakazakii could be amplified, and that other bacteria could not be amplified. The results show that the method has good specificity for detecting the Cronobacter sakazakii.
Example 3:
an RPA detection kit for cronobacter sakazakii, comprising the primer pair mngB-F/mngB-R in example 1, buffer and sterile double distilled water. Wherein the buffer solution is specifically 29.4 mu L of Abuffer and B buffer 2.5 mu L; the concentration of the upstream primer and the downstream primer in the primer pair mngB-F/mngB-R is 10 mu M, and the volumes are 2 mu L; the volume of sterile double distilled water was 13.1. Mu.L.
In some other embodiments, the above-described kits may further comprise a gene extraction reagent and/or positive genomic DNA.
It will be apparent that the described embodiments are only some, but not all, of the embodiments of the present application. Reference herein to "an embodiment" means that a particular feature or characteristic described in connection with the embodiment may be included in at least one embodiment of the application for the embodiment. The appearances of such phrases in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments. Those of skill in the art will explicitly and implicitly understand that the embodiments described herein may be combined with other embodiments. All other embodiments, which can be made by one of ordinary skill in the art without undue burden from the present disclosure, are within the scope of the present disclosure.
While embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that: many changes, modifications, substitutions and variations may be made to the embodiments without departing from the spirit and principles of the invention, the scope of which is defined by the claims and their equivalents.
Claims (10)
1. The application of the target gene fragment in detecting the cronobacter sakazakii is characterized in that the nucleotide sequence of the target gene fragment is shown as SEQ ID NO. 1.
2. A primer pair for detecting cronobacter sakazakii, characterized in that the primer pair is designed according to the target gene fragment of claim 1, and comprises an upstream primer mngB-F and a downstream primer mngB-R, wherein the sequence of the upstream primer mngB-F is shown as SEQ ID No.2, and the sequence of the downstream primer mngB-R is shown as SEQ ID No. 3.
3. A kit for detecting cronobacter sakazakii, comprising the primer pair of claim 2.
4. The kit of claim 3, further comprising a buffer and sterile double distilled water.
5. The kit of claim 4, further comprising a gene extraction reagent and/or positive genomic DNA.
6. A method for detecting cronobacter sakazakii for non-disease diagnosis or treatment purposes, comprising the steps of: the primer pair as set forth in claim 2 is used in RPA reaction of DNA in sample to be tested, and after the reaction, the presence of positive stripe is detected.
7. The method of claim 6, wherein the reaction system of the RPA reaction comprises: reaction buffer Abuffer 29.4. Mu.L, reaction buffer B buffer 29.4. Mu.L, 10. Mu.M of the upstream primer 2. Mu.L, 10. Mu.M of the downstream primer 2. Mu.L, sterile double distilled water 13.1. Mu.L, and 1. Mu.L of sample DNA to be tested.
8. The method of claim 7, wherein the RPA reaction conditions are: 37-42 ℃ for 15-40 min.
9. The method of claim 6, wherein the presence or absence of a positive band is detected by agarose gel electrophoresis.
10. Use of the method of claim 6 in the field of food detection.
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| CN117887873B (en) * | 2024-03-14 | 2024-06-11 | 国家食品安全风险评估中心 | Primer probe combination for specifically detecting Cronobacter and detection method |
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