A primer pair, kit and method for detecting Cronobacter sakazakii

Abstract

The present invention belongs to the field of molecular biology application technology, and specifically relates to a primer pair, a kit and a method for detecting Cronobacter sakazakii. The nucleotide sequence of the target gene fragment is shown in SEQ ID NO.1; the primer pair is designed according to the target gene fragment, including an upstream primer mngB-F and a downstream primer mngB-R, and their sequences are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively. The primer pair and RPA detection method of the target gene fragment based on the new target provided by the present invention solve the problem that the target gene types of the existing Cronobacter sakazakii nucleic acid detection are few and the specificity is not strong, and has the advantages of strong broad spectrum, good specificity, high sensitivity, high accuracy, simple operation and low cost, and has good application prospects in the field of food detection.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology application, and in particular relates to a primer pair, a kit and a method for detecting Cronobacter sakazakii. Background Art

[0002] Cronobacter sakazakii, formerly known as Enterobacter sakazakii, is a facultative anaerobic Gram-negative bacillus that exists in a variety of foods such as infant formula, infant cereals, meat, water, and vegetables. Infants and young children are at high risk of infection with Cronobacter sakazakii. Once an infant is infected with this bacteria, it may cause bacteremia, meningitis, necrotizing enterocolitis, etc., and the mortality rate of related diseases is as high as 40%-80%. Cronobacter sakazakii has a strong proliferation ability, strong resistance to environmental factors, and has high heat resistance, acid and alkali resistance, and extremely strong drying resistance. Therefore, establishing a rapid and specific detection method for Cronobacter sakazakii is the key to effectively prevent and control the contamination of this bacterium in food.

[0003] The traditional method for detecting Cronobacter is to enrich and culture the bacteria, separate them on a color plate, and then perform biochemical identification. However, this detection method has problems such as cumbersome steps and a long process. Therefore, establishing an accurate, sensitive, rapid, and specific detection method can play an important role in food safety testing. At present, a variety of rapid detection methods have been developed, which are mainly polymerase chain reaction (PCR) technology, loop-mediated isothermal method (LAMP) technology, and recombinase polymerase amplification (RPA) technology. Recombinase polymerase amplification (RPA) is an isothermal nucleic acid amplification technology developed by TwistDx in the UK in 2006.

[0004] Existing detection technologies for Cronobacter sakazakii generally target α-glucosidase (gluA) gene, outer membrane protein A (ompA) gene, 16S rRNA gene, etc. Compared with other foodborne pathogens such as Salmonella, basic research on specific genes of Cronobacter sakazakii is insufficient. The prevalent strains of Cronobacter sakazakii vary from place to place, and their genes mutate rapidly. The commonly used detection target genes are not specific enough. It is necessary to explore new specific target genes to improve the specificity and broad spectrum of detection technology. Summary of the invention

[0005] Existing RPA detection technology generally targets the gluA gene, ompA gene, 16S rRNA gene, etc. of Cronobacter sakazakii, but there are many types of Cronobacter sakazakii and many gene variations. It is necessary to explore new specific target genes to improve the specificity and broad spectrum of detection technology. The first purpose of the present invention is to provide a new target for detecting Cronobacter sakazakii with strong specificity.

[0006] The present invention systematically analyzes the specific genes of Cronobacter sakazakii based on comparative genomics, discovers a new target glycosyl hydrolase gene mngB, and provides a nucleotide sequence (target gene fragment) in the gene for use in detecting Cronobacter sakazakii. The nucleotide sequence of the target gene fragment is shown in SEQ ID NO.1.

[0007] The inventors found that all second-generation and third-generation whole genome sequencing strains of Cronobacter sakazakii in the NCBI database have high homology. The lowest similarity of the ompA gene sequence of different strains of Cronobacter sakazakii is only about 96%, the lowest similarity of the gluA gene sequence is only about 97%, and the lowest similarity of the mngB gene sequence of different strains of Cronobacter sakazakii can reach about 98%. Compared with the ompA and gluA genes, the mngB homology of different strains of Cronobacter sakazakii is higher, so the method of the present invention has excellent broad spectrum in identifying and distinguishing Cronobacter sakazakii.

[0008] In addition, the mngB gene of Cronobacter sakazakii has very low homology with other closely related bacteria such as Cronobacter zurichensis and Cronobacter malonaticus. The ompA and gluA genes of Cronobacter sakazakii have high homology with other closely related bacteria. The method based on the target gene fragment of the present invention has excellent specificity in distinguishing Cronobacter sakazakii from its closely related species.

[0009] Based on the target gene fragment of the above-mentioned new target, the second object of the present invention is to provide an RPA primer pair for detecting Cronobacter sakazakii, wherein the primer pair is designed according to the above-mentioned target gene fragment, and comprises an upstream primer mngB-F and a downstream primer mngB-R, wherein the sequence of the upstream primer mngB-F is shown in SEQ ID NO.2, and the sequence of the downstream primer mngB-R is shown in SEQ ID NO.3.

[0010] The above-mentioned primer pair can be used to prepare identification reagents and kits for Cronobacter sakazakii. Based on this, the three objects of the present invention are to provide an RPA detection kit for Cronobacter sakazakii that is accurate, rapid, highly specific and broad-spectrum, comprising the above-mentioned primer pair; the kit also includes a buffer solution and sterile double distilled water; and may also include a gene extraction reagent and / or positive genomic DNA.

[0011] Based on the above primer pairs, the fourth object of the present invention is to provide a method for detecting Cronobacter sakazakii for non-disease diagnosis or treatment purposes, comprising the following steps: using the above primer pairs to perform RPA reaction on the DNA of the sample to be tested, and detecting whether there is a positive band after the reaction is completed.

[0012] Preferably, the reaction system of the RPA reaction includes: reaction buffer A buffer 29.4μL, reaction buffer B buffer 29.4μL, 10μM upstream primer 2μL, 10μM downstream primer 2μL, sterile double distilled water 13.1μL, and sample DNA 1μL to be tested. The preferred RPA reaction conditions are: 37℃-42℃, 15min-40min (more preferably 37℃, 30min). The domestic TwistAmp Basic kit can be used for implementation. The above reaction system and reaction conditions are the optimal conditions finally determined by the inventors after continuous software analysis and experiments, which can better achieve the effect.

[0013] During the detection, the presence of a positive band can be detected by agarose gel electrophoresis; or the product can be purified and then agarose gel electrophoresis can be used to detect the presence of a positive band.

[0014] The above method can be applied to the detection of Cronobacter sakazakii in foods such as infant formula, infant rice cereal, vegetables and meat, and has a wide range of applicability.

[0015] The beneficial effects of the present invention are as follows: the present invention provides a new target, and proposes a primer pair and RPA detection method based on the target gene fragment, which solves the problem that the existing target gene types of Cronobacter sakazakii nucleic acid detection are few and the specificity is not strong. It has the advantages of strong broad spectrum, good specificity, high sensitivity, high accuracy, simple operation and low cost, and has good application prospects in the field of food detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Shown is the result of sequence homology comparison of the Cronobacter sakazakii mngB gene in the NCBI database;

[0017] Figure 2 Shown is the result of sequence homology comparison of the ompA gene of Cronobacter sakazakii in the NCBI database;

[0018] Figure 3 Shown is the result of sequence homology comparison of Cronobacter sakazakii gluA gene in NCBI database;

[0019] Figure 4 Shown is the result of the mngB primer detection sensitivity experiment;

[0020] Figure 5 Shown is the result of the mngB primer detection specificity experiment. DETAILED DESCRIPTION

[0021] The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments and drawings to fully understand the purpose, scheme and effects of the present invention.

[0022] Example 1: Screening of new targets for Cronobacter sakazakii and design of RPA primer pairs

[0023] (1) Screening of new targets for Cronobacter sakazakii

[0024] Cronobacter sakazakii JXES-28 (NCBI accession number: CP098777.1) was selected as the model strain, and its whole genome sequence was compared with Salmonella strain A7 (NCBI accession number: CP084001) by Blastn. The non-homologous segments were intercepted by its Graphics results, and then the intercepted non-homologous segments were compared with the RAST annotation results, and the repeated sequences and hypothetical protein genes were removed. The functional genes located in the non-homologous segments were screened out and sorted out. Then the functional gene sequences were entered into the NCBI database for Blastn comparison in turn, and the conserved sequences with high homology to Cronobacter sakazakii (i.e., E<10-200) (the matching length with non-Cronobacter sakazakii was <30bp) were retained. These functional genes were initially selected as specific detection targets for Cronobacter sakazakii.

[0025] This method was used to screen out the new target mngB gene for Cronobacter sakazakii detection. The full length of the mngB gene and the commonly used target ompA and gluA genes of Cronobacter sakazakii were input into the NCBI data for blast comparison. The sequence similarity of the mngB gene in different Cronobacter sakazakii strains was as low as about 98% (e.g. Figure 1 The ompA gene sequence similarity is the lowest at only about 96% (as shown in Figure 2 The gluA gene sequence has the lowest similarity of only 97% (as shown in Figure 3 shown).

[0026] Except for Cronobacter sakazakii, the mngB gene homology of other closely related bacteria is very low (such as Cronobacter zurichensis (C. turicensis), Cronobacter malonaticus (C. malonaticus), such as Figure 1 The ompA gene of Cronobacter sakazakii has a sequence similarity of 96% with that of the closely related bacterium Cronobacter dublinensis. The gluA gene of Cronobacter sakazakii has a sequence similarity of 95% with that of Cronobacter malonaticus (as shown in Figure 2 and Figure 3 Compared with ompA and gluA, mngB gene has better broad spectrum and specificity as a new target for Cronobacter sakazakii detection.

[0027] (2) Design of RPA primer pairs

[0028] Multiple mngB genes of Cronobacter sakazakii were downloaded from the NCBI database, multiple sequence alignment was performed, and RPA primers were designed in the sequence conserved region. The primers were then input into NCBI for blast comparison and the specificity of the primers was analyzed. Finally, the primer pairs disclosed in the present invention were selected. The primers were synthesized by Shanghai Biotech Co., Ltd., and are as follows:

[0029] Upstream primer mngB-F: 5′-GCTGGATCTACTGGAAAAGGATGCGGATTT-3′ (SEQ ID NO. 2).

[0030] Downstream primer mngB-R: 5′-TAGTTGGTGGCGATGGATTTCTGGTTTGGT-3′ (SEQ ID NO. 3).

[0031] Example 2: RPA method for detecting Cronobacter sakazakii

[0032] An RPA method for detecting Cronobacter sakazakii comprises the following steps:

[0033] S1. Prepare DNA samples of samples to be tested;

[0034] S2. Perform RPA reaction on the DNA sample using the primer pair mngB-F / mngB-R obtained in Example 1;

[0035] S3. After the RPA reaction is completed, agarose gel electrophoresis is directly performed to detect whether there are positive bands.

[0036] The RPA reaction system was as follows: 29.4 μL A buffer; 2.5 μL B buffer; 2 μL each of 10 μM upstream and downstream primers; 13.1 μL sterile double distilled water; 1 μL DNA sample. The reaction conditions were: 37°C, 30 min. The domestic TwistAmpBasic kit (Anpu Future (Changzhou) Biotechnology Co., Ltd.) was used for the reaction.

[0037] After the reaction, 5 μL of the reaction solution was subjected to 1.5% agarose gel electrophoresis, and Goldview was added to the electrophoresis gel for staining. After the electrophoresis, the product was observed and photographed under a UV projector. In other embodiments, a PCR product kit can be used to purify the product, and then agarose gel electrophoresis and photographic observation can be performed.

[0038] The above method was used to evaluate the effects of various strains, as follows:

[0039] (1) Experimental strains

[0040] Eleven reference strains, including Cronobacter sakazakii JXES-28 (NCBI accession number: CP098777.1), Cronobacter sakazakii JXES-5, Cronobacter sakazakii JXES-14, Cronobacter sakazakii JXES-17, Cronobacter sakazakii JXES-19, Cronobacter sakazakii JXES-30, Escherichia coli ATCC 25922, Aeromonas 429, Listeria monocytogenes ATCC19114, Proteus mirabilis 012 (NCBI accession number: CP062146), and Salmonella ATCC 14028, are preserved in the laboratory of the Food Safety Team of Wuhan University of Technology.

[0041] (2) Preparation of bacterial DNA template

[0042] Extract the genomic DNA of the strain to be tested by boiling method: culture the strain overnight in LB liquid medium, take 1mL of bacterial solution and centrifuge at 12000rpm for 5min, and discard the supernatant; wash the cell pellet with 1mL of sterile water, centrifuge at 12000rpm for 5min, and discard the supernatant. Add 100μL of sterile deionized water to the pellet to form a suspension, boil for 10min, ice bath for 5min, centrifuge at 12000rpm for 5min, and the supernatant is the genomic DNA, which is used as the template for RPA amplification.

[0043] In other embodiments, strain DNA can also be extracted using commercial kits or organic reagents.

[0044] (3) Sensitivity test experiment

[0045] The DNA of Cronobacter sakazakii JXES-28 was extracted and the concentration was determined to be 146 ng / μL. It was diluted ten-fold with sterile water and used as a detection template. 1 μL of each concentration gradient was added to the RPA reaction system for sensitivity testing.

[0046] The results are as follows Figure 4 As shown, lanes 1-8 are the electrophoresis results of JXES-28RPA amplification products of different concentrations, including lanes 1-2: 14.6 ng / μL; lanes 3-4: 1.46 ng / μL; lanes 5-6: 0.146 ng / μL; lanes 7-8: 0.0146 ng / μL; lane 9: blank control without DNA template.

[0047] Depend on Figure 4 The sensitivity verification results showed that the RPA technology of the present invention can detect Cronobacter sakazakii with a DNA concentration as low as 0.146 ng / μL.

[0048] (4) Specificity detection experiment

[0049] Ten bacteria were selected for RPA specificity experiments. Figure 5 As shown, lanes 1-10 are Cronobacter sakazakii JXES-5, Cronobacter sakazakii JXES-14, Cronobacter sakazakii JXES-17, Cronobacter sakazakii JXES-19, Cronobacter sakazakii JXES-30, Escherichia coli ATCC 25922, Aeromonas 429, Listeria monocytogenes ATCC19114, Proteus mirabilis 012 (NCBI accession number: CP062146), and Salmonella ATCC 14028, respectively; lanes 11-12 are blank controls without adding DNA template.

[0050] Depend on Figure 5 The specificity verification results showed that only Cronobacter sakazakii could be amplified, and other bacteria could not be amplified. The results showed that the method of the present invention has good specificity for detecting Cronobacter sakazakii.

[0051] Embodiment 3:

[0052] An RPA detection kit for Cronobacter sakazakii, comprising the primer pair mngB-F / mngB-R in Example 1, and also comprising a buffer and sterile double distilled water. The buffer is specifically 29.4 μL Abuffer and 2.5 μL B buffer; the concentration of the upstream primer and the downstream primer in the primer pair mngB-F / mngB-R are both 10 μM, and the volume is both 2 μL; the volume of the sterile double distilled water is 13.1 μL.

[0053] In some other embodiments, the above-mentioned kit may further include a gene extraction reagent and / or a positive genomic DNA.

[0054] Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Mentioning "embodiment" in this article means that the specific features or characteristics described in conjunction with the embodiment may be included in at least one embodiment of the present embodiment application. The appearance of this phrase in various positions in the specification is not necessarily the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It can be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0055] Although the embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the claims and their equivalents.

Claims

1. Application of a target gene fragment in detecting Cronobacter sakazakii, characterized in that: The nucleotide sequence of the target gene fragment is shown in 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 according to claim 1, and includes an upstream primer mngB-F and a downstream primer mngB-R, the sequence of the upstream primer mngB-F is shown in SEQ ID NO.2, and the sequence of the downstream primer mngB-R is shown in SEQ ID NO.

3.

3. A kit for detecting Cronobacter sakazakii, characterized in that: Comprising the primer pair described in claim 2.

4. The kit according to claim 3, characterized in that The kit also includes a buffer and sterile double distilled water.

5. The kit according to claim 4, characterized in that: The kit also includes a gene extraction reagent and / or positive genomic DNA.

6. A method for detecting Cronobacter sakazakii for purposes other than diagnosis or treatment of a disease, characterized in that: The method comprises the following steps: using the primer pair described in claim 2 to perform RPA reaction on the DNA of the sample to be tested, and detecting whether there is a positive band after the reaction is completed.

7. The method according to claim 6, characterized in that The reaction system of the RPA reaction includes: 29.4 μL of reaction buffer Abuffer, 29.4 μL of reaction buffer B buffer, 2 μL of the 10 μM upstream primer, 2 μL of the 10 μM downstream primer, 13.1 μL of sterile double distilled water, and 1 μL of the sample DNA to be tested.

8. The method according to claim 7, characterized in that The RPA reaction conditions are: 37°C-42°C, 15min-40min.

9. The method according to claim 6, characterized in that The presence of positive bands was detected by agarose gel electrophoresis.

10. Application of the method according to claim 6 in the field of food testing.

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