CN110964786B

CN110964786B - Rapid constant-temperature detection method, primer group and kit for cronobacter sakazakii

Info

Publication number: CN110964786B
Application number: CN202010004054.0A
Authority: CN
Inventors: 曹永梅; 李园园; 李雪玲; 刘伟; 韦朝春; 陆长德; 李亦学; 贾犇
Original assignee: SHANGHAI INDUSTRIAL TECHNOLOGY INSTITUTE; Shanghai Wangwang Food Group Co ltd
Current assignee: SHANGHAI INDUSTRIAL TECHNOLOGY INSTITUTE; Shanghai Wangwang Food Group Co ltd
Priority date: 2015-09-02
Filing date: 2016-08-30
Publication date: 2022-08-26
Anticipated expiration: 2036-08-30
Also published as: CN111020047B; CN106434882B; CN111334595A; CN106434883B; CN106434888A; CN111020047A; CN111100906A; CN111057781B; CN106434889A; CN106434895A; CN106434891B; CN110938678A; CN110938676A; CN106434897A; CN106434899B; CN106434896A; CN111020009A; CN106434886B; CN106434898A; CN111041113A

Abstract

The invention discloses a rapid constant-temperature detection method, a primer group and a kit for cronobacter sakazakii. The method comprises the following steps: extracting genome DNA from a sample to be detected; performing constant-temperature amplification reaction in an enzyme reaction system by using the genome DNA as a template and a primer group capable of amplifying a specific sequence of Cronobacter sakazakii as a primer; and determining whether the sample to be detected has cronobacter sakazakii or not by judging whether the reaction result is positive or not. The detection method has the advantages of high sensitivity and high specificity, short detection time, simple result judgment, convenient operation, low cost and wide application prospect.

Description

Rapid constant-temperature detection method, primer group and kit for cronobacter sakazakii

The application is filed on 2016, 8, 30, and has the application number of 201610767389.1 and the name of the invention: the divisional application of the Chinese invention patent application of 'method, primer and application for rapid constant temperature detection of Cronobacter sakazakii'; the parent application claims the priority of the Chinese patent application with the application date of 2015, 9 and 2, the application number of 201510556917.4, named as 'method, primer and kit for rapid isothermal detection of cronobacter sakazakii'.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method, primers and a kit for rapidly detecting cronobacter sakazakii at a constant temperature.

Background

Sakazakii (Cronobacter sakazakii) is a gram-negative, peritrichous, motile, sporular facultative anaerobic bacterium, named as atypical flavochrome-producing enterobacter cloacae before 1980, which was named enterobacter sakazakii after 1980, and reclassified in 2008, and classified as Cronobacter sakazakii of enterobacteriaceae. The cronobacter sakazakii is widely existed in the natural world, is an important conditioned pathogen which harms the health of infants through the formula powder, can cause serious clinical symptoms of the infants, such as cerebral abscess, meningitis, necrotizing enterocolitis, systemic septicemia and the like, and has the lethality rate of 40-80%. Both newborn infants and premature infants are at risk of infection with cronobacter sakazakii strains by consumption of infant formula contaminated with cronobacter sakazakii. Compared with other food-borne pathogenic microorganisms, the cronobacter sakazakii has a low infection rate but has a high mortality rate for special people, and the host and the propagation model of the cronobacter sakazakii are not clear yet. Therefore, it is particularly important to prevent and detect the bacteria.

At present, the detection method for cronobacter sakazakii at home and abroad still uses a conventional culture method as a standard, but the detection period is longer (up to 5-7 days), the operation is relatively complex, the detection efficiency is lower, and the requirements of modern society on high flux, high sensitivity, high specificity, rapidness and convenience in the detection process of food-borne pathogenic bacteria are difficult to meet. Researchers developed detection means of PCR and fluorescence PCR technologies successively, and the detection means is also brought into the industry standard of 'detection method of Cronobacter sakazakii in milk powder' in China, but the two methods need special detection instruments, and therefore, the method is not suitable for being widely applied to basic detection departments, particularly real-time field detection in enterprise production lines.

Loop-mediated isothermal amplification (LAMP) is a novel isothermal Nucleic acid amplification method developed in recent years, which designs 4 specific primers (including upstream and downstream outer primers F3 and B3, and upstream and downstream inner primers FIP and BIP, wherein FIP is composed of F1C and F2, and BIP is composed of B1C and B2) for 6 regions of a target sequence, and completes the Nucleic acid amplification reaction by incubating for about 60min at an isothermal condition, and generates a visible reaction by-product, white magnesium pyrophosphate precipitate (see Notomi T, Oyayaama H, Masubuchi H, Yonekawa T, Watanabe K, Nuino N, Hase T. loop-mediated isothermal amplification (8512, 2000, 63). The technology can be completed at a constant temperature without a PCR instrument or a fluorescent quantitative PCR instrument, can judge the reaction result by naked eyes, and has the advantages of high sensitivity, strong specificity, short reaction time, convenient operation, low cost and the like.

Primer design is the most critical step in LAMP technology, and the conventional method is to introduce the acknowledged specific gene of a certain organism to be detected into an online website (http:// primer explorer. jp/e) designed by LAMP primers, and set relevant parameters to generate a primer group. That is, the user must first ensure that the target gene is a specific sequence of the species to be tested. Taking the invention patents ZL201010614700.1 and CN 101319249B as examples, the OmpA gene and the 18S rDNA sequence, which are specific genes of Cronobacter sakazakii and are reported in the literature, are respectively used for detecting the Cronobacter sakazakii by adopting LAMP technology. However, the so-called "recognized specific gene" is often based on the knowledge of hysteresis and is not necessarily updated based on the ever-increasing genome data of microorganisms, so that a primer obtained based on the sequence of the target gene is not necessarily able to ensure its specificity in practical use. The present invention, as shown in Table 1, shows the problems of insufficient primer specificity and unsuitability of versatility in the prior art. That is, the cronobacter sakazakii detection sequence used in the prior art method is not actually specific to cronobacter sakazakii, that is, it is possible that cronobacter sakazakii is erroneously identified as cronobacter sakazakii. Meanwhile, the cronobacter sakazakii detection sequence used in the prior art method is not common to cronobacter sakazakii, that is, there is a possibility that a part of strains of cronobacter sakazakii is missed. Therefore, a cronobacter sakazakii detection method capable of ensuring specificity and universality is urgently needed in the industry, and meanwhile, the requirements of the primary detection department on rapidness and convenience are met, and real-time field detection can be conveniently carried out in an enterprise production line. In order to ensure the safety of food, a rapid, simple and accurate method for detecting cronobacter sakazakii in the food is urgently needed.

Disclosure of Invention

The invention aims to overcome the defects of insufficient primer universality and specificity in the primer design of the prior LAMP technology, fully utilizes abundant microbial genome sequence information in the current public data resources and corresponding sequence analysis tools, designs a primer group for specifically identifying the cronobacter sakazakii, and forms a high-sensitivity and high-specificity detection kit on the basis. The invention provides a method, a primer group and a kit for detecting cronobacter sakazakii by rapid isothermal amplification, which are used for designing cronobacter sakazakii LAMP primers based on microbial genome data resources (data 8/5/8/2013) in a GenBank database. The detection method for detecting the cronobacter sakazakii has the advantages of high sensitivity and specificity, short detection time, simple result judgment, convenient operation and low cost.

The invention provides a method for rapidly detecting cronobacter sakazakii strains, which comprises the following steps:

(1) extracting genome DNA from a sample to be detected;

(2) carrying out constant-temperature amplification reaction in an enzyme reaction system by taking the genome DNA as a template and a primer group capable of amplifying the specific base sequence of the Cronobacter sakazakii genome as a primer;

(3) and determining whether the sample to be detected has cronobacter sakazakii or not by judging whether the reaction result is positive or not.

The method for detecting the cronobacter sakazakii strain at constant temperature extracts genome DNA from a sample to be detected, takes the genome DNA as a template and takes a cronobacter sakazakii specific amplification primer group as a primer to carry out constant temperature amplification reaction, and then determines whether the cronobacter sakazakii exists in the sample to be detected or not by judging whether the reaction result is positive or not. Wherein, the enzyme reaction system includes but is not limited to a DNA polymerase reaction system.

In the invention, the genome specific alkali sequence of the cronobacter sakazakii is the sequence of 1080296-1081222 bp bits of the cronobacter sakazakii with the GI number of 156932229.

In the present invention, the primer set capable of amplifying the nucleotide sequence specific to the Cronobacter sakazakii genome is a part of the nucleotide sequence of 1080296 to 1081222bp of the genome (GI No. 156932229) or a part of the complementary strand thereof. Wherein the sakazakii genome-specific nucleotide sequence is a nucleotide sequence unique to the sakazakii genome, and is not contained in the genome of another microorganism.

Wherein the primer set capable of amplifying the specific nucleotide sequence of the Cronobacter sakazakii genome includes, but is not limited to, any one selected from the following primer sets A to D, or any one selected from the primer sets having a homology of 50% or more with a single sequence in the sequence of the primer set or the complementary strand sequence thereof.

Primer set a:

upstream outer primer F3_ a: 5'-GGCGGGTTTATACTGGAT-3' (SEQ ID NO: 1);

downstream outer primer B3_ a: 5'-CACTGAAATGTCACAAGCTA-3' (SEQ ID NO: 2);

upstream inner primer FIP _ a: 5'-CGGCGTATCTAAATCAAATGCCCTTAGCCCAACACGATTC-3' (SEQ ID NO: 3);

the downstream inner primer BIP _ A: 5'-GCGACGTTATCTAATATTGTAAGGCGCCAGAAACCAAATCATTG-3' (SEQ ID NO: 4);

primer set B:

upstream outer primer F3_ B: 5'-ATAGTGTTTTGTTTTCCGGG-3' (SEQ ID NO: 5);

downstream outer primer B3_ B: 5'-ACGAGGGTAAAGGACAAA-3' (SEQ ID NO: 6);

upstream inner primer FIP _ B: 5'-GCTTCAGTTTGGTTGATGGGAAAAGAAGTGGTAAGGAAGCT-3' (SEQ ID NO: 7);

the downstream inner primer BIP _ B: 5'-TTTCTCCGTGACAGTGAATACGAAACTGCCAGTGAAGTGA-3' (SEQ ID NO: 8);

primer set C:

the upstream outer primer F3_ C: 5'-GGATCATGGAGCAATTTCC-3' (SEQ ID NO: 9);

downstream outer primer B3 — C: 5'-TCATTTTCGCACAAAGCC-3' (SEQ ID NO: 10);

upstream inner primer FIP _ C: 5'-AAGAACCAGACTGGCGTAACTTTTTCAGTCCTTTCTCCG-3' (SEQ ID NO: 11);

a downstream inner primer BIP _ C: 5'-CCCTCGTAAGTAGCGAATTTAGCCGATTTCCTTGAAGCAGTG-3' (SEQ ID NO: 12);

primer set D:

upstream outer primer F3_ D: 5'-GCGAATTTAGCACCCAAA-3' (SEQ ID NO: 13);

downstream outer primer B3_ D: 5'-GGACATCAGCAAACCTTC-3' (SEQ ID NO: 14);

upstream inner primer FIP _ D: 5'-GACGCAGGCTATTCATTTTCGTTTTGACCATTTACCACTGC-3' (SEQ ID NO: 15);

the downstream inner primer BIP _ D: 5'-CATAGTTCAAAAAGCCTGTGTCAAGAGCTGATACTGCTACTGC-3' (SEQ ID NO: 16).

In the present invention, the primer set capable of amplifying the specific nucleotide sequence of the cronobacter sakazakii genome may further include a primer set having a homology of 50% or more with a single sequence in each of the aforementioned primer set sequences or the complementary strand sequences thereof, and the primer set includes, but is not limited to, any one of the following primer sets E to H:

primer set E:

the upstream outer primer F3_ E: 5'-GGCGGGTTTATACTGGAT-3' (SEQ ID NO: 17);

downstream outer primer B3_ E: 5'-TCAATGGCACTGAAATGTC-3' (SEQ ID NO: 18) (60% homology to primer B3_ A5 '-CACTGAAATGTCACAAGCTA-3');

upstream inner primer FIP _ E: 5'-CGGCGTATCTAAATCAAATGCCCTTAGCCCAACACGATTC-3' (SEQ ID NO: 19);

the downstream inner primer BIP _ E: 5'-GCGACGTTATCTAATATTGTAAGGCGAGCAATAGCCAGAAACC-3' (SEQ ID NO: 20);

a primer set F:

upstream outer primer F3 — F: 5'-TACCCTCGTAAGTAGCGA-3' (SEQ ID NO: 21) (50% homology to the complementary strand 5'-TTTGTCCTTTACCCTCGT-3' of primer B3_ B);

downstream outer primer B3 — F: 5'-GGACATCAGCAAACCTTC-3' (SEQ ID NO: 22);

upstream inner primer FIP _ F: 5'-CGCACAAAGCCAATTTCGATTTTAGCACCCAAATTTTGACC-3' (SEQ ID NO: 23);

the downstream inner primer BIP _ F: 5'-CATAGTTCAAAAAGCCTGTGTCAAGAGCTGATACTGCTACTGC-3' (SEQ ID NO: 24);

primer set G:

upstream outer primer F3_ G: 5'-AGCAATTTCCCATCAACC-3' (SEQ ID NO: 25) (homology 52.6% to primer F3_ C5 '-GGATCATGGAGCAATTTCC-3');

downstream outer primer B3_ G: 5'-GGGTGCTAAATTCGCTAC-3' (SEQ ID NO: 26);

upstream inner primer FIP _ G: 5'-AAGAACCAGACTGGCGTAACTGAAGCCATTTTTCAGTCC-3' (SEQ ID NO: 27);

the downstream inner primer BIP _ G: 5'-CCGTTTTTAATGTCACTTCACTGGTACGAGGGTAAAGGACAAA-3' (SEQ ID NO: 28);

a primer set H:

upstream outer primer F3 — H: 5'-ATAGTGTTTTGTTTTCCGGG-3' (SEQ ID NO: 29);

downstream outer primer B3 — H: 5'-ATGGTCAAAATTTGGGTGC-3' (SEQ ID NO: 30) (50% homology to the complementary strand 5'-TTTGGGTGCTAAATTCGC-3' of primer F3_ D);

upstream inner primer FIP _ H: 5'-GAGAAAGGACTGAAAAATGGCTTCTAAGGAAGCTCTGGGGAT-3' (SEQ ID NO: 31);

the downstream inner primer BIP _ H: 5'-CCAGTCTGGTTCTTCCGTTTACGAGGGTAAAGGACAAA-3' (SEQ ID NO: 32).

In the method of the present invention, the primer set capable of amplifying the genomic specific nucleotide sequence of cronobacter sakazakii may or may not contain a loop primer. The loop primer may be one or more, including primers LF and/or LB. The primer group capable of amplifying the specific base sequence of the cronobacter sakazakii genome is selected from any one of the following primer groups A ', B ', E ', G ' and H '; or any one selected from the group consisting of primer sets having a homology of 50% or more with respect to a single sequence in the sequences of the primer sets A ', B ', E ', G ', H ' or the complementary strand sequences thereof:

a primer set A':

upstream outer primer F3_ a: 5'-GGCGGGTTTATACTGGAT-3', respectively;

downstream outer primer B3_ a: 5'-CACTGAAATGTCACAAGCTA-3', respectively;

upstream inner primer FIP _ A: 5'-CGGCGTATCTAAATCAAATGCCCTTAGCCCAACACGATTC-3', respectively;

the downstream inner primer BIP _ A:

5’-GCGACGTTATCTAATATTGTAAGGCGCCAGAAACCAAATCATTG-3’；

upstream loop primer LF _ a: 5'-TAAGGCAGCGAAGATACAGC-3' (SEQ ID NO: 33);

a primer set B':

upstream outer primer F3_ B: 5'-ATAGTGTTTTGTTTTCCGGG-3';

downstream outer primer B3_ B: 5'-ACGAGGGTAAAGGACAAA-3', respectively;

an upstream inner primer FIP _ B: 5'-GCTTCAGTTTGGTTGATGGGAAAAGAAGTGGTAAGGAAGCT-3', respectively;

the downstream inner primer BIP _ B: 5'-TTTCTCCGTGACAGTGAATACGAAACTGCCAGTGAAGTGA-3', respectively;

downstream loop primer LB _ B: 5'-CATTGACGTTACGCCAGTCT-3' (SEQ ID NO: 34);

a primer set E':

the upstream outer primer F3_ E: 5'-GGCGGGTTTATACTGGAT-3', respectively;

downstream outer primer B3_ E: 5'-TCAATGGCACTGAAATGTC-3', respectively;

upstream inner primer FIP _ E: 5'-CGGCGTATCTAAATCAAATGCCCTTAGCCCAACACGATTC-3';

the downstream inner primer BIP _ E: 5'-GCGACGTTATCTAATATTGTAAGGCGAGCAATAGCCAGAAACC-3', respectively;

upstream loop primer LF _ E: 5'-TAAGGCAGCGAAGATACAGC-3' (SEQ ID NO: 35);

a primer group G':

upstream outer primer F3_ G: 5'-AGCAATTTCCCATCAACC-3', respectively;

downstream outer primer B3_ G: 5'-GGGTGCTAAATTCGCTAC-3', respectively;

upstream inner primer FIP _ G: 5'-AAGAACCAGACTGGCGTAACTGAAGCCATTTTTCAGTCC-3', respectively;

the downstream inner primer BIP _ G: 5'-CCGTTTTTAATGTCACTTCACTGGTACGAGGGTAAAGGACAAA-3';

upstream loop primer LF _ G: 5'-CGTATTCACTGTCACGGAGAA-3' (SEQ ID NO: 36);

a primer set H':

upstream outer primer F3 — H: 5'-ATAGTGTTTTGTTTTCCGGG-3', respectively;

downstream outer primer B3 — H: 5'-ATGGTCAAAATTTGGGTGC-3', respectively;

upstream inner primer FIP _ H: 5'-GAGAAAGGACTGAAAAATGGCTTCTAAGGAAGCTCTGGGGAT-3', respectively;

the downstream inner primer BIP _ H: 5'-CCAGTCTGGTTCTTCCGTTTACGAGGGTAAAGGACAAA-3';

upstream loop primer LF _ H: 5'-GGTTGATGGGAAATTGCTCCAT-3' (SEQ ID NO: 37);

and/or, the downstream loop primer LB _ H: 5'-TAATGTCACTTCACTGGCAGTT-3' (SEQ ID NO: 38).

In specific embodiments, for example, the primer set H' may include only one forward loop primer, only one downstream loop primer, or both a forward loop primer and a downstream loop primer.

In a specific embodiment (including a loop primer), the enzyme reaction system for isothermal amplification is as follows: 1 XBst DNA polymerase reaction buffer, 2-9mmol/L Mg ²⁺ (MgSO ₄ Or MgCl ₂ ) 1.0-1.6mmol/L dNTP, 0.8-2.0 mu mol/L FIP and BIP primers, 0.15-0.3 mu mol/L F3 and B3 primers, 0.4-1.0 mu mol/L LF and/or LB primers, 0.16-0.64U/mu L Bst DNA polymerase and 0-1.5mol/L betaine. In another embodiment (without loop primer), the enzyme reaction system for isothermal amplification is: 1 XBst DNA polymerase reaction buffer solution, 2-9mmol/L Mg ²⁺ (MgSO ₄ Or MgCl ₂ ) 1.0-1.6mmol/L dNTP, 0.8-2.0 mu mol/L FIP and BIP primers, 0.15-0.3 mu mol/L F3 and B3 primers, 0.16-0.64U/mu L Bst DNA polymerase and 0-1.5mol/L betaine. The loop primer contributes to the improvement of the reaction efficiency. For example, 1 XBst DNA polymerase reaction buffer can be 1 × Thermopol reaction buffer containing 20mmol/L Tris-HCl (pH8.8), 10mmol/L KCl, 10mmol/L (NH4) ₂ SO4，0.1％Triton X-100，2mM MgSO ₄ . MgSO in 1 XBst DNA polymerase reaction buffer ₄ And magnesium ion Mg in enzyme reaction system ²⁺ And (6) merging.

In the method, the reaction procedure of the constant-temperature amplification reaction is incubation at 60-65 ℃ for 10-90 min, preferably 10-60 min; ② terminating the reaction for 2-20 min at 80 ℃. The invention is not limited to the implementation of the detection method of the invention by other suitable reaction procedures.

In the method of the present invention, the detection method includes, but is not limited to, electrophoresis detection, turbidity detection, color detection, or the like. The electrophoresis detection is preferably a gel electrophoresis detection method, and may be agarose gel or polyacrylamide gel. In the electrophoresis detection result, if the electrophoresis chart shows a characteristic step-shaped strip, the sample to be detected is positive to the cronobacter sakazakii and contains the cronobacter sakazakii; if the electrophoretogram does not present a characteristic ladder-shaped strip, the sample to be detected is negative to cronobacter sakazakii. The turbidity detection is to detect turbidity by naked eye observation or a turbidity meter, and if the detection tube is turbid, the sample to be detected is positive to the cronobacter sakazakii and contains the cronobacter sakazakii; if no turbidity is found, the sample to be tested is negative to cronobacter sakazakii. Or the reaction tube bottom can be visually observed whether the precipitate exists after the centrifugation, if the precipitate exists at the reaction tube bottom, the sample to be detected is positive to the cronobacter sakazakii and contains the cronobacter sakazakii; if the reaction tube bottom is not precipitated, the sample to be tested is negative to cronobacter sakazakii.

The color development detection is to add color development reagent, including but not limited to calcein (50 μ M) or SYBR Green I (30-50 ×), or hydroxynaphthol blue (i.e. HNB, 120-. When calcein or SYBR Green I is used as a color developing agent, if the color is orange after reaction, the sample to be detected is negative to cronobacter sakazakii; if the color after the reaction is green, the sample to be detected is positive to the cronobacter sakazakii and contains the cronobacter sakazakii. When hydroxyl naphthol blue is used as a color developing agent, if the color after reaction is violet, the sample to be detected is negative to cronobacter sakazakii; if the color after the reaction is sky blue, the sample to be detected is positive to the cronobacter sakazakii. The chromogenic detection can be used for detecting the reaction result in real time or at an end point through a detection instrument besides observing the reaction result through naked eyes, and by reasonably setting a threshold value of negative reaction, when the reaction result of the sample to be detected is lower than or equal to the threshold value, the sample to be detected is negative to the sakazakii; and when the reaction result of the sample to be detected is greater than the threshold value, determining that the sample to be detected is positive to the cronobacter sakazakii. The detection instrument comprises but is not limited to a fluorescence spectrophotometer, a fluorescence quantitative PCR instrument, a constant temperature amplification microfluidic chip nucleic acid analyzer, a Genie II isothermal amplification fluorescence detection system and the like.

In the color development detection, if calcein or hydroxynaphthol blue is used as a color developing agent, the color developing agent can be added before the constant-temperature amplification reaction, or can be added after the constant-temperature amplification reaction is completed, preferably before the constant-temperature amplification reaction, so that the possibility of reaction pollution can be effectively reduced. If SYBR Green I is adopted as a color developing agent, the SYBR Green I is added after the isothermal amplification reaction is finished. If calcein is used as color-developing agent, 50 μ M calcein is added into enzyme reaction system, and 0.6-1mM [ Mn ] is added ²⁺ ]For example, 0.6-1mM of MnCl ₂ . The invention also provides a primer used in the method for detecting the cronobacter sakazakii strain at constant temperature. The primer comprises a primer group capable of amplifying specific base sequences of a cronobacter sakazakii genome, and the primer comprises but is not limited to a part of a nucleic acid sequence with the GI number of 156932229 at the 1080296-1081222 bp position of the cronobacter sakazakii genome or a part of a complementary strand thereof.

Wherein the primer group capable of amplifying the specific nucleotide sequence of the cronobacter sakazakii genome is selected from any one of the following primer groups, or is selected from any one of the primer groups having homology of 50% or more with a single sequence in the sequence of each primer group or the sequence of the complementary strand thereof. Wherein the primer set includes, but is not limited to, any one of the following primer sets A to D. The primer set having a homology of 50% or more with respect to a single sequence in the aforementioned primer set sequence or the complementary strand sequence thereof includes, but is not limited to, any one of the following primer sets E to H.

Primer set a:

upstream outer primer F3_ a: 5'-GGCGGGTTTATACTGGAT-3';

downstream outer primer B3_ a: 5'-CACTGAAATGTCACAAGCTA-3', respectively;

the downstream inner primer BIP _ A:

5’-GCGACGTTATCTAATATTGTAAGGCGCCAGAAACCAAATCATTG-3’；

primer set B:

the upstream outer primer F3_ B: 5'-ATAGTGTTTTGTTTTCCGGG-3', respectively;

downstream outer primer B3_ B: 5'-ACGAGGGTAAAGGACAAA-3', respectively;

upstream inner primer FIP _ B: 5'-GCTTCAGTTTGGTTGATGGGAAAAGAAGTGGTAAGGAAGCT-3', respectively;

a downstream inner primer BIP _ B: 5'-TTTCTCCGTGACAGTGAATACGAAACTGCCAGTGAAGTGA-3', respectively;

primer set C:

upstream outer primer F3_ C: 5'-GGATCATGGAGCAATTTCC-3', respectively;

downstream outer primer B3 — C: 5'-TCATTTTCGCACAAAGCC-3', respectively;

upstream inner primer FIP _ C: 5'-AAGAACCAGACTGGCGTAACTTTTTCAGTCCTTTCTCCG-3', respectively;

the downstream inner primer BIP _ C: 5'-CCCTCGTAAGTAGCGAATTTAGCCGATTTCCTTGAAGCAGTG-3', respectively;

primer set D:

upstream outer primer F3_ D: 5'-GCGAATTTAGCACCCAAA-3';

downstream outer primer B3_ D: 5'-GGACATCAGCAAACCTTC-3';

upstream inner primer FIP _ D: 5'-GACGCAGGCTATTCATTTTCGTTTTGACCATTTACCACTGC-3', respectively;

the downstream inner primer BIP _ D: 5'-CATAGTTCAAAAAGCCTGTGTCAAGAGCTGATACTGCTACTGC-3', respectively;

primer set E:

upstream outer primer F3_ E: 5'-GGCGGGTTTATACTGGAT-3', respectively;

downstream outer primer B3_ E: 5'-TCAATGGCACTGAAATGTC-3', respectively;

upstream inner primer FIP _ E: 5'-CGGCGTATCTAAATCAAATGCCCTTAGCCCAACACGATTC-3', respectively;

a primer set F:

upstream outer primer F3 — F: 5'-TACCCTCGTAAGTAGCGA-3', respectively;

downstream outer primer B3 — F: 5'-GGACATCAGCAAACCTTC-3', respectively;

upstream inner primer FIP _ F: 5'-CGCACAAAGCCAATTTCGATTTTAGCACCCAAATTTTGACC-3', respectively;

the downstream inner primer BIP _ F: 5'-CATAGTTCAAAAAGCCTGTGTCAAGAGCTGATACTGCTACTGC-3', respectively;

primer set G:

the upstream outer primer F3_ G: 5'-AGCAATTTCCCATCAACC-3';

downstream outer primer B3_ G: 5'-GGGTGCTAAATTCGCTAC-3';

the downstream inner primer BIP _ G: 5'-CCGTTTTTAATGTCACTTCACTGGTACGAGGGTAAAGGACAAA-3', respectively;

a primer set H:

upstream outer primer F3 — H: 5'-ATAGTGTTTTGTTTTCCGGG-3', respectively;

downstream outer primer B3 — H: 5'-ATGGTCAAAATTTGGGTGC-3', respectively;

the downstream inner primer BIP _ H: 5'-CCAGTCTGGTTCTTCCGTTTACGAGGGTAAAGGACAAA-3' are provided.

In the primer used in the method for detecting cronobacter sakazakii at constant temperature, the primer group capable of amplifying the specific base sequence of the cronobacter sakazakii genome may or may not comprise one or more loop primers; the loop primer is LF and/or LB. The primer group capable of amplifying the specific base sequence of the sakazakii cronobacter genome is selected from any one of the following primer groups A ', B ', E ', G ' and H '; or any one selected from the group consisting of primer sets having a homology of 50% or more with respect to a single sequence in the sequences of the primer sets A ', B ', E ', G ', H ' or the complementary strand sequences thereof:

primer set a':

upstream outer primer F3_ a: 5'-GGCGGGTTTATACTGGAT-3', respectively;

downstream outer primer B3_ a: 5'-CACTGAAATGTCACAAGCTA-3', respectively;

the downstream inner primer BIP _ A:

5’-GCGACGTTATCTAATATTGTAAGGCGCCAGAAACCAAATCATTG-3’；

upstream loop primer LF _ a: 5'-TAAGGCAGCGAAGATACAGC-3';

a primer set B':

the upstream outer primer F3_ B: 5'-ATAGTGTTTTGTTTTCCGGG-3', respectively;

downstream outer primer B3_ B: 5'-ACGAGGGTAAAGGACAAA-3', respectively;

downstream loop primer LB _ B: 5'-CATTGACGTTACGCCAGTCT-3', respectively;

a primer set E':

upstream outer primer F3_ E: 5'-GGCGGGTTTATACTGGAT-3';

downstream outer primer B3_ E: 5'-TCAATGGCACTGAAATGTC-3', respectively;

an upstream inner primer FIP _ E: 5'-CGGCGTATCTAAATCAAATGCCCTTAGCCCAACACGATTC-3', respectively;

upstream loop primer LF _ E: 5'-TAAGGCAGCGAAGATACAGC-3', respectively;

a primer set G':

upstream outer primer F3_ G: 5'-AGCAATTTCCCATCAACC-3', respectively;

downstream outer primer B3_ G: 5'-GGGTGCTAAATTCGCTAC-3', respectively;

upstream loop primer LF _ G: 5'-CGTATTCACTGTCACGGAGAA-3', respectively;

a primer set H':

upstream outer primer F3 — H: 5'-ATAGTGTTTTGTTTTCCGGG-3', respectively;

downstream outer primer B3 — H: 5'-ATGGTCAAAATTTGGGTGC-3', respectively;

the downstream inner primer BIP _ H: 5'-CCAGTCTGGTTCTTCCGTTTACGAGGGTAAAGGACAAA-3', respectively;

upstream loop primer LF _ H: 5'-GGTTGATGGGAAATTGCTCCAT-3', respectively;

and/or, the downstream loop primer LB _ H: 5'-TAATGTCACTTCACTGGCAGTT-3' are provided.

In a specific embodiment, the primer set H' may include only one upstream loop primer, only one downstream loop primer, or both an upstream loop primer and a downstream loop primer. In a specific embodiment, the primers are respectively FIP, BIP, F3, B3, LF and LB primers or primers with homology of 50% or more with the above primer sequence or single primer in the complementary strand sequence.

The invention also provides a kit used in the method for detecting the Cronobacter sakazakii strain at the constant temperature, which comprises the primer group capable of amplifying the specific base sequence of the Cronobacter sakazakii genome. In the kit of the present invention, the primer set capable of amplifying the nucleotide sequence specific to the Cronobacter sakazakii genome includes, but is not limited to, a primer sequence including a part of the nucleic acid sequence at the 1080296 to 1081222bp position of the genome (GI No: 156932229) or a part of the complementary strand thereof; the primer includes, but is not limited to, any one of the primer set A, the primer set B, the primer set C, the primer set D, and the like. But not limited to, a primer set having a homology of 50% or more with the aforementioned primer sequence or a single sequence in the complementary strand sequence thereof; including but not limited to primer set E, primer set F, primer set G, primer set H, etc.

In the kit of the present invention, the primer set capable of amplifying the sakazakii genome-specific base sequence may or may not comprise one or more loop primers; the loop primer serves as an optional component. The loop primer is LF and/or LB. The primer set comprising the loop primer LF and/or LB includes, but is not limited to, the primer sets A ', B ', E ', G ', H ', etc. In a specific embodiment, the kit of the present invention may comprise 0.4-1.0. mu. mol/L of LF and/or LB loop primers. In a specific embodiment, the sequences of the primer sets are respectively the primers shown by FIP, BIP, F3, B3, LF and LB, or the primers with 50% or more homology to the single primer of the aforementioned sequence or its complementary strand sequence.

The kit also comprises BstDNA polymerase buffer solution, BstDNA polymerase, dNTP solution and Mg ²⁺ (MgSO ₄ Or MgCl ₂ ) And betaine. In a specific embodiment, the enzyme reaction system of the kit comprises 1 XBst DNA polymerase reaction buffer solution and 2-9mmol/L Mg ²⁺ (MgSO ₄ Or MgCl ₂ ) 1.0-1.6mmol/L dNTP, 0.8-2.0 mu mol/L FIP and BIP primers, 0.15-0.3 mu mol/L F3 and B3 primers, 0.16-0.64U/mu L Bst DNA polymerase and 0-1.5mol/L betaine. For example, 1 XBst DNA polymerase reaction buffer can be 1 × Thermopol reaction buffer containing 20mmol/L Tris-HCl (pH8.8), 10mmol/L KCl, 10mmol/L (NH4) ₂ SO4，0.1％Triton X-100，2mM MgSO ₄ . MgSO in 1 XBst DNA polymerase reaction buffer ₄ And magnesium ion Mg in enzyme reaction system ²⁺ And (6) merging.

The kit of the invention also comprises a positive control template. In a specific embodiment, the positive control template includes, but is not limited to, whole genomic DNA, partial genomic DNA of cronobacter sakazakii, or a vector comprising whole genomic DNA or partial genomic DNA of cronobacter sakazakii.

The kit of the invention further comprises a negative control template, and the negative control template comprises but is not limited to double distilled water.

The kit further comprises a color developing agent, wherein the color developing agent comprises but is not limited to calcein, SYBR Green I or hydroxynaphthol blue. When the color developing agent is calcein, the kit also comprises [ Mn ²⁺ ]For example, MnCl ₂ 。

The kit of the invention also comprises double distilled water.

The kit of the invention also comprises a nucleic acid extraction reagent.

The invention also provides a vector, which comprises any one primer selected from the primer groups A-D, E-H, A ', B ', E ', G ' and H '. The vector contains a DNA sequence with specificity of the cronobacter sakazakii, so the vector can be applied to the research fields of microbial taxonomy, comparative genomics, evolution and the like and the application field of microbial detection and the like. The vector may be, but is not limited to, a plasmid vector (e.g., pBR322, pUC18, pUC19, pBluescript M13, Ti plasmid, etc.), a viral vector (e.g., lambda phage, etc.), and an artificial chromosome vector (e.g., bacterial artificial chromosome BAC, yeast artificial chromosome YAC, etc.). For example, vector pBR322-A containing any one primer of primer set A, vector pBR322-B containing any one primer of primer set B, vector pBR322-H 'containing any one primer of primer set H' … …, and the like. A vector lambda phage-A containing any one of the primers of the primer set A, a vector lambda phage-B containing any one of the primers of the primer set B, … … a vector lambda phage-H 'containing any one of the primers of the primer set H', and the like.

The invention also provides application of the primers selected from any one of the primer groups A-D, E-H, A ', B ', E ', G ' and H ' in detecting the cronobacter sakazakii at constant temperature.

The invention also provides application of the kit in constant-temperature detection of Cronobacter sakazakii.

The invention also provides application of the vector in constant temperature detection of Cronobacter sakazakii.

The invention provides a simple, rapid and sensitive method, primer/primer group and detection reagent/kit for detecting cronobacter sakazakii in the technical field of food safety detection, and has great significance for food safety in China. The beneficial effects of the invention include: the method for detecting the cronobacter sakazakii has the advantages of strong specificity, high sensitivity, short detection time, simple result judgment, convenient operation, low cost and the like. Compared with the current common detection method, the isothermal amplification method adopted by the invention can be carried out under the isothermal condition, only a simple isothermal device is needed, expensive instruments in PCR experiments are not needed, and steps such as electrophoresis detection and the like on amplification products are not needed, so the method is very suitable for being widely applied to various social fields including basic food safety detection departments to be popularized and used, and can be fully applied even under the environment with relatively insufficient professional knowledge and skill base in molecular biology. Any combination of the above preferred conditions is within the scope of the present invention based on the general knowledge in the art.

Drawings

FIG. 1 shows the specificity of the isothermal detection method of Cronobacter sakazakii of example 7 of the present invention.

FIG. 2 shows the sensitivity of the Cronobacter sakazakii detection method of example 8 of the present invention.

Detailed Description

The present invention will be described in further detail with reference to the following specific examples and drawings, and the present invention is not limited to the following examples. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected. The procedures, conditions, reagents, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art, except for the contents specifically mentioned below, and the present invention is not particularly limited.

Example 1-6 Cronobacter sakazakii constant temperature reaction System and detection method

The detection is carried out according to the following steps (1) to (3):

(1) extraction of genomic DNA

The cronobacter sakazakii strain for detection is derived from the collection management center of the chinese industrial microbial strains under the number cic 21560 (ATCC 29544). 1mL of the bacterial culture was used to extract genomic DNA and DNA OD using a bacterial nucleic acid extraction kit from Beijing Tiangen bioengineering Co ₂₆₀ /OD ₂₈₀ At a concentration of 1.8, 288 ng/. mu.L.

(2) The method comprises the steps of taking genomic DNA of cronobacter sakazakii to be detected as a template, respectively adopting self-prepared kits (shown in tables 2 and 3) and preparing a reaction system according to the conditions in the table 3, and taking a cronobacter sakazakii specific amplification primer group as a primer to carry out constant-temperature amplification reaction. The primers used in examples 1 to 6 were primer set A, B ', H' (2 loop primers), H '(1 loop primer), H, and G', respectively.

(3) The amplification results were confirmed by electrophoresis, turbidity or color development under the conditions shown in Table 3.

As can be seen from Table 3, the detection method and the primer set and the reaction system adopted by the detection method can well amplify the specific fragment of Cronobacter sakazakii and obtain the detection result. In addition, when the detection is performed by using a detector, the detection effect is good when the reaction time is shortened to 10min (as in example 6). Therefore, the invention can be applied to detecting whether the sample contains cronobacter sakazakii.

According to the method of the above embodiment, the specific fragment of Cronobacter sakazakii can be amplified well and the detection result can be obtained by using the primer sets B to D, the primer sets E to G, and the primer sets A 'and E', respectively.

Example 7 Cronobacter sakazakii specific detection

The non-sakazakii 28 strains (1 to 22, 24 to 29 in table 4 and fig. 1) were collected, and these strains were cultured separately from the sakazakii strain (23 in table 4 and fig. 1), and 1mL of the bacterial solution was taken, and bacterial DNA was extracted using the kit IA, and LAMP amplification (primer set a) and color developer addition observation were performed separately with reference to the reaction system and conditions of example 1.

The results are shown in Table 4 and FIG. 1, in FIG. 1, 1-22 are respectively Staphylococcus aureus, Staphylococcus aureus Kindoderma subspecies, Staphylococcus epidermidis, Rhodococcus equi, Bacillus cereus, Bacillus mycoides, Listeria monocytogenes, Listeria Ennok, Listeria Iseli, Salmonella enterica subspecies, Salmonella enteritidis, Salmonella typhimurium, Salmonella paratyphi B, Shigella dysenteriae, Shigella boydii, Shigella flexneri, Escherichia coli (containing Clostridium botulinum type A gene), pathogenic Escherichia coli, Escherichia coli diarrheal, enterotoxigenic Escherichia coli, Escherichia coli hemorrhagic, and 24-29 are respectively Yersinia enterocolitica, Yersinia pseudotuberculosis, Vibrio vulnificus, Vibrio parahaemolyticus, Yersinia enterocolitica, Yersinia pseudotuberculosis, Vibrio parahaemolyticus, Vibrio, Vibrio freundii and vibrio cholerae, NTC: negative control, 23: cronobacter sakazakii. In fig. 1, only the product after the amplification reaction of the cronobacter sakazakii strain appeared bright green, which was a positive result, as shown in tube No. 23. The other sakazakii strains and the products obtained after the negative control amplification reaction are orange, which are negative results, as shown in tubes No. 1-22 and NTC negative control tube.

As can be seen from the results in fig. 1 and table 4, the detection kit and the detection method of the present invention have good specificity for the cronobacter sakazakii strain, that is, only the cronobacter sakazakii strain is amplified positively, and the other cronobacter sakazakii strains are not amplified negatively.

Preparing a detection kit, wherein the primers adopted in the kit are respectively primer groups B-D, primer groups E-H, primer groups A ', B ', E ', G ', H ' according to the specificity detection method, and the same detection results are respectively obtained, namely, the products after the amplification reaction of the non-sakazakii strains and the negative control are negative results, and the products after the amplification reaction of the sakazakii strains are positive results.

In addition, theoretical analysis was performed on the specificity of each of the primer sets a to D, the primer sets E to H, and the primer sets a ', B ', E ', G ', H ' according to the method described in table 1, and as a result, it was found that, in the case where at most 2 mismatches were allowed in each of the primers, each of the primers in each of the primer sets could not be simultaneously aligned to cronobacter sakazakii, indicating that the specificity of each of the primer sets was good.

Example 8 sensitivity detection

DNA of the bacterium CICC21560 was extracted by the method of example 2, and LAMP amplification (primer set B') and visualization by adding a color-developing agent were carried out by using the kit IIB and by gradient addition of 50ng, 5ng, 500pg, 50pg, 5pg, 500fg and 50fg DNA according to the method of example 2 of Table 3 under other reaction conditions. As shown in fig. 2, 1 to 7 are 50ng, 5ng, 500pg, 50pg, 5pg, 500fg and 50fg, respectively, NTC: and (5) negative control. In FIG. 2, the reaction products of 50ng, 5ng, 500pg, 50pg, and 5pg treatments appeared bright green and as positive results, the reaction products of 500fg and 50fg treatments and the negative control appeared orange and as negative results. The detection results show that DNA of a minimum of 5pg (about 1000 bacteria) can be detected in each reaction tube, and the sensitivity is high.

According to the detection method, other steps and conditions are the same, the primer groups A-D, the primer groups E-H and the primer groups A ', E', G 'and H' are respectively used, DNA as low as 5 pg-500 fg in each reaction tube can still be detected, and the detection sensitivity is higher.

Example 9 commonality testing

Theoretical analysis of the versatility of the primer sets a to D, E to H, and a pair of primer sets a ', B ', E ', G ', H ' was conducted according to the method described in table 1, and as a result, it was found that the primer regions of the respective primer sets completely match the genomic sequences of the three sakazakii strains (GI nos. 156932229, 449306535, and 389839000, respectively), and that the primers can be theoretically used for the detection of the three sakazakii strains, indicating that the versatility of the respective primer sets is good.

TABLE 1 analysis of the versatility and specificity of primers in the existing detection method of Cronobacter sakazakii

Note: a) the sequence between primers F3 and B3 in the patent was Bowtie aligned with three genomes of Cronobacter sakazakii (GI Nos. 156932229, 449306535 and 389839000, respectively) to determine the position of the detection region in the GI No. 156932229 genome (asterisk indicates that the sequence between primers F3 and B3 can be aligned to 7 repetitive regions in the GI No. 156932229 genome, and only 1 region is listed in the table). The amplification regions of the four sets of primers in patent ZL201310287426.5 cannot be located, and the genes of the amplified target genes are not shown in the text. b) And performing Blast comparison on the detection region sequences in public database resources, wherein the primer regions are completely matched and have good universality. c) And performing Blast comparison on the detection region sequences in public database resources, wherein the higher the matching degree of the primer regions is, the poorer the specificity is.

TABLE 2 types and major components of kit for isothermal detection of cronobacter sakazakii

Table 3 examples 1 to 6 reaction conditions and results of detection in the method for isothermal detection of cronobacter sakazakii according to the present invention

TABLE 4 strains used in the test and the results

Note: a) CGMCC: china general microbiological culture Collection center, CICC: china center for preservation and management of industrial microbial strains, CMCC: china center for preservation and management of bacterial strains. b) +: positive result, -: and (5) negative result.

Sequence listing

<110> Shanghai Wangwang food group Co., Ltd., Shanghai Marine industry and technology research institute

Rapid constant-temperature detection method, primer group and kit for sakazakii

<160> 38

<170> SIPOSequenceListing 1.0

<210> 1

<211> 18

<212> DNA

<213> Artificial sequence ()

<400> 1

ggcgggttta tactggat 18

<210> 2

<211> 20

<212> DNA

<213> Artificial sequence ()

<400> 2

cactgaaatg tcacaagcta 20

<210> 3

<211> 39

<212> DNA

<213> Artificial sequence ()

<400> 3

cggcgtatct aaatcaaatg cccttagccc aacacgatt 39

<210> 4

<211> 44

<212> DNA

<213> Artificial sequence ()

<400> 4

gcgacgttat ctaatattgt aaggcgccag aaaccaaatc attg 44

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence ()

<400> 5

atagtgtttt gttttccggg 20

<210> 6

<211> 18

<212> DNA

<213> Artificial sequence ()

<400> 6

acgagggtaa aggacaaa 18

<210> 7

<211> 41

<212> DNA

<213> Artificial sequence ()

<400> 7

gcttcagttt ggttgatggg aaaagaagtg gtaaggaagc t 41

<210> 8

<211> 40

<212> DNA

<213> Artificial sequence ()

<400> 8

tttctccgtg acagtgaata cgaaactgcc agtgaagtga 40

<210> 9

<211> 19

<212> DNA

<213> Artificial sequence ()

<400> 9

ggatcatgga gcaatttcc 19

<210> 10

<211> 18

<212> DNA

<213> Artificial sequence ()

<400> 10

tcattttcgc acaaagcc 18

<210> 11

<211> 39

<212> DNA

<213> Artificial sequence ()

<400> 11

aagaaccaga ctggcgtaac tttttcagtc ctttctccg 39

<210> 12

<211> 42

<212> DNA

<213> Artificial sequence ()

<400> 12

ccctcgtaag tagcgaattt agccgatttc cttgaagcag tg 42

<210> 13

<211> 18

<212> DNA

<213> Artificial sequence ()

<400> 13

gcgaatttag cacccaaa 18

<210> 14

<211> 18

<212> DNA

<213> Artificial sequence ()

<400> 14

ggacatcagc aaaccttc 18

<210> 15

<211> 41

<212> DNA

<213> Artificial sequence ()

<400> 15

gacgcaggct attcattttc gttttgacca tttaccactg c 41

<210> 16

<211> 43

<212> DNA

<213> Artificial sequence ()

<400> 16

catagttcaa aaagcctgtg tcaagagctg atactgctac tgc 43

<210> 17

<211> 18

<212> DNA

<213> Artificial sequence ()

<400> 17

ggcgggttta tactggat 18

<210> 18

<211> 19

<212> DNA

<213> Artificial sequence ()

<400> 18

tcaatggcac tgaaatgtc 19

<210> 19

<211> 40

<212> DNA

<213> Artificial sequence ()

<400> 19

cggcgtatct aaatcaaatg cccttagccc aacacgattc 40

<210> 20

<211> 43

<212> DNA

<213> Artificial sequence ()

<400> 20

gcgacgttat ctaatattgt aaggcgagca atagccagaa acc 43

<210> 21

<211> 18

<212> DNA

<213> Artificial sequence ()

<400> 21

taccctcgta agtagcga 18

<210> 22

<211> 18

<212> DNA

<213> Artificial sequence ()

<400> 22

ggacatcagc aaaccttc 18

<210> 23

<211> 41

<212> DNA

<213> Artificial sequence ()

<400> 23

cgcacaaagc caatttcgat tttagcaccc aaattttgac c 41

<210> 24

<211> 43

<212> DNA

<213> Artificial sequence ()

<400> 24

catagttcaa aaagcctgtg tcaagagctg atactgctac tgc 43

<210> 25

<211> 18

<212> DNA

<213> Artificial sequence ()

<400> 25

agcaatttcc catcaacc 18

<210> 26

<211> 18

<212> DNA

<213> Artificial sequence ()

<400> 26

gggtgctaaa ttcgctac 18

<210> 27

<211> 39

<212> DNA

<213> Artificial sequence ()

<400> 27

aagaaccaga ctggcgtaac tgaagccatt tttcagtcc 39

<210> 28

<211> 43

<212> DNA

<213> Artificial sequence ()

<400> 28

ccgtttttaa tgtcacttca ctggtacgag ggtaaaggac aaa 43

<210> 29

<211> 20

<212> DNA

<213> Artificial sequence ()

<400> 29

atagtgtttt gttttccggg 20

<210> 30

<211> 19

<212> DNA

<213> Artificial sequence ()

<400> 30

atggtcaaaa tttgggtgc 19

<210> 31

<211> 42

<212> DNA

<213> Artificial sequence ()

<400> 31

gagaaaggac tgaaaaatgg cttctaagga agctctgggg at 42

<210> 32

<211> 38

<212> DNA

<213> Artificial sequence ()

<400> 32

ccagtctggt tcttccgttt acgagggtaa aggacaaa 38

<210> 33

<211> 20

<212> DNA

<213> Artificial sequence ()

<400> 33

taaggcagcg aagatacagc 20

<210> 34

<211> 20

<212> DNA

<213> Artificial sequence ()

<400> 34

cattgacgtt acgccagtct 20

<210> 35

<211> 20

<212> DNA

<213> Artificial sequence ()

<400> 35

taaggcagcg aagatacagc 20

<210> 36

<211> 21

<212> DNA

<213> Artificial sequence ()

<400> 36

cgtattcact gtcacggaga a 21

<210> 37

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 37

ggttgatggg aaattgctcc at 22

<210> 38

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 38

taatgtcact tcactggcag tt 22

Claims

1. A rapid constant temperature detection method aiming at Cronobacter sakazakii and not used for diagnosis is characterized by comprising the following steps:

(1) extracting genome DNA from a sample to be detected;

(2) taking the genome DNA as a template, taking a primer group capable of amplifying the specific base sequence of the Cronobacter sakazakii genome as a primer, and carrying out constant-temperature amplification reaction in an enzyme reaction system;

(3) determining whether the sample to be detected has cronobacter sakazakii or not by judging whether the reaction result is positive or not;

wherein the cronobacter sakazakii genome specific alkali sequence is a sequence of 1080296-1081222 bp bits of the cronobacter sakazakii genome with the GI number of 156932229;

wherein the primer group capable of amplifying the specific base sequence of the cronobacter sakazakii genome is selected from the primer group E or E';

primer set E:

the upstream outer primer F3_ E: 5'-GGCGGGTTTATACTGGAT-3' (SEQ ID NO: 17);

downstream outer primer B3_ E: 5'-TCAATGGCACTGAAATGTC-3' (SEQ ID NO: 18);

a downstream inner primer BIP _ E: 5'-GCGACGTTATCTAATATTGTAAGGCGAGCAATAGCCAGAAACC-3' (SEQ ID NO: 20);

a primer set E':

upstream outer primer F3_ E: 5'-GGCGGGTTTATACTGGAT-3', respectively;

downstream outer primer B3_ E: 5'-TCAATGGCACTGAAATGTC-3', respectively;

the downstream inner primer BIP _ E: 5'-GCGACGTTATCTAATATTGTAAGGCGAGCAATAGCCAGAAACC-3';

upstream loop primer LF _ E: 5'-TAAGGCAGCGAAGATACAGC-3' (SEQ ID NO: 35).

2. The method of claim 1, wherein in step (2), the enzymatic reaction system comprises: 1 XBst DNA polymerase reaction buffer, 2-9mmol/L Mg ²⁺ 1.0-1.6mmol/L dNTP, 0.8-2.0 μmol/L FIP _ E and BIP _ E primers, 0.15-0.3 μmol/L F3_ E and B3_ E primers, 0.16-0.64U/μ L Bst DNA polymerase, 0-1.5mol/L betaine, and 0.4-1.0 μmol/L LF _ E primer is included or excluded.

3. The method of claim 1, wherein the isothermal amplification reaction is performed by a reaction sequence comprising: incubating for 10-90 min at 60-65 ℃; ② terminating the reaction for 2-20 min at 80 ℃.

4. The primer in the rapid constant temperature detection method for the cronobacter sakazakii is characterized by comprising a primer group capable of amplifying a specific base sequence of the cronobacter sakazakii genome, wherein the specific base sequence of the cronobacter sakazakii genome is a part of a nucleic acid sequence with the GI number of 156932229 at the 1080296-1081222 bp position of the cronobacter sakazakii genome or a part of a complementary strand thereof;

wherein the primer group capable of amplifying the Cronobacter sakazakii genome-specific base sequence is selected from the primer group E or E';

primer set E:

upstream outer primer F3_ E: 5'-GGCGGGTTTATACTGGAT-3' (SEQ ID NO: 17);

downstream outer primer B3_ E: 5'-TCAATGGCACTGAAATGTC-3' (SEQ ID NO: 18);

a primer set E':

upstream outer primer F3_ E: 5'-GGCGGGTTTATACTGGAT-3', respectively;

downstream outer primer B3_ E: 5'-TCAATGGCACTGAAATGTC-3', respectively;

upstream loop primer LF _ E: 5'-TAAGGCAGCGAAGATACAGC-3' (SEQ ID NO: 35).

5. A rapid isothermal detection kit for Cronobacter sakazakii, comprising the primer of claim 4.

6. The kit of claim 5, further comprising Bst DNA polymerase reaction buffer, Bst DNA polymerase, dNTP solution, Mg ²⁺ And one or more of betaine.

7. The kit of claim 5, wherein the enzymatic reaction system of the kit comprises: 1 XBst DNA polymerase reaction buffer, 2-9mmol/L Mg ²⁺ 1.0-1.6mmol/L dNTP, 0.8-2.0 μmol/L FIP _ E and BIP _ E primers, 0.15-0.3 μmol/L F3_ E and B3_ E primers, 0.16-0.64U/μ L Bst DNA polymerase, 0-1.5mol/L betaine, and 0.4-1.0 μmol/L LF _ E primer is included or excluded.

8. Use of a primer for isothermal detection of cronobacter sakazakii for non-diagnostic purposes, wherein the primer is according to claim 4.

9. Use of a kit according to any one of claims 5 to 7 for isothermal detection of cronobacter sakazakii for non-diagnostic purposes.